EBMT 2008 Handbook Haematopoirtic stem cells transplantation

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APPENDIX

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EBMT Officers

EBMT Coordination Offices

President Dietger Niederwieser

EBMT Secretariat & JACIE Accreditation Office

Division of Haematology & Oncology University Hospital Leipzig Johannisallee 32 A 04103 Leipzig GERMANY Tel: +49 341 971 3050 Fax: +49 341 971 3059 [email protected]

Hospital Clínic Villarroel 170, 08036 Barcelona SPAIN Tel: +34 93 454 9543 Fax: +34 93 453 1263 [email protected] [email protected]

Secretary Per Ljungman

12th Floor Tower Wing Guy's Hospital Great Maze Pond London SE1 9RT UNITED KINGDOM

Department of Haematology Karolinska University Hosp/Huddinge 141 86 Stockholm SWEDEN Tel.: +46 8 585 80000 Fax: +46 8 774 8725 [email protected]

Treasurer (2002 – 2008) Harry Schouten University Hospital Maastricht Dept. Hematology P.O. Box 58000 6202 AZ Maastricht The Netherlands Tel.: +31 43 3 877025 Fax: +31 43 3 875006 [email protected]

EBMT Clinical Trials & Registry Office

Registry Tel: +44 207 188 8408 Fax: +44 207 188 8411 [email protected]

Clinical Trials Tel: +44 207 188 8402 Fax: +44 207 188 8406 [email protected]

EBMT Data & Study Office Faculté de Médecine Saint-Antoine 27, rue Chaligny, 75571 Paris Cedex 12 FRANCE Tel: +33 1 40 46 95 07 Fax: +33 1 40 46 96 07 [email protected]

EBMT Clinical Trials & Study Office Department of Medical Statistics & Bioinformatics, Postzone S-05-P LUMC, PO Box 9600 2300 RC Leiden THE NETHERLANDS [email protected] HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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EBMT Committees Cord Blood Committee Chair: Eliane Gluckman

Outreach Committee Co-Chairs: Vladimir Koza

[email protected]

[email protected]

Eliane Gluckman Developmental Committee Co-Chairs: Katarina Leblanc Willem Fibbe

Prospective Clinical Trials Chair: Gérard Socié

[email protected]

[email protected]

Education Committee Chair: Tamás Masszi

Quality Assessment of Autografts Chair: Francesco Lanza

[email protected]

[email protected]

JACIE Executive Committee President: Ineke Slaper-Cortenbach

Registry Committee Chair: Carmen Ruiz de Elvira

[email protected]

[email protected]

[email protected]

Vice-President: Jane Apperley [email protected] Nuclear Accident Committee Chair: Ray Powles [email protected]

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2008 REVISED EDITION

Statistical Committee Chair: Myriam Labopin [email protected]

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EBMT Working Parties

Please refer to the following website address:

http://www.ebmt.org/5WorkingParties/wparties1.html

EBMT Nurses Group Board President Erik Aerts

Secretary Michelle Davies

University Hospital Zürich Dept. of Medicine 8091 Zürich SWITZERLAND Tel.: +41 44 255 2633 Fax: +41 44 255 9781 [email protected]

Adult Leukaemia Unit Christie Hospital Wilmslow Road Manchester M20 4BX UNITED KINGDOM [email protected]

President Elect Arno Mank Academic Medical Centre Dept. Hematology Meibergreef 9, NL-1105 AZ Amsterdam NETHERLANDS Tel: +31 20 566 7905 Fax: +31 20 566 9030 [email protected]

Treasurer Joachim Blankart AK St. Georg Dept. of Hematology G3, STE Lohmuehlenstrasse, 5 20099 Hamburg GERMANY [email protected]

HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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Affiliated Organisations/Websites International Organisations: ASBMT ASCO ASH BMDW EBMT EHA EORTC ESH EUROCORD IBMTR ISCT Europe ISEH NETCORD WMDA

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www.asbmt.org www.asco.org www.hematology.org www.bmdw.org www.ebmt.org www.ehaweb.org www.eortc.be www.esh.org www.eurocord.org www.ibmtr.org www.celltherapysociety.org/Related_Organizations/ ISCT_Europe/ www.iseh.org www.netcord.org www.worldmarrow.org


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Published by

Via Martin Piaggio, 17/6 16122 Genoa, Italy tel +39 10 83794229 - fax +39 10 83794260 [email protected] - www.accmed.org

© 2008 EBMT and ESH Printed by Litoprint (Genoa, Italy)

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CHAPTER 1

Stem cell transplant organisations

J. Apperley, A. Keating

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CHAPTER 1 • Transplant societies

Haematopoietic stem cell transplantation (HSCT) is a professional medical activity and like other medical specialities, lends itself to the development of groups of individuals with similar and/or overlapping interests. It is therefore not surprising that there are a number of national and international HSCT societies that carry out activities with common aims, i.e. the continuous development of their speciality and improvement in outcome for patients. Such activities usually revolve around one or more annual meetings at which new information can be presented, discussed and disseminated. The organisations may have accompanying infrastructures for education, professional standards, production of guidelines and lobbying. However there is one additional characteristic of the HSCT societies that sets them apart from many other organisations and that is the collection, analysis and reporting of outcome of all transplants performed by their member centres. Worldwide there are three international HSCT societies - European Group for Blood and Marrow Transplantation (EBMT) - Center for International Blood and Marrow Transplantation Research (CIBMTR) - Asia Pacific Blood and Marrow Transplantation (APBMT). There are several national HSCT societies in Europe (Table 1) and there are welldeveloped national groups elsewhere including those in Australia, Canada, Japan, New Zealand and North America. The largest of these is the ASBMT in the USA and their official journal is Biology of Blood and Marrow Transplantation.

Table 1: National HSCT Societies and Registries in Europe Country

E-mail address

Austrian SCT Registry

[email protected]

Czech SCT Registry

[email protected]

Societé Francaise de Greffe de Moelle et de Thérapie Cellulaire

[email protected]

Deutsches Register für Stammzelltransplantationen (DRST)

[email protected]

Italian National BMT Registry (GITMO)

[email protected]

Dutch National Registry (TYPHON)

[email protected]

Spanish Haematopoietic Transplantation Group & Cellular Therapy (GETH)

[email protected]

Swiss National Stem Cell Transplant Registry (STABMT)

[email protected]

Turkish Transplant Registry

[email protected]

British Society of Blood and Marrow Transplant

[email protected]


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Laboratory processing of haematopoietic stem cells typically involves a group of professionals with skills that are complementary to those of the average transplant physician and they are represented by the International Society of Cellular Therapy. There is clearly a considerable overlap with the interests of the physician orientated HSCT societies and indeed these professional groups work closely together, most notably in the development of an accreditation system that has established and inspects against, a series of standards for the clinical, collection and laboratory facilities, i.e. FACT-JACIE. The principles of this accreditation system will be discussed in Chapter 4 and will not be discussed in detail in this Chapter. All these societies (EBMT, CIBMTR, APBMT, ISCT) have a similar structure in which a Board of Directors (usually elected by the professional membership) provide and manage the infrastructure, which in turn permits the work of the society. Although the terminology may differ a number of committees or working groups oversee the scientific work and parallel groups of individuals focus on policy issues such as education, meeting organisation, legal and regulatory matters etc. In addition the provision of donors (bone marrow, peripheral blood derived stem cells, cord blood, cellular sub-populations) has also necessitated the development of local and national donor organisations, whose focus is rather different from the professional societies. They have established mechanisms for the recruitment, assessment, provision and follow-up of donors, and are all members of an umbrella organisation, the World Marrow Donor Association (WMDA), that oversees the development of standards and guidelines for the management of these volunteer individuals. NETCORD was developed to share best practice in cord blood procurement. In order to facilitate rapid identification of suitable HLA-matched unrelated donors for individual patients, all the donor registries submit their typing data to an organisation known as Bone Marrow Donors Worldwide (BMDW).

1. European Group for Blood and Marrow Transplantation (EBMT) 1.1. History Over the past three decades the EBMT has grown from a small group of enthusiasts to a society representing more than 500 teams from 57 countries. In 1975 teams from Paris, Leiden, London and Basel met for the first time to discuss general problems and individual patients, and to draw up protocols with a view to improving results. They continued to meet regularly in the Swiss or French Alps and the group, then called the European Cooperative Group for Bone Marrow Transplantation, attracted an ever-increasing number of participants. In 1979, the European Foundation for Bone Marrow Transplantation (EBMT) was formally established with legal status in 20

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the Netherlands. Four Working Parties were created, each with its own registry, i.e. Aplastic Anaemia, Acute Leukaemia, Inborn Errors and Transplantation Immunology. In 1989, a new constitution created the “European Group for Bone Marrow Transplantation”, the abbreviation EBMT being retained. Subsequently, the working parties were restructured and new ones added, i.e. Acute Leukaemia, Aplastic Anaemia, Autoimmune Disease, Chronic Leukaemia, Immunobiology, Inborn Errors, Infectious Diseases, Late Effects, Lymphoma, Nursing, Paediatrics and Solid Tumours. In 1995 the name of the group was changed once more to “European Group for Blood and Marrow Transplantation”, though the acronym “EBMT” again remained. 1.2. Aims The overall objective of the EBMT is to improve the outcome of HSCT by a number of connected activities. The most long-standing of these activities is the collection, validation and analysis of outcome data of all transplants performed by member teams. However the work of the group has expanded to include accreditation, education and outreach, prospective clinical trials and regulatory affairs. 1.3. Organisation EBMT members may be full members, associate members, individual members or corporate patrons. Full and associate memberships are granted to teams rather than individuals. EBMT members have the right to propose and elect office holders, stand for election, attend annual meetings and participate in the Working Parties and EBMT studies. Full membership implies a duty to report individual transplant data to the EBMT registries. The EBMT Board is composed of elected individuals including the President, Secretary, Treasurer and the Working Party Chairpersons. The Board co-ordinates EBMT activities and is responsible for the budget. The organisational structure is shown in Figure 1. The Working Parties are the heart of EBMT, performing retrospective and prospective studies relating to their particular disease or other interest. However the Board of the EBMT have long recognised the need for activities that cross Working Party activities and this has led to the development of EBMT committees. Unlike the Working Party Chairs, who are elected by the member centres and who have permanent positions on the EBMT Board, the Chairs of the committees are appointed by the Board. Committees are designed to be responsive to the needs of the organisation and can be dissolved which there is no longer any requirement for the particular activity. The journal Bone Marrow Transplantation is the official journal of the EBMT.


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Figure 1: Organisational structure of EBMT EBMT Board Working Parties

Committees

Registries

Administration

Acute Leukaemia Aplastic Anaemia Autoimmune Chronic Leukaemias Immunobiology Infectious Diseases Inborn Errors Late Effects Lymphoma Nurses group Paediatrics Solid tumours

Accreditation Clinical Trials Education Developmental Graft Engineering Nuclear Accidents Outreach Statistical

Data Managers Study Coordinators Statisticians

EBMT Secretary EBMT Treasurer Executive Secretary JACIE Office

1.4. Committees The current committees are shown in Table 2. The work of some of these committees will be discussed in more detail below. 1.4.1. Education and Outreach The Education committee was established in 1995 with the intention to provide a more structural approach to training in HSCT. Together with the European School for Haematology (ESH) they have successfully organised the annual residential training course in which senior transplant physicians and scientists interact in formal and informal settings with less experienced colleagues. This committee have also issued this ESH-EBMT transplant manual, now in its fifth edition. Most recently this committee has developed an Outreach project to assist transplant units in economically disadvantaged countries. The EBMT are not alone in this concept and are actively integrating their ideas with the ESH, and are aware of similar programmes within the American Society of Hematology (ASH), CIBMTR, the European Hematology Association (EHA) and the World Health Organisation (WHO). This project supports educational initiatives including training courses in clinical and laboratory techniques, exchange programmes, fellowships, clinical trial participation and twinning programmes.

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Table 2: EBMT Committees Sub-committee

Responsibilities

Accreditation

JACIE, audit and activity survey

Developmental

Emerging stem cell therapies for nonhaematological disorders

Education

Training courses, handbook (jointly with ESH), outreach initiatives

Nuclear accident

Establishment of network of units with haematological expertise to respond to the need for care of neutropenic patients in the event of nuclear accidents

Outreach

Dissemination of excellence in HSCT

Prospective clinical trials

Infrastructure for prospective clinical trials

Quality assessment of autografts

Standardisation of assessment of haematopoietic cell product quality

Statistics

Ensure statistical accuracy within EBMT studies. Develop new statistical tools, production of guidelines

1.4.2. Prospective Clinical Trials This committee was established in 2003 to assist the EBMT in their progress from a retrospective study organisation to a prospective clinical trials group. The introduction into European Legislation of the EU Directive on Clinical Trials (2001/20/EC) has highlighted the necessity of creating an appropriate infrastructure for the conduct of these trials. Most recently the EBMT together with the CIBMTR and the University of Central Lancashire (UCLAN) have been successful in obtaining funding from the European Commission to develop international prospective clinical trials within the transplant community, a project that has the acronym CLINT. 1.4.3. Accreditation The Accreditation committee has a number of diverse functions, which have developed over many years. The committee was originally responsible for a simple form of accreditation for allogeneic sibling transplant in which centres requested accreditation on the basis of performance of a minimum number of transplants annually. Each year, one-third of centres is chosen at random and alerted to the possibility of an audit of data and centre validation. Later, a proportion of these “at risk” centres are visited by senior transplant physicians and data are checked at source.


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One of the most valuable activities of this committee and indeed of EBMT is also one of the simplest. In 1990, Professor Alois Gratwohl began to collect information relating to the annual transplant activity of all transplant centres in Europe (including those centres that are not EBMT members). As a result the EBMT now hold important data relating to activity, geographical variation, changing trends and the development of new technologies. Activity is published annually and has proved invaluable to the funding agencies (public and private) in identifying transplant needs. More recently the bulk of the work if the accreditation committee has been the development and introduction of the FACT-JACIE accreditation programme for all transplant units. This is discussed in more detail in Chapter 4. 1.5. Data collection and data-flow Since its inception the essence of EBMT has been to evaluate and optimise the outcome of patients treated by HSCT. In order to do this it was always deemed necessary to collect patient data, and so the EBMT registry was formed. As of today the registry holds information on almost 300,000 transplants performed by the member teams, and this has proved an invaluable resource in the analysis of factors affecting transplant outcome, in identifying complications and their optimal treatment, in determining trends in management and in evaluating new technologies. Member teams are required to report all their transplant activity. “Minimal Essential Data” (MED) are collected on a number of different forms. MED-A data form the basis of the registry. Relatively simple and limited data collected in this way allow the analyses of overall survival, disease free survival, transplant related mortality, and relapse risk. These data are identical to those collected by the CIBMTR on their Transplant Essential Data (TED) form, and the facility exists to allow cross reporting to both registries. More detailed information is collected on MED-B forms which relate to the specific disease and to the procedure (allograft or autograft). The submission of these data by members is voluntary as opposed to MED A data which are obligatory. Information relating to specific studies is collected via MED-C forms. Until recently the term “form” literally referred to hard copy in virtually all cases. Paper forms were sent to the EBMT and transcribed onto a central electronic database, but the complexity of the programme and the relative lack of familiarity with computers at the time precluded widespread use of this approach. The EBMT has now developed an on-line Internet based reporting system, Project Manager Internet Server or ProMISe. Data reported in this way are received simultaneously by the EBMT registries in Genoa, London, Leiden and Paris but can also be downloaded for local use. This system has revolutionised data reporting with increasing numbers of centres adopting ProMISe. In 2006, 458 users from 364 centres accessed the database to enter data for more than 25,000 transplants. 24

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The EBMT has welcomed the development of national transplant societies and has understood the desire of some of these groups to have their own national databases. In the past, for member centres in countries with national registries, data flowed from the centre to the national registry and then to EBMT. Centres in countries without a national registry reported directly to EBMT. Now if the centre is utilising ProMISe, the data are deposited simultaneously in the national and EBMT registries.

2. Center for International Blood and Marrow Transplantation Research (CIBMTR) 2.1. History The CIBMTR is a relatively new organisation having developed in 2004 from an affiliation of the International Blood and Marrow Transplant Registry (IBMTR), a sister organisation of EBMT based at the Medical College of Wisconsin, and the research interests of the US National Marrow Donor Program (NMDP). The IBMTR was established in 1972 to link transplant centres internationally and to collect data derived from allogeneic transplants for the purposes of analysis and reporting. As autografting became more extensively used these transplants were added to the IBMTR databases but collection was confined to procedures performed in North and South America. The NMDP was established in 1987 to facilitate the search, identification, and procurement of volunteer unrelated donor haematopoietic stem cells. At that time the NMDP also had the remit to collect and analyse data emanating from these alternative donor transplants, a task which has now been subsumed into the activities of CIBMTR. The NMDP, like several of the unrelated donor registries, also maintains a tissue biobank of donor and recipient samples. The CIBMTR now has 459 members in 52 countries. Based on data collected in the Centers for Disease Control Hospital Surveys and the US Government Accounting Office and worldwide surveys of transplant activity, approximately 40% of allogeneic transplants worldwide and about 50% of autografts performed in North and South America are registered with the CIBMTR. 2.2. Aims The CIBMTR is a clinical research programme whose major mission is to provide a resource of data and statistical expertise to the HSCT community. They aim to define key areas for future research, secure funding for research, design and implement clinical studies and make available their resources including the clinical database and the tissue bank.


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2.3. Organisation The organisational structure of the CIBMTR is shown in Figure 2. The Affiliation Board and Executive Director have managerial oversight. The Chief Scientific Director has primary responsibility for administrative and scientific operations. CIBMTR Scientific Working, Executive and Advisory Committees provide policy and scientific oversight for this work. The activities of the CIBMTR are funded primarily by grants and contracts from the US government. The CIBMTR Assembly is the voting membership, comprised of a single representative from each CIBMTR Research Center. The CIBMTR Advisory Committee meets biannually to review scientific and other activities of the CIBMTR. The CIBMTR Executive Committee is a subcommittee of the Advisory Committee that provides ongoing advice and counsel to the CIBMTR Statistical Center between meetings of the Advisory Committee.

Figure 2: Organisational structure of CIBMTR

NMDP - Research

Affiliation Committee

Assembly

Executive Director

Advisory Committee

Executive Committee

MCW - IBMTR

Senior Research Advisor

Chief Scientific Director Working/Steering Committees Statistical Methodology

Clinical Trials Support

BMT CTN*

Immunobiology

RCI BMT

Observational Research

Clinical Outcomes

Health Policy

* The BMT CTN Data and Coordinating Center is a Collaboration of CIBMTR, NMDP and the EMMES Corporation; NMDP: National Marrow Donor Program; IBMTR: International Bone Marrow Transplant Registry; MCW: Medical College of Wisconsin; BMT CTN: Blood and Marrow Transplant Clinical Trials Network; RCI BMT: Resource of Clinical Investigations in Blood and Marrow Transplantation 26

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2.4. Committees The Scientific Working Committees are analogous to the EBMT Working Parties and set priorities for observational studies. There are 17 Working Committees: Acute Leukaemia; Chronic Leukaemia; Lymphoma; Plasma Cell Disorders; Solid Tumours; Paediatric Cancer; Non-malignant Marrow Disorders; Immune Deficiencies/Inborn Errors of Metabolism; Autoimmune Diseases; Graft Sources and Manipulation; Graft versus Host Disease; Late Effects and Quality of Life; Immunobiology; Infection and Immune Reconstitution; Regimen-related Toxicity and Supportive Care; Health Services and Psychosocial Issues; Donor Health and Safety. Membership on CIBMTR Working Committees is open to anyone willing to take an active role in studies using CIBMTR data and/or resources. The CIBMTR also functions as the Data and Coordinating Centre of the US Blood and Marrow Transplant Clinical Trials Network (BMT CTN). The BMT CTN was established by the National Heart, Lung, and Blood Institute (NHLBI) and the National Cancer Institute (NCI) in order to design, develop, and execute prospective clinical trials to improve the safety, applicability, and efficacy of HSCT. Sixteen core centres were awarded cooperative agreements with the NHLBI/NCI and a broader network of over 60 transplant centres have participated in eight prospective trials enrolling over 1,500 patients in the first four years of activity. 2.5. Data collection The CIBMTR collects data at two levels. Registration data (Transplant Essential Data–TED) are identical to the Med-A data of the EBMT and the facility exists to allow cross reporting to both registries. All CIBMTR teams contribute registration data. Research data are collected on subsets of registered patients and includes comprehensive pre- and post-transplant clinical information.

3. The Asia Pacific Blood and Marrow Transplantation (APBMT) The Asia Pacific Blood and Marrow Transplantation (APBMT) group is an organisation, akin to EBMT and CIBMTR, which allows HSCT physicians in Asian countries to share their experience and develop co-operative studies. The APBMT group was established in 1990 and initial meetings were held in China and Japan. Since 1994 annual meetings have been an integral part of the organisation. The APBMT aims to promote all aspects associated with HSCT including basic and clinical research. A registry to collect the results of HSCT performed in member centres began in 2006.

4. Worldwide Network for Blood and Marrow Transplantation (WBMT) In 2007 the concept developed of an organisation that would unite the three


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international HSCT societies (EBMT, CIBMTR and APBMT) together with the WMDA with the aim of unifying data collection and reporting systems, developing harmonised guidelines, providing an effective professional lobby to the various national and international regulatory agencies and strengthening the research database. The name Worldwide Network for Blood and Marrow Transplantation was adopted and the constitution is currently in development.

5. International Society for Cellular Therapy (ISCT) 5.1. History ISCT was established in 2002 as a professional organisation for those working or interested in cell-based research, processing, manipulation, and clinical translation. It evolved from the US and European branches of the International Society for Hematotherapy and Graft Engineering (ISHAGE) when the change in name and focus was considered essential to encompass the use of cellular therapy for a wider range of diseases and applications. ISHAGE had itself been established in 1991 when it had become increasingly clear that an organisation was required to represent the interests and needs of those involved in graft manipulation. 5.2. Aims The mission of ISCT is to provide a global forum for the development and validation of standardised technology and for representation of the membership to other professional organisations and regulatory and governmental bodies. They also have a strong commitment to education and training. 5.3. Organisation In contrast to the other HSCT organisations, the membership of the ISCT is largely composed of individuals although there are arrangements for laboratory and corporate membership. In 2007, the Society had over 1200 members from 6 continents. Leadership of the ISCT is provided by an Executive Committee which manages the affairs of the Society and oversees the work of the committees and working groups. An advisory committee, composed largely of previous elected officers, provides input from the membership to the Executive Committee and also advise on the long-term strategy of the Society. ISCT is composed of several standing committees, representing all facets of ongoing activities of the Society, and short-term working groups, designed to address immediate concerns within cell therapy, drafting position statements and guidance for broad dissemination or submission to the FDA. The current committees and groups are shown in Table 3. 28

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Table 3: Working Parties and Standing Committees of ISCT Working Parties

Standing Committees

- Microbial testing - Storage & transportation - Devices - Facility sanitisation - Homologous use - Release testing

- Cell therapy commercialisation - Cell and tissue evaluation - Education and publication - Gene therapy - Haematopoietic stem cells - Immunotherapy & dendritic cells - Laboratory practices - Regulatory affairs - Europe - Regulatory affairs - America - Regulatory affairs - Asia - Mesenchymal and tissue stem cells

ISCT organises an annual meeting of diverse groups of investigators, practitioners, and technologists with a focus on research and education. Cytotherapy is the official journal of ISCT.

6. World Marrow Donor Association (WMDA) 6.1. History The WMDA was created in 1994 to address obstacles faced when transplantation involved donors and recipients in different countries. It is a voluntary organisation of representatives of HSCT donor registries, cord blood banks, and other institutions and individuals involved in the use of unrelated donors. Currently the WMDA represents 11.8 million donors in 67 stem cell registries and 292,000 units in 47 cord blood banks from 48 countries. The individual donor registries are too numerous to mention here but include the National Marrow Donor Program (NMDP) in the United States, the Anthony Nolan Trust in the UK and the German. 6.2. Aims To improve and simplify stem cell donation for donors and patients. 6.3. Organisation (Figure 3) The membership of the WMDA is comprised of both donor (blood, marrow and cord blood) organisations and individuals. Much of the work of the WMDA is done through its working groups (Figure 3), which are focussed on developing best practice in the medical, ethical, technical, quality and financial activities of international HCT as they relate to volunteer donors. Importantly, the WMDA established an


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Figure 3: Organisational structure of WMDA President Vice Presidents Past President Chief President Elect President Emeritus North & South America Operating Asia, Australia, Officer Secretary General Pacific Islands, Europe Treasurer

Working Groups Clinical Registries Information Technology Ethics Quality Assurance

Accreditation Committee

Secretaries North & South America Asia, Australia, Pacific Islands, Europe

WMDA Administrative Office Leiden, NL

accreditation program for donor registries in 2003, to certify adherence to WMDA standards for registry operation. These standards cover the general organisation of the registry, donor recruitment and consent procedures, donor characterisation and evaluation, information technology, facilitation of search requests, second and subsequent donations, graft procurement and transport, assessment of donor and transplant outcomes and financial and legal liabilities. The standards are exacting and to date 12 registries have received accreditation. The aim is clearly to extend the process to all their registry members.

7. Alliance for harmonisation of cellular therapy accreditation (AHCTA) The process of HSCT has recently come under extensive scrutiny by national and international regulatory agencies and this has resulted in increasing stringency in the legislation regarding the procurement and use of stem cells. In 2006 it became clear that there was a real risk that regulations could be introduced nationally that would conflict with processes elsewhere in the world. This is particularly relevant to the requirements for import and export of cells. As it is common practice in alternative donor transplantation to use stem cells collected in one country for a patient treated in another country there was some urgency 30

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to present our concerns in a unified manner. Furthermore there was clearly a need for the relevant parties to agree common policies, procedures and protocols. AHCTA was developed to harmonise the standards of all the involved organisations (Figure 4) with the objective of creating a single set of quality, safety and professional requirements for cellular therapy. These standards will comprehensively cover all aspects of the process from assessment of donor eligibility to transplantation and clinical outcome. AHCTA regards regulatory authorities as partners in the application of these global standards, essential to their successful adoption and will endeavour to inform and support these authorities in the area of cellular therapy regulation.

Figure 4: Membership of AHCTA European Federation for Immunogenetics

European Group for Blood & Marrow Transplantation

Foundation for the Accreditation of Cellular Therapy

Joint Accreditation Committee ISCT-EBMT

International NETCORD Foundation

World Marrow Donor Association

International Society for Cellular Therapy (Europe)

8. Bone Marrow Donors Worldwide (BMDW) 8.1. History BMDW began as an initiative of the Immunobiology Working Party of the EBMT in 1988. It represents a voluntary collaborative effort of the stem cell donor registries and cord blood banks to provide centralised information on the HLA phenotypes and other relevant data of unrelated stem cell donors and cord blood units and to make this information easily accessible to the physicians of patients in need of HSCT. The first edition of a publication containing the donor files of eight registries with a total of 155,000 volunteer stem cell donors was


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issued in 1988. The current number of donors and cord blood units in the BMDW database is almost 12 million! 8.2. Aims The original goal was to collect the HLA phenotypes of volunteer stem cell donors and cord blood units, and to maximise the chance of finding a stem cell donor or cord blood unit by providing access to all stem cell donors and cord blood units available in the world. 8.3. Organisation The BMDW Editorial Board consists of one representative of each stem cell donor registry or cord blood bank participating in BMDW, and meets twice a year to discuss achievements and necessary improvements. The office of BMDW remains in Leiden in the Netherlands and the service is provided and managed by Europdonor Foundation. The HLA types and matching programmes are available online. More recently programmes for mismatched donors and cord blood donors have been added together with a haplotype frequency analysis on phenotype data submitted by the participating registries. Only HSCT professionals who have received authorisation and participating registries can access these services. A username and password are required, and a secure protocol has been implemented to encrypt all transmitted data.

9. NETCORD 9.1. History NETCORD is a global non-profit organisation, which was established in 1998 to connect the existing cord blood banks in order to share best practice in this rapidly evolving field. 9.2. Aims The goal of the organisation is to maintain compliance with quality standards so as to ensure uniformity and quality assurance of the cord blood units supplied by their members. In addition they provide access for registered transplant units to a centralised web-based search process, support research to constantly improve the processes of procurement and processing and develop educational programmes to increase the donor pool and the subsequent use of the products. 9.3. Organisation Member cord blood banks are expected to have inventories of at least 1000 cord 32

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blood units of documented high quality. Thus far, NETCORD has 23 members and as of late 2007 has an inventory of 153,830 units, an increase of nearly 30,000 units or 23% in the past 12 months. NETCORD has facilitated the use of over 3,000 cord blood units worldwide both for children and adults. In order to ensure high and uniform quality of all cord blood (CB) units, NETCORD established quality standards in collaboration with FACT for the collection, cryopreservation, storage and release of units. All NETCORD banks are expected to obtain accreditation by NETCORD-FACT and to comply with their quality standards. To facilitate direct searches for CB units by transplant centres and to avoid allocation conflicts for patients, NETCORD developed the Virtual Office (VO), which contains a real-time search programme for compatible and available units. Details include high resolution HLA typing, volume, cell number and infectious disease markers. By submitting a search request to the VO, transplant centres no longer have to search multiple databases of the different cord blood banks, but receive a single unified search report on all acceptable units that are contained in the inventory. The collection of clinical outcome data is coordinated by CIBMTR or Eurocord. The Eurocord registry has operated on behalf of EBMT since 1995. Thanks to the NETCORD-Eurocord collaboration, 3286 cord blood transplantations have been reported to the Eurocord registry from 177 EBMT (64% of cases) and 187 non-EBMT (36% of cases) centers from 43 European and nonEuropean countries. In summary, the intense activity within all these societies reflects the on-going enthusiasm for HSCT and cellular therapy. Further information can be obtained from the specific websites (Table 4).

Table 4: Web addresses of transplant related organisations EBMT

www.ebmt.org

ESH

www.esh.org

CIBMTR

www.cibmtr.org

APBMT

www.apbmt.org

ISCT

www.celltherapysociety.org

WMDA

www.worldmarrow.org

AHCTA

www.ahcta.org

BMDW

www.bmdw.org

NETCORD

www.netcord.org

Eurocord

www.eurocord.org


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*

CHAPTER 2

Biological properties of haematopoietic stem cells

A. Wodnar-Filipowicz

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CHAPTER 2 • Haematopoiesis

1. Introduction Until a few years ago, an interest in stem cells, mostly of haematopoietic origin, was limited to a relatively small representation of scientists and clinicians seeking to understand the role of these rare cells in tissue homeostasis and to utilise their remarkable potential to regenerate an adult tissue following transplantation. Over the last few years, stem cells have captured the imagination of scientists, clinicians and the lay public alike with the promise of representing a future remedy for the major degenerative diseases of our civilisation and even dysfunctions associated with normal ageing. The progress in technologies to detect and enumerate stem cells in vivo led to discovery of stem cells residing in most mammalian tissues, contributing to their generation, homeostasis and probably repair. A major breakthrough has been achieved in the development of methods to propagate human embryonic stem (ES) cells in culture and to drive their in vitro differentiation into specialised human tissues. The concept of cancer stem cells has emerged, as cells responsible for generation and persistence of tumours. Here we discuss the stem cell field by summarising the current knowledge of the phenotype of these cells, their interactions with the microenvironment in which they reside, and the mechanisms regulating their functions. The central place will be taken by haematopoietic stem cells (HSCs), representing the first-discovered, the best-understood and, at present, the only clinically-applicable population of stem cells. We also summarise the current state of knowledge on the functional plasticity of somatic tissue stem cells and on human ES cells. The therapeutic relevance gained from the basic research findings will be emphasised.

2. Stem cell definition Stem cells are defined as a population of undifferentiated cells with the capacity to divide for indefinite periods, to self-renew and to generate a functional progeny of highly specialised cells. This common definition includes cells present in different physical locations and having fundamentally different proliferative properties and functions (Table 1). A fertilised egg (zygote) represents a totipotent stem cell, a cell with unrestricted differentiation potential and the only cell with the capacity to give rise to all cells necessary for the development of foetal and adult organs. ES cells forming a cluster of cells inside the blastocyst are pluripotent stem cells, capable of generating a variety of specialised cell types, but limited in their differentiation potential by the inability to support the development of a foetus. Further specialisation results in generation of multipotent stem cells residing in adult somatic tissues. Their physiological functions are to replenish mature cell populations of the given tissue


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Table 1: Definition of stem cell types Stem cell

Developmental properties

Fertilised egg

- Totipotent - Unrestricted differentiation potential

ES cells

- Pluripotent - Give rise to a variety of specialised cell types

Adult somatic stem cells

- Multipotent - Limited to specific tissues

or organ, and to respond to stress by repairing the damage. HSCs represent the prototype of multipotent adult tissue stem cells (1). In humans, HSCs can be found in cord blood as a result of stem cell migratory properties during foetal development, whereas post-natally, the only organ harbouring HSCs and pursuing active multilineage haematopoiesis is the bone marrow.

3. Characteristics of stem cells in the bone marrow More than 40 years of research on bone marrow-derived stem cells, initiated in the 1960s by Till and McCulloch, marked an ongoing improvement in methods to quantitate and isolate these cells. Assays for clonogenic precursors of the myeloerythroid lineages in vitro, defined as long term culture-initiating cells (LTCICs) and committed colony forming units (CFUs) were followed by the development of a model of immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, which allows the study of the repopulating ability of human haematopoietic cells in vivo (2). These functional assays are paralleled by progress in the phenotypic characterisation of haematopoietic cells by flowcytometry, owing to monoclonal antibodies specifically recognizing cell surface molecules (Figure 1). The phenotypic properties of murine HSCs have been precisely defined as cells devoid of lineage markers and expressing the stem cell antigen (sca1) and the receptor c-kit. The Lin-sca1+c-kit+ (LSK) cell population has self-renewing and long-term repopulating activity in vivo. Other cell surface markers defining the HSC compartment in mice include the tie2 and flt3 receptors and CD150. Characteristically, c-kit, flt3 and tie2 function as receptors for early-acting haematopoietic growth factors: stem cell factor, flt3 ligand and angiopoietin, which act as key positive regulators of haematopoiesis (3). The most primitive human HSCs express CD34 and lack CD38 cell surface antigens and have the capacity to reconstitute a sublethally irradiated NOD/SCID host. The CD34+CD38- HSC compartment, which constitutes ±0.1% of bone marrow cells, is 36

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Figure 1: The immunophenotypic characteristics of human and mouse long-term repopulating haematopoietic stem cells (LT-HSC)

Human HSC

Mouse HSC

LinCD34+ CD38c-kit+ CD133+ SP

Linc-kit+ sca1+ Tie2+ CD150+ CD244CD48SP

LT-HSC

Lin: lineage; SP: side population cells

heterogeneous and contains also c-kit-, flt3-, and CD133-expressing cells. Both mouse and human HSCs are present among the side population (SP) cells which express the drug transporter protein Abcg2 and therefore have the ability to actively efflux the DNA-intercalating dye Hoechst. Human HSCs remain at present less well defined than murine HSCs. Despite the availability of methods which greatly facilitate and enhance the precision of studies with well defined cell populations, it is most likely that pure human HSCs have not yet reached the hands of the scientists. Even less defined remain the properties of stem cells from tissues other than bone marrow, primarily because the isolation of stem cells of skin, muscle, brain or liver remains difficult. Nevertheless, the flow cytometry-based characterisation of somatic tissue stem cells indicates that several cell surface markers are shared, including CD34, c-kit, sca1 and CD133, underlying common features of these rare and not easilyaccessible stem cell populations. Haematopoiesis occurs in close physical contact with stroma lining the bone marrow niches. Recently, much attention has focused on the differentiation properties of stroma cells themselves. A marrow stromal cell population, termed mesenchymal stem cells, has been shown to give rise to numerous tissue types, including cartilage, bone, fat, and muscle (4). In addition, a minor population of adherent multipotent adult progenitor cells (MAPCs) was found to be capable of differentiation into functional hepatocytes, endothelial cells, skeletal myeloblasts, osteoblasts, chondrocytes and, importantly, haematopoietic cells (5). This suggests the capacity of the bone marrow for multilineage tissue regeneration is present in


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both haematopoietic and non-haematopoietic stem cell populations. The therapeutic implications of non-haematopoietic bone marrow cells have already been clinically tested in the treatment of osteogenesis imperfecta in transplanted children (6) (see also Chapter 34). Improvement of clinical parameters suggests that bone marrow derived mesenchymal progenitor cells carry a potential for repair of bone and cartilage tissue.

4. Stem cell niches The concept of the stem cell niche defines a microenvironment, where important interactions between adhesion molecules and their ligands, and between cytokines, chemokines and their corresponding receptors control the fate of stem cells and their progeny (7). In the adult bone marrow, HSCs are located in the trabecular endosteum, where osteoblastic cells are critical components sustaining the quiescence or selfrenewal of HSCs, the properties essential for long-term haematopoiesis (8). Both intrinsic and extrinsic mechanisms define the state of either quiescence or cycling and differentiation of HSCs (Figure 2). The intrinsic mechanisms include transcription Figure 2: The mechanisms regulating the HSC niche

Extrinsic mechanisms PTH

nerves Osteoblast

Cadherins VCAM - VLA ICAM - LFA SDF-1 - CXCR4

cell-cell adhesion migration

quiescence self-renewal expansion

SCF - c-kit Flt3-L - Flt3 Ang-1 - tie2 Jagged/Delta - Notch Wnt - Frizzled

Signal transduction Gene expression HSC

Intrinsic mechanisms

A graphical representation of extrinsic and intrinsic mechanisms in the niche based on the functional interaction of the parathyroid hormone (PTH) and nervous system with osteoblasts, the homing of HSCs in response to chemokines (SDF-1), the physical interactions involving the cadherins and integrins (VLA), and regulation of HSC quiescence, self-renewal and expansion by cytokines: stem cell factor (SCF), Flt3 ligand (Flt3-L), angiopoietin-1 (ang-1), notch ligands (jagged and delta) and wnt ligands, followed by signaling downstream from the cognate receptors and initiating the gene expression 38

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factors and epigenetic regulators acting through chromatin remodelling. The extrinsic mechanisms are dictated by the environment of stromal and osteoblastic cells. Chemokines are responsible for HSC homing into the niche. The direct physical interaction between HSCs and the niche cells are mediated by adhesion molecules, such as integrins and cadherins. The membrane-bound and locally secreted cytokines define the HSC fate by initiating specific signalling pathways within the cell. The most prominent examples are stem cell factor, flt3 ligand, angiopoietin, Notch ligands and wnt ligands, which act synergistically. Also ex vivo, these cytokines allow an expansion of HSC numbers. According to the most recent findings, the extrinsic environmental cues in the HSC niche include hormonal regulators, such as parathyroid hormone, and the influence of a sympathetic nervous system, both signalling through the osteoblastic niche component, as well as the regulation by oxidative conditions in the niche. The information on the structure and cellular composition of the niche is only beginning to be revealed, and particularly in the human system the knowledge is only just emerging.

5. Leukaemic stem cells The available data suggest that leukaemia is a stem cell disease, in which the stem cell self-renewal mechanisms are preserved but the tight growth control is lost due to malignant transformation. As an additional mechanism, the oncogenic events might be imposing the self-renewal capacity at the level of committed progenitor cells (9). Consequently, leukaemic stem cells (LSCs) share many molecular mechanisms that regulate the function of normal HSCs. At present, phenotypic features specific for LSCs are not defined. LSCs are thought to reside within the CD34+CD38- cell population, which contains transplantable cells giving rise to human leukaemia in NOD/SCID mice. HSC-characteristic adhesion molecules, such as integrins, are involved in LSC interaction with stroma. Hence, dissimilarities between normal and leukaemic cells most likely have their origin in differences in intracellular signal transduction pathways (10). As an example, oncogenic lesions in the cytokine receptors, c-kit and flt3, are responsible for constitutive activation of downstream signalling in cells at the earliest stages of haematopoietic differentiation, resulting in HSC to LSC transition. A better understanding of the properties of LSCs is of major therapeutic relevance for the design of LSC-targeted therapies (Figure 3). Conventional chemotherapy-based treatment of leukaemia, and cancer in general, is primarily directed against the bulk of malignant cells, and thus does not eliminate the abnormal stem cells. These cells are the origin of cancer recurrence and are responsible for relapse. Current efforts are focussed on the development of methodology to isolate these cells to better


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Figure 3: The therapeutic relevance of LSCs

Conventional cancer therapy

Specific cancer stem cell therapy

Leukaemia

Leukaemia relapse

Leukaemia regression

Leukaemic blasts are depicted in grey, and LSCs in blue

homogeneity, and to dissect the differences in molecular mechanisms used by normal HSC and LSC for their self-renewal and interaction with the microenvironment in the bone marrow (11).

6. Embryonic stem cells Human ES cells can be isolated from the blastocyst 4–5 days after fertilisation, and cultured in vitro to give rise to immortalised cell lines. Depending on the culture conditions, differentiation into cells bearing characteristics of various somatic tissue types including haematopoietic, neural, muscle and other tissues, can be achieved. This work, initiated in 1998 by Thompson et al. (12) who defined methods to isolate and propagate human ES cells from the fertilised oocyte at the 30 cell stage, is of significant value for studies on human developmental biology. Importantly, in vitro cultured human ES cells represent valuable tools for drug screening. The most publicised and controversial aspect of ES-related research is associated with the origin of these cells, being a donated surplus human blastocyst from in vitro fertilisation procedures. Destruction of human embryos in order to obtain the ES cells has been of serious ethical concern. Novel findings describe derivation of human ES cell lines from single blastomeres at the 8-cell stage, without embryo destruction. ES cells are the subject of intense research which aims at understanding the molecular basis of “stemness”. In parallel, cellular biology techniques have been

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developed which allow manipulations such as nuclear transfer from a somatic cell to the enucleated oocyte, and the further generation of ES cell lines bearing genetic information defined by the donated nucleus. The multilineage developmental potential of human ES cells opens new therapeutic avenues for the restoration of damaged or diseased tissue. The possibility to provide these cells with genetic information from the patient by nuclear transfer, is an approach which – in the future – might yield transplantable tissue of a full immunological compatibility. Ultimately, the choice of an approach will require the consideration of advantages and disadvantages associated with the cell source for transplantation (Table 2).

Table 2: Potential therapeutic value of ES cells and adult somatic stem cells Stem cell source

Advantages

Disadvantages

ES cells

- perfect plasticity - easy to programme - stable in culture - no risk of infection transmission

- ethical concern - limited source - rejection danger - carcinogenicity?

Adult somatic stem cells

- accessible source (HSCs) - no rejection (autologous) - no ethical concern - no carcinogenicity

- less/no plasticity?

7. Stem cell plasticity The term “adult stem cell plasticity” defines the ability of tissue-specific stem cells to acquire, under certain microenviromental conditions, the fate of cell types different from the tissue of origin and belonging to all three germ layers, i.e. similar to the differentiation ability of ES cells. Traditionally, the development of adult stem cells has been depicted along a well-defined path of a linear and irreversible progression concluding in terminally differentiated cell types. Furthermore, the differentiation and regenerative potential of adult stem cells has been regarded as restricted to tissues in which they reside. These traditional concepts have been challenged in the recent years by numerous studies performed by transplantation of stem cells derived from bone marrow and other organs, and which demonstrated the presence of cells of interest in tissues other than those in which they normally reside. The findings from murine studies, which suggested that stem cells may be recruited out of a circulation and engaged in regeneration of diverse tissues at distal sites, have initiated a search for “unusual” locations of donor-derived stem cells


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in patients receiving organ transplants. In some cases transitions have been documented, and are likely to reflect a healing response by cells summoned to the site of injury and instructed by the local environment of the damaged tissue. However, donor-host cell fusions rather than functional stem cell plasticity may represent the underlying mechanism (13). This concept of plasticity of somatic tissue stem cells has a potential clinical impact and may revolutionise tissue transplantation therapies and regenerative medicine. According to this novel view, at least a subset of stem cells may alter their fate in a manner that is more plastic and dynamic then previously thought, causing a fascination with these cells that has spread to nearly all clinical disciplines. There are more questions raised then answers. Following a phase of excitement and rapid accumulation of results in favour of stem cell plasticity, research in this field is now going through a less spectacular phase of verification of the existing data with refined techniques. It is too soon to discard the basic paradigm of developmental biology of the mesodermal, endodermal and ectodermal germ layer origin of mammalian organs, but a need for possible revisions to the unidirectional view of cell fate in post-embryonic development may arise.

8. Conclusions and future perspectives The ultimate goal for regenerative medicine is to channel the multipotent and/or pluripotent stem cells with high proliferative capacity into specified differentiation programs within the body for a multitude of therapeutic uses. These envisaged uses may include the generation of neurons for treatment of Alzheimer’s disease, Parkinson’s disease or spinal cord injuries, the generation of insulin-secreting pancreatic cells for the treatment of diabetes, or the generation of heart muscle cells for treatment of congenital disorders or heart attacks. Recent major technical advancements in the isolation, expansion and controlled differentiation of human ES cells and adult stem cells from at least some tissues, and additionally, the establishment of nuclear transfer techniques from the somatic cell to an enucleated donor oocyte opened a number of potential new therapeutic approaches for the restoration of damaged or diseased tissue. The new challenge in stem cell biology is related to understanding the molecular and the functional programmes of leukaemic versus normal stem cells. The principle of self-renewal and lineage-making decisions of stem cells of different tissue-origin requires understanding in molecular terms. Gene expression profiles bring evidence of the overlapping genetic programs of ES cells, haematopoietic and other adult tissue stem cells, both normal and transformed (14). Determining how epigenetic features relate to the transcriptional signatures of ES and various types 42

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of adult stem cells, is a new key challenge for the future (15). Understanding of the stem cell niche is essential for advancing the approaches to control developmental pathways by both cell-autonomous and microenvironmental cues. All this information will be used as guidance for specifically targeting the fate of normal and malignant stem cells in clinical settings.

Acknowledgments This work has been supported by the Swiss National Science Foundation grant 3100-110511.

References 1. Bryder D, Rossi DJ, Weissman IL. Hematopoietic stem cells: The paradigmatic tissue-specific stem cell. Am J Pathol 2006; 169: 338-346. 2. Bhatia M, et al. A newly discovered class of human hematopoietic cells with SCIDrepopulating activity. Nat Med 1998; 4: 1038-1045. 3. Arai F, SudaT. Maintenance of quiescent hematopoietic stem cells in the osteoblastic niche. Ann NY Acad Sci 2007; 1106: 41-53. 4. Pittenger MF, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143-147. 5. Jiang Y, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418: 41-49. 6. Horwitz EM, et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 2001; 97: 1227-1231. 7. Scadden DT. The stem-cell niche as an entity of action. Nature 2006; 441: 1075-1079. 8. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Imunol 2006; 6: 93-106. 9. Huntly BJ, Gilliland DG. Leukaemia stem cells and the evolution of cancer-stem-cell research. Nat Rev Cancer 2005; 5: 311-321. 10.Rizo A, et al. Signaling pathways in self-renewing hematopoietic and leukemic stem cells: Do all stem cells need a niche? Hum Mol Genet 2006; 15 Spec No 2: R210-9. 11.Clarke MF, et al. Cancer Stem Cells – Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Res 2006; 66: 9339-9344. 12.Thomson JA, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145-1147. 13.Serafini M, Verfaillie CM. Pluripotency in adult stem cells: State of the art. Semin Reprod Med 2006; 24: 379-388. 14.Forsberg EC, Bhattacharya D, Weissman IL. Hematopoietic stem cells: Expression profiling and beyond. Stem Cell Rev 2006; 2: 23-30. 15.Spivakov M, Fisher AG. Epigenetic signatures of stem-cell identity. Nat Rev Genet 2007; 8: 263-271.


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Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Haematopoiesis takes place in the following adult human organs: a) Bone marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Peripheral blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Spleen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The stem cell compartment in the bone marrow consists of: a) Clonogenic CFU and LTC-IC progenitors only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Haematopoietic, mesenchymal and endothelial cell progenitors . . . . . . . . . . c) NOD/SCID repopulating cells only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Haematopoietic, liver and neural stem cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell surface antigen CD34 is expressed on: a) Long-term repopulating haematopoietic stem cells . . . . . . . . . . . . . . . . . . . . . . . . b) Short-term repopulating haematopoietic stem cells . . . . . . . . . . . . . . . . . . . . . . . . c) Haematopoietic and non-haematopoietic stem and progenitor cells . . . . . . d) Lineage-committed progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Embryonic stem cells are characterised by: a) Lineage-restricted differentiation potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Potential to become a variety of specialised cell types . . . . . . . . . . . . . . . . . . . . c) Ability to generate placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Ability to form a blastocyt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Osteoblast components of the stem cell niche are involved in: a) Supporting stem cell self-renewal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Supporting the stem cell differentiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Inhibiting the bone marrow stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Promoting the stem cell exit to the peripheral blood . . . . . . . . . . . . . . . . . . . . . .

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NOTES


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*

CHAPTER 3

Immunogenetics of allogeneic HSCT

*

3.1

The role of HLA in HSCT J.M. Tiercy

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CHAPTER 3.1 • HLA and donor matching

1. Introduction Tissue compatibility is determined by genes of the major histocompatibility complex (MHC), known as the HLA system in man, that are clustered on the short arm of chromosome 6. The HLA region is a multigenic system that encodes structurally homologous cell surface glycoproteins characterised by a high degree of allelic polymorphism in human populations. Immune responses against incompatible HLA antigens represent a major barrier to haematopoietic stem cell transplantation (HSCT). The accuracy of histocompatibility testing and matching criteria will therefore have important consequences on transplant outcome. This is particularly true in the case of transplantation with HSC from unrelated donors, where serologically hidden incompatibilities may account for the increased rate of post-transplant complications.

2. HLA antigens The homologous HLA Class I (HLA-A, -B, -C) and Class II (HLA-DR, -DQ, -DP) antigens are codominantly expressed and differ in their structure (Figure 1), tissue distribution and characteristics in peptide presentation to T-cells (1). The biological function of HLA molecules is to present peptide antigens to T-cells, thereby playing a central role in T-cell-mediated adaptive immunity. HLA Class I molecules, which are expressed on most nucleated cells, are composed of an a-chain (encoded in the MHC) non covalently associated with b2-microglobulin (encoded on chromosome 15) (Figure 1). The two outermost a1 and a2 domains of the heavy chain form the peptide binding site. Peptides (usually of 8–10 amino acids) presented by Class I

Figure 1: Schematic representation of HLA Class I and Class II molecules

The two polymorphic domains a1/a2 of HLA Class I are encoded by exons 2 and 3, respectively, and exon 3 codes for the most proximal a3-domain that interacts with b2-microglobulin (b2m) and CD8. The HLA Class I b-chain is non covalently associated with b2m. The Class II molecules are heterodimers composed of an a and a b-chain. The most distal domains of each of the two chains form the peptidebinding site


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molecules are derived from proteolytic degradation of cytoplasmic proteins by the proteasome. These are transported across the endoplasmic reticulum where they bind to Class I antigens. Pathogen-derived peptides presented to Class I antigens are usually recognised by CD8+ cytotoxic T-lymphocytes (CTL) (1). HLA Class II antigens are expressed on a subset of cells of the immune system comprising dendritic cells, B cells, activated T-cells, macrophages, collectively referred to as antigen presenting cells (APC). They are heterodimers composed of the two membrane-bound a- and b-chains that are encoded by two genes that colocalise in the MHC. The peptide-binding pocket is formed by the most distal domains of the two chains. Extracellular antigens internalised by endocytosis/phagocytosis are degraded in an endocytic compartment into peptides of 10–30 amino acids that bind Class II molecules. HLA Class II-peptide complexes expressed on the membrane are usually recognised by CD4+ T-helper cells (1). Peptide-HLA complexes are the ligands of clonally distributed T-cell receptors (TCRs). TCRs are also able to recognise allogeneic HLA molecules at a high frequency, so that 1–10% of the peripheral blood lymphocytes of a donor can respond to a given allo-MHC antigen (2). Immune responses against incompatible HLA antigens may be extreme, such as in the case of graft-versus-host disease (GvHD) mediated by alloreactive cytotoxic T-lymphocytes (CTL), and thus represent a major barrier to HSCT.

3. Genomic organisation of the HLA system The MHC comprises 12 classical HLA genes located on a 3.6 Mb segment of the short arm of chromosome 6. Three HLA Class I genes (A, B, C) (Figure 2) encode for the heavy chains of HLA-A, B and -C antigens. Polymorphic residues are essentially located in the a1- and a2-domains encoded by exons 2 and 3, respectively, which form the peptide binding site. HLA Class II antigens (DR, DQ, DP) are heterodimers encoded by an a-chain and a b-chain gene (e.g. DRA/DRB1 or DQA1/DQB1) that co-localise at the centromeric part of the MHC (Figure 2) (1, 3). Essentially all of the polymorphism is located on exon 2 of b-chain genes, whereas the DRA gene is non polymorphic, and DQA1 and DPA1 loci exhibit a lower level of polymorphism (Figure 1). The HLA-DR sub-region presents an additional level of complexity since a second polymorphic DRB gene may be present, i.e. DRB3 in DR11/DR12/DR13/DR14/DR17/DR18 haplotypes, DRB4 in DR4/DR7/DR9 haplotypes, and DRB5 in DR15/DR16 haplotypes (Figure 2). Because of the codominant expression of HLA genes, a heterozygous individual may therefore express up to 12 different HLA antigens.

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Figure 2: HLA as a multigenic and polymorphic system

Schematic representation of the 12 HLA Class I and II loci in the MHC that comprises >200 genes on the short arm of chromosome 6. The corresponding number of antigens (as defined by serology) and alleles (as defined by nucleotide sequence) are indicated for each locus (3). About 10% of the alleles assigned by the HLA Nomenclature Committee are characterised by silent substitutions, and 2% are null alleles (3). Lower part of the figure: the HLA-DR subregion presents an additional level of complexity, with the presence of a second DRB gene in most haplotypes: DRB3, DRB4 or DRB5, which encode respectively the DR52, DR53, and DR51 antigens. For the nomenclature: see Appendix 1

4. Allelic polymorphism HLA genes are the most polymorphic loci of the human genome accounting for a total of 2584 alleles currently identified in worldwide populations (Figure 2) (3). Polymorphism of the HLA system was initially detected by serology, i.e. by using typing reagents derived from sera of multiparous women, or individuals who have been immunised by multiple transfusions. In the early 1980’s molecular cloning of the first HLA genes opened the way for a complete understanding of the molecular basis of HLA diversity and for the development of a variety of DNA-based typing techniques. Most of the serologically defined specificities are now subdivided into numerous alleles, and this number is still continuously growing (3). A regular update of the new alleles at the various HLA loci is available on the web (www.ebi.ac/imgt/hla) (Figure 2). Appendix 1 lists the HLA-A/B/C/DR/DQ serotypes and the corresponding groups of alleles detected by high resolution typing. The variability within a given serotype was recognised by cellular assays long before the DNA sequencing era. For example CTLs were shown to discriminate between two HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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HLA-A2 serologically identical individuals. Figure 3 shows a schematic alignment of the a1/a2 domain sequences with unique residues shared by all A2 alleles (G62, T142-H145). These residues are recognised by the monospecific and/or polyspecific allo-antisera used as typing reagents. Each of the 103 different A2 alleles expressed at the cell surface differs by a very limited number of residues, usually 1–4, and such allele mismatches can be efficiently recognised by CTLs (4–7). Although many of the Class I and II antigens comprise a large number of alleles worldwide, a limited number of alleles are found in any given population at a gene frequency >0.1% (8, 9). The combination of HLA alleles inherited on the same segment of chromosome 6 is referred to as a haplotype, for example the A1-B8-DR3 or the DRB1*1501DQB1*0602 haplotypes. Because of linkage disequilibrium HLA disparities at a given locus will frequently be associated with incompatibilities at an adjacent locus, as observed in many instances for B-Cw and DRB1-DQB1 bi-locus groups.

Figure 3: Schematic representation of HLA-A2 alleles showing common residues on the a1/a2-domains that are recognised by alloantisera

Open boxes mark residues that differ between the 0201-0207, 9201, and 9215 alleles. For example 0201 and 0206 alleles differ at one single position (Phe9 vs. Tyr9). Such a serologically silent mismatch is recognised by CTLs (see Figure 4) 50

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5. HLA typing: Methods and resolution levels Serology (microlymphocytotoxicity) is still a method of choice for low resolution typing in most laboratories, at least for HLA-AB, due to its simplicity and low cost. However the lack of monospecific alloantisera, the low-resolution power of the method and the requirement for viable lymphocytes, all contributed to the development of genomic DNA typing techniques, initially for Class II, later on for Class I typing. DNA typing techniques are based on the nucleotide sequence information of the polymorphic DNA segments, using PCR technology. A number of HLA typing methods based on DNA sequence variations have been developed, mainly using PCR-SSP (sequence-specific primers) amplification, or reverse PCR-SSOP (sequence-specific oligonucleotide probes) hybridisation on solid support (e.g. microbead arrays), or direct sequencing (8, 10). Three different levels of resolution for HLA DNA typing are usually recognised (Table 1). Low-resolution, also referred to as generic typing or 2-digit typing, corresponds to the identification of broad families of alleles that cluster into serotypes (e.g. A*02), and is thus the equivalent of serological typing (A2). High resolution, or 4-digit

Table 1: HLA nomenclature and levels of resolution HLA (a)

Definition

Resolution

A2

Refers to the A2 antigen defined by monospecific/ polyspecific antisera Any of the A*02 alleles (A*0201-0299 and A*9201-9215) Either one of these 4 alleles, other DRB1*11 alleles are excluded Allele defined Allele with a substitution in the coding sequence that leads to a stop codon (null allele), this allele is not expressed 2 DRB1*0301 alleles that differ by a silent substitution in exon 2, this difference is functionally silent, but may influence DNA typing A*2402 allele with low expression due to a mutation in intron 2 sequence, this allele is expressed at a level that is undetectable by serological typing

low

A*02 DRB1*1102/1103/ 1111/1114 A*0201 A*0232N DRB1*030101 and DRB1*030102 A*24020102L

low intermediate high high high

high

(a) DNA nomenclature: the first 2 digits refer to the serotype (A*02), the 3rd and 4th digits define substitution(s) in the coding sequence (A*0201), the 5th and 6th digits describe synonymous substitution(s), and the 7th and 8th 2 digits refer to substitution(s) in intron or 5’ or 3’ sequences. N and L mark alleles with respectively no or low surface expression. The suffix S means an allele encoding a protein that is expressed as a secreted molecule only and the suffix Q means an allele with a mutation that has been shown previously to have a significant effect on cell surface expression but without confirmation, and therefore with a questionable expression status


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typing, allows the discrimination of the individual alleles within each serotype (e.g. A*0201). Intermediate resolution HLA typing of a potential donor for this patient might give as a result A*0205/08/22: that means that the donor can be either A*0205, or A*0208, or A*0222, but definitively not A*0201 or any of the other A2 alleles. This level of typing results from the fact that these 3 alleles share common sequence determinants and are therefore identified in the same hybridisation group pattern. It is obviously very practical for rapid donor selection. In the example given above, such a donor typed as A*0205/08/22 would be disregarded for further analysis, whereas a parallel donor with the HLA type A*0201/01L/09/43N/66/75 would be selected and tested further in order to disclose compatibility at the allele level with the A*0201-positive patient. The accreditation program for the histocompatibility laboratories set up by the European Federation for Immunogenetics (EFI) has defined minimal criteria for HLA typing in related and unrelated HSCT (www.efiweb.org/standards.html).

6. HLA matching in related HSCT The best donor is a HLA genotypically matched sibling as identified by family typing. The family study allows not only the identification of a potential related donor, but also the confirmation of the patient’s genotype, which is important if an unrelated donor search is initiated. HLA-A, B, DR low resolution typing (serology or 2-digit DNA typing) is able in most cases to determine the paternal and maternal haplotypes present in the patient and a potential sibling donor. Thus ABDR typing can confirm genotypic identity for the whole set of HLA genes, i.e. a 12/12 match (Figure 4). Because of weak linkage disequilibrium between DP and the DR/DQ loci, a low level of DP-mismatched sibling donors (1–2%) are identified due to recombination. An HLA-A/B or B/DRB1 recombination event is detected by routine HLA-A/B/DR typing in about 2% of families. For a given patient the probability of having a genotypically identical sibling donor is 25% for each sibling, whereas the probability of having a haploidentical donor (i.e. with one shared haplotype) is 50%. When the patient has a very frequent ABDR haplotype it may be worth searching for a phenotypically identical donor in the blood-related members of the extended family, particularly when information on consanguinity is provided, for example when there is sound information that there has been intermarrying between aunts/uncles on paternal and aunts/uncles on maternal side, so that cousins may be HLA genotypically identical. In some families, apparent homozygosity of one of the parents may prevent formal identification of genotypic identity. As illustrated in the example shown in Table 2, both siblings 1 and 2 are phenotypically identical to the patient, based on ABDR 52

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Figure 4: Matching criteria in related and unrelated HSCT

By definition an HLA-genotypically identical sibling donor is compatible at the allele level at all loci on both chromosomes (12/12 match). In unrelated HSCT, matching for A/B/C/DRB1/DQB1 loci is usually searched for (10/10 match). In addition to DRB1 compatibility, some centres also consider DRB3/DRB5 polymorphism. DRB3 mismatches occur frequently in DR13 haplotypes. Because of strong linkage disequilibrium with DRB1 (at least in Caucasoids), the DRB5 locus is usually not tested. In DR15/16 haplotypes, DRB5 mismatches usually co-occur with DRB1 disparities. Searching for a 12/12 match implies DPB1 typing. Donors with an 8/8 match (not shown) or a 6/6 match apply, respectively, when HLAC/DP, or HLA-C/DQ/DP are not tested

low resolution typing. However typing for HLA-C and HLA-DRB1 at the allele level shows that sibling 1 is actually compatible with the patient, whereas sibling 2 has inherited a different maternal haplotype with 2 mismatches. In case of mismatched related HSCT, the risk of GvHD (recipient alleles absent in the donor) and graft failure (donor alleles absent in the recipient) increase with the number of HLA disparities on the non-shared haplotype (reviewed in ref. 11). A differential effect of Class I and Class II mismatches has been described, in which aGvHD risk was associated with Class II disparities (12).

7. HLA matching in unrelated HSC transplantation When no HLA-genotypically identical sibling donor is available, transplantation with HSC from HLA-ABCDRB1/DQB1-allele matched unrelated donors can result in comparable disease-free survival rates, notably for good-risk patients (11). Donor


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Table 2: Low versus high resolution HLA typing in homozygous haplotypes Haplotype

Low resolution typing

High resolution typing

father

a b

A1-B8-DR3 A31-B18-DR15

A*0101-B*0801-Cw*0701-DRB1*0301 A*3101-B*1801-Cw*1203-DRB1*1501

mother

c d

A2-B51-DR11 A2-B51-DR11

A*0201-B*5101-Cw*0501-DRB1*1101 A*0201-B*5101-Cw*1203-DRB1*1104

patient

a c

A1-B8-DR3 A2-B51-DR11

A*0101-B*0801-Cw*0701-DRB1*0301 A*0201-B*5101-Cw*0501-DRB1*1101

sibling 1

a c

A1-B8-DR3 A2-B51-DR11

A*0101-B*0801-Cw*0701-DRB1*0301 A*0201-B*5101-Cw*0501-DRB1*1101

sibling 2

a d

A1-B8-DR3 A2-B51-DR11

A*0101-B*0801-Cw*0701-DRB1*0301 A*0201-B*5101-Cw*1203-DRB1*1104

High resolution typing may disclose hidden incompatibilities in families where the 4 parental genotypes cannot be unambiguously determined. In this example the mother is ABDR homozygous by serology, but in fact has 2 different haplotypes on the basis of high resolution typing

identification has been largely facilitated by the Bone Marrow Donor Worldwide (BMDW) Registry (www.bmdw.org) which provides access to >11 million HLA-typed donors and cord blood units available in the national registries. Compared to HSCT from HLA genotypically identical sibling donors, unrelated HSCT is associated with an increased frequency of post-transplant complications, which are mainly due to undisclosed HLA incompatibilities not detected by serology (8, 13). Allele mismatches usually involve difference(s) in the peptide-binding site (Figure 5) that influence

Figure 5: Examples of common serologically hidden HLA Class I incompatibilities that involve single amino acid mismatches recognised by CTLs

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T-cell recognition either by direct contact with TCR or indirectly by modulating the repertoire of peptides bound by the HLA molecule. 7.1. Matching criteria for unrelated donors Whereas genotypically identical siblings share by definition the same alleles at all loci, the degree of allele matching in two unrelated individuals strictly depends on how many loci are considered for high resolution typing. Figure 4 illustrates the three most common situations: the gold standard matching comprises the analysis of HLAA, -B, -C, -DRB1, and -DQB1 loci. When a patient and a donor share the same 5 alleles on both haplotypes the situation is referred to as a 10/10 match. If HLA-C or -DQB1 typing is omitted, this will be an 8/8 match, and if only the A/B/DRB1 loci are considered, it becomes a 6/6 match. Obviously because of the multiple possible BC and DRB1-DQB1 associations, a 6/6 match may in fact correspond to a 6/10 match, i.e. to an incompatible combination (Table 3). In the case of non-malignant diseases the graft versus leukaemia effect that may be mediated by DP incompatibilities is not necessary and DPB1 matching might be considered when several HLA-A/B/C/DR/DQ-compatible donors are available (12/12 match).

Table 3: Hidden incompatibilities in HLA-ABDRB1 allele-matched donors Matching

6/6

8/10

A*

B*

Cw*

DRB1*

DQB1*

patient

0201/1101

0801/3503

NT

0301/1101

NT

donor

0201/1101

0801/3503

NT

0301/1101

NT

patient

0201/1101

0801/3503

0701/0401

0301/1101

0201/0301

donor

0201/1101

0801/3503

0701/1203

0301/1101

0201/0501

7.2. Allele matching: Relative importance of individual loci In the early 1990’s the role of HLA matching was hampered by the poor resolution achieved by HLA typing, particularly for HLA Class I alleles. Based on high resolution typing methods recent studies (14–18) have reached the almost general consensus that allele-level matching does improve transplant outcome after both myeloablative and reduced-intensity conditioning regimen (reviewed in ref. 11). The HLA effect on transplant outcome is modulated by the disease risk with an effect of single disparities reported to be significant for low risk disease patients (16). However the relative importance of individual loci still remains an open issue. Whereas


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the effect of A/B/C/DRB1 mismatches have been well documented by most retrospective large scale studies, the role of DQ and DP incompatibilities appear more controversial (reviewed in ref. 11). The National Marrow Donor program (NMDP) Histocompatibility Committee has recommended allele-level typing for HLAA/B/C/DRB1 (8/8 match) (13). There is however some evidence suggesting a trend of an additive HLA-DQ effect in HSCT from donors with multiple mismatches (16). Recent data on T-cell-depleted unrelated HSCT have documented that HLA-DPB1 matching was associated with increased risk of relapse, irrespective of the compatibility status for the other loci (19). A comparison of serological versus allele class I mismatches in CML patients suggested that qualitative differences may influence the risk of graft failure, with a higher risk in serotype-mismatched patients (15, 20). Contrasting outcomes have been reported in studies with patients from different ethnic backgrounds: in the Japanese Marrow Donor Program (JMDP) study (14), HLAA/B/C/DRB1 mismatches were found to be significant risk factors for grades III–IV acute GvHD, whereas the U.S. National Marrow Donor program (NMDP) data revealed a DRB1 effect with no contribution of HLA-DQ/DP or HLA-B/C (15) mismatches. HLAA/B/C/DRB1, but not DQ/DP mismatches decreased survival in the NMDP study (15), whereas in the JMDP study only A/B/DRB1 disparities were associated with mortality (14). Differences between studies may involve selection criteria of each transplant centre, patients age or other pre-transplant risk factors, experience in treating GvHD, as well as the relevance of the GvL effect in CML patients. Also when comparing studies from varying population groups, major differences between mismatched allele combinations may contribute to contrasting clinical outcomes. Selection of cord blood units is generally based on HLA-A/B-low and -DRB1-high resolution matching, taking into account that cell dose is the most important parameter affecting outcome. Data on the effect of HLA matching in cord blood transplantation are difficult to interpret, since most reports lack HLA-C/DQ and high resolution HLA-AB typing results. Nevertheless cord blood appears less HLA restricted than adult HSCT, in other words HLA incompatibilities may be less detrimental (21).

8. Probability estimates of finding a matched unrelated donor Identification of an unrelated HLA allele-matched HSC donor is a costly and time consuming procedure. To improve search efficiency, an estimate of the probability of identifying a 10/10 HLA matched donor allows an early decision to transplant with HSC from an alternative donor (haploidentical, cord blood, autologous), or to accept varying degrees of allele-mismatches for searches with a low probability of success, depending on the clinical context and local guidelines. Based on the current 56

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knowledge of allele and haplotype frequencies in different populations the probability of identifying a 10/10 matched donor at the start of the search is highly predictable (22). The number of ABDR-matched (low resolution) donors available in the Registry for a given patient often reflects the chance of finding a donor compatible at the allele level. However, the following parameters have a negative impact on this probability: • presence of a rare allele in the patient (2 alleles occurring at a frequency >10% of a given serotype in the population, e.g. B35, B44, DR4, DR11, DR13 • presence of B*2705, B18, B*4402, B*4403, B51 (higher risk of HLA-C mismatch) • 2 x 107/kg and there are no more than 2 HLA mismatches (4/6).

10. Conclusions The HLA system, with 100 serologically defined specificities and over 2500 alleles, represents a major barrier to HSC transplantation. There is now a broad consensus that selection of unrelated HSC donor by high resolution molecular typing technology contributes to a better clinical outcome. However the relative importance of individual loci still remain to be better defined, and multicentre studies should contribute to resolving this issue. The difficulty in reaching clear consensus among 58

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the clinical studies, particularly with respect to the role of single locus mismatches, possibly results from subtle differences in patients groups with respect to patient and donor selection criteria, pre-transplant risk factors or GvHD prophylaxis. Additional immunogenetic factors such as minor histocompatibility antigens, cytokine and chemokine genes, or activating/inhibitory killer immunoglobulin-like receptor (KIR) genes, may play a role in determining transplant outcome. Donor HLA matching criteria should take into account parameters such as the time frame allowed by the patient’s disease and the probability of identifying a well matched donor based on the patient’s HLA phenotype.

References 1. Klein J, Sato A. The HLA system. First of two parts. New Engl J Med 2000; 343: 702-709. 2. Afzali B, Lechler RI, Hernandez-Fuentes MP. Allorecognition and the alloresponse: Clinical implications. Tissue Antigens 2007; 69: 545-556. 3. Marsh SGE, Albert ED, Bodmer WF, et al. Nomenclature for factors of the HLA system, 2002. Tissue Antigens 2002; 60: 407-464. 4. Fleischhauer K, Kernan NA, O'Reilly RJ, et al. Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44. N Engl J Med 1990; 323: 1818-1822. 5. Rufer N, Tiercy J-M, Breur-Vriesendorp B, et al. Histoincompatibilities in ABDR-matched unrelated donor recipient combinations. Bone Marrow Transplantation 1995; 16: 641-646. 6. Oudshoorn M, Doxiadis II, van den Berg-Loonen PM, et al. Functional versus structural matching: Can the CTLp test be replaced by HLA allele typing? Hum Immunol 2002; 63: 176-184. 7. Scott I, O’Shea J, Bunce M, et al. Molecular typing reveals a high level of HLA class I incompatibility in serologically well matched donor/recipient pairs - Implications for unrelated bone marrow donor selection. Blood 1988; 92: 4864-4871. 8. Tiercy JM, Villard J, Roosnek E. Selection of unrelated bone marrow donors by serology, molecular typing and cellular assays. Transpl Immunol 2002; 10: 215-221. 9. Hurley CK, Fernandez-Vina M, Hildebrand WH, et al. A high degree of HLA disparity arises from limited allelic diversity: Analysis of 1775 unrelated bone marrow transplant donorrecipient pairs. Human Immunol 2007; 68: 30-40. 10.Little AM, Marsh SG, Madrigal JA. Current methodologies of human leukocyte antigen typing utilized for bone marrow donor selection. Curr Opin Hematol 1998; 5: 419-428. 11.Petersdorf EW. Risk assessment in hematopoietic stem cell transplantation. Best Pract Res Clin Haematol 2007; 20: 155-170. 12.Ottinger HD, Ferencik S, Beelen DW, et al. Hematopoietic stem cell transplantation: Contrasting the outcome of transplantations from HLA-identical siblings, partially HLAmismatched related donors, and HLA-matched unrelated donors. Blood 2003; 102: 11311137. 13.Hurley CK, Wagner JE, Setterholm MI, Confer DL. Advances in HLA: Practical implications for selecting adult donors and cord blood units. Biol Blood Marrow Transplant 2006; 12


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(1 Suppl 1): 28-33. 14.Morishima Y, Sasazuki T, Inoko H, et al. The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors. Blood 2002; 99: 4200-4206. 15.Flomenberg N, Baxter-Lowe LA, Confer D, et al. Impact of HLA class I and class II high resolution matching on outcomes of unrelated donor bone marrow transplantation. Blood 2004; 104: 1923-1930. 16.Petersdorf EW, Anasetti C, Martin PJ, et al. Limits of HLA mismatching in unrelated hematopoietic cell transplantation. Blood 2004; 104: 2976-2980. 17.Chalandon Y, Tiercy J-M, Schanz U, et al. Impact of high resolution matching in allogeneic unrelated donor stem cell transplantation in Switzerland. Bone Marrow Transplant 2006; 37: 906-916. 18.Carreras E, Jiminez M, Gomez-Garcia V, et al. Donor age and degree of HLA matching have a major impact on the outcome of unrelated donor hematopoietic cell transplantation for chronic myeloid leukemia. Bone Marrow Transplant 2006; 37: 33-40. 19.Shaw BE, Marsh SG, Mayor NP, et al. HLA-DPB1 matching status has significant implications for recipients of unrelated donor stem cell transplants. Blood 2006; 107: 1220-1226. 20.Petersdorf EW, Hansen JA, Martin PJ, et al. Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation. New Engl J Med 2001; 345: 17941800. 21.Gluckman E, Rocha V. Donor selection for unrelated cord blood transplants. Curr Op Immunol 2006; 18: 565-570. 22.Tiercy J-M, Nicoloso de Faveri G, Passweg J, et al. The probability to identify a 10/10 HLA allele-matched unrelated donor is highly predictable. Bone Marrow Transplantation, 2007; 40: 515-522. 23.Tiercy J-M, Bujan-Lose M, Chapuis B, et al. Bone marrow transplantation with unrelated donors: What is the probability of identifying an HLA-A/B/Cw/DRB1/B3/B5/DQB1matched donor? Bone Marrow Transpl 2000; 26: 437-441.

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Appendix 1. List of HLA-A, -B, -Cw, -DR and -DQ serotypes with their corresponding groups of alleles (June 2007) serotype A1 A2 A203 A210 A3 A11 A23 (9) A24 (9) A2403 A25 (10) A26 (10) A29 (19)

alleles A* 0101-0125 0201-0299, 9201-9215 0203 0210 0301-0329 1101-1130 2301-2315 2402-2476 2403, 2410 2501-2506 2601-2634 2901-2916

B7 B703 B8 B13 B14 B15 B18 B27 B2708 B35 B37 B38 (16) B39 (16) B3901 B3902 B40 B4005 B41 B42 B44 B45

alleles B* 0702-0754 0703 0801-0833 1301-1317 1401-1407N 1501-1599, 9501-9529 1801-1826 2701-2737 2708 3501-3575 3701-3712 3801-3816 3901-3941 3901 3902 4001-4074 4005 4101-4108 4201-4209 4402-4453 4501-4507, 5002

serotype A30 (19) A31 (19) A32 (19) A33 (19) A34 (10) A36 A43 (10) A66 (10) A68 (28) A69 (28) A74 (19) A80

B53 B54 (22) B55 (22) B56 (22) B57 (17) B58 (17) B60 (40) B61 (40) B62 (15)

B63 (15) B64 (14) B65 (14) B67 B70 (15) B71 (70)

alleles A* 3001-3021 3101-3117 3201-3215 3301-3310 3401-3408 3601-3604 4301 6601-6606 6801-6838 6901 7401-7412N 8001 alleles B* 5301-5312 5401-5411 5501-5526 5601-5620 5701-5712 5801-5815 4001,4007,4010,4014, 4031,4034,4048,4054 4002-4004,4006,4009, 4016,4027,4029 1501,1504-07,1515, 1520,1524,1525,1527, 1528,1530,1532,1535, 1539,1545,1548,1570, 1571,1573,1582,1584, 1516-1517 1401 1402 6701-6702 1509,1537,1551 1510,1518,1580,1593 HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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B46 B47 B48 B49 (21) B50 (21) B51 (5) B5102 B5102 B52 (5)

4601-4610 4701-4705 4801-4816 4901-4905 5001-5002,5004 5101-5148 5102 5102 5201-5211

B72 (70) B73 B75 (15)

serotype Cw1 Cw2 Cw3 Cw4 Cw5 Cw6 Cw7 Cw8

alleles Cw* 0101-0118 0202-0218 0302-0340 0401-0427 0501-0516 0602-0616N 0701-0748 0801-0814

serotype Cw9 Cw10 Cwx Cwx Cwx Cwx

DR1 DR1 DR15 (2) DR16 (2) DR3 DR4 DR7 DR8

alleles DRB1* 0101-0110 0101-0116 1501-1522 1601-1611 0302-0335 0401-0464 0701, 0703-0712 0801-0832

DR52

DR53 blank

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DRB3*0101-01 DRB3*0201-0218 DRB3*0301-0303 DRB4*0101-0106 DRB4*01030102N, 0201N,0301N


B76 (15) B77 (15) B78 B81 B82 B83

1503,1546 7301 1502,1508,1511,1521, 1531 1512,1514 1513 7801-7805 8101-8102 8201-8202 8301

Cwx Cwx

alleles Cw* Cw*0303 Cw*0302,0304 1202-1221 1402-1408 1502-1520 16011602,1604, 1606-1609 1701-1704 1801-1803

DR9 DR10 DR11 (5) DR12 (5) DR13 (6) DR14 (6)

alleles DRB1* 0901-0906 1001 1101-1162 1201-1215 1301-1379 1401-1466

DR51

DRB5*0101-0112 DRB5*0201-0205

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DQ2 DQ3 DQ4 DQ5 (1)

alleles DQB1* 0201-0205 0301-0320 0401-0402 0501-0505

DQ6 (1) DQ7 (3) DQ8 (3) DQ9 (3)

alleles DQB1* 0601-0630 0301, 0304 0302, 0305, 0310 0303


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Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. The biological function of HLA molecules is to present peptide antigens to T-cells. The peptide binding site of HLA Class I molecules is composed of: a) An a-chain and a b-chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) The proximal part of the a-chain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The distal part of the a-chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The a-chain and the b2-microglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. A heterozygous individual can express at the cell surface a maximum of: a) 10 different HLA Class I and Class II antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) 12 different HLA Class I and Class II antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) 13 different HLA Class I and Class II antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) 14 different HLA Class I and Class II antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. A HLA-ABDR haplotype is: a) A combination of alleles encoded on the same chromosome . . . . . . . . . . . . . . b) A combination of alleles shared by two unrelated individuals . . . . . . . . . . . . . c) A combination of alleles that differ between a patient and a partially incompatible sibling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) A combination of alleles that are frequent in a given population . . . . . . . . . 4. In which patient/donor combination would you expect a higher risk of HLA-C mismatches? a) Two HLA-ABDR phenotypically identical siblings. . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Two HLA-ABDR phenotypically identical unrelated individuals . . . . . . . . . . . . c) Two HLA-ABDR phenotypically identical cousins . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Two HLA-ABDR genotypically identical siblings . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5. The HLA-ABDR typing of a patient’s family leads to the identification of a potential sibling donor who differs from the patient by one single DR antigen. The father is homozygous for HLA-AB antigens and heterozygous for HLA-DR. The possible reason for the DR-incompatibility is: a) A recombination event between HLA-A and -B in one paternal haplotype. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) A recombination event between HLA-B and -DR in one paternal haplotye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Inheritance of 2 different paternal HLA-ABDR haplotypes . . . . . . . . . . . . . . . . . d) Either of the 3 possibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


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*

CHAPTER 3

Immunogenetics of allogeneic HSCT

*

3.2

KIR: Beneficial effects of natural killer cell alloreactivity in haploidentical HSCT A. Velardi, L. Ruggeri, A. Mancusi, F. Aversa, M. F. Martelli

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CHAPTER 3.2 • NK cells in haploidentical transplantation

1. Introduction Recent studies have shown natural killer (NK) cells influence the outcome of haploidentical haematopoietic transplantation in a remarkable way, with a favourable outcome where NK cells are alloreactive in the donor-recipient direction (1-4). NK cell activation is regulated by a balance between inhibitory and activating receptors, called killer-cell Ig-like receptors (KIRs). In humans, currently 16 inhibitory KIR genes and pseudo-genes are known, which codify for inhibitory and activating KIRs. Inhibitory KIRs recognise amino acids in the COOH-terminal portion of the MHC class I a1 helix (reviewed in refs. 3, 5, 6). They possess two (KIR2D) or three (KIR3D) extra-cellular C2-type Ig-like domains and a long cytoplasmic tail (L) containing immunoreceptor tyrosine-based inhibition motifs (ITIM) which recruit and activate SHP-1 and SHP-2 phosphatases for inhibitory signal transduction. KIR2DL1 recognises HLA-C alleles characterised by a Lys80 residue (HLACw4 and related, “Group 2” alleles). KIR2DL2 and KIR2DL3 (which are allele variants) recognise HLA-C with an Asn80 residue (HLA-Cw3 and related, “Group 1” alleles). KIR3DL1 is the receptor for HLA-B alleles sharing the Bw4 supertypic specificity (Table 1). Another type of human NK cell inhibitory receptor involved in HLA recognition is CD94-NKG2A. It binds to the non-conventional class I molecule HLA-E. Several HLA class I alleles provide signal sequence peptides that bind HLA-E and allow its expression at the cell surface. Consequently, it is expressed in every individual. Inhibitory KIRs, CD94/NKG2 and HLA-class I genes determine individual NK cell repertoires during development. As they are located on different chromosomes, receptors and ligands segregate independently in human pedigrees. The HLA class I genotype selects a self-tolerant repertoire by dictating which KIR and/or NKG2A

Table 1: HLA-class I allele specificity of the main KIRs expressed by human NK cells KIR gene

Encoded protein

HLA specificity

KIR2DL1

P58.1 receptor

HLA-C group 2 (e.g., -Cw2, -Cw4, -Cw5, -Cw6) (Lys80)

KIR2DL2/3

P58.2 receptor

HLA-C group 1 (e.g., -Cw1, -Cw3, -Cw7, -Cw8) (Asn80)

KIR3DL1

P70/NKB1 receptor

Bw4 alleles (e.g., HLA-B27)


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receptor combinations are to be used as inhibitory receptors for self HLA class I (7). Consequently, every functional NK cell in the mature repertoire expresses at least one inhibitory receptor for self HLA: co-expression of two or more receptors is less frequent.

2. Inhibitory KIRs and alloreactivity Since inhibitory KIRs recognise specific groups of HLA class I molecules, i.e., HLAC group 1, HLA-C group 2, HLA-Bw4 alleles, NK cells with the potential to exert alloreactions use KIRs as inhibitory receptors for self (1-6). NK cells which express, as their only inhibitory receptor for self, a KIR for the HLA class I group which is absent on allogeneic targets, sense the missing expression of the self class I KIR ligand and mediate alloreactions (“missing self” recognition). Importantly, most individuals possess a full complement of inhibitory KIR genes and can exert NK cell alloreactions (3, 4, 6). In particular, 100% of individuals possess the KIR2DL2 and/or KIR2DL3 receptors for HLA-C group 1 alleles. If they have HLA-C group 1 allele(s) in their HLA type, these individuals possess HLA-C1-specific NK cells which are alloreactive against cells from individuals who do not express HLA-C group 1 alleles. Ninety-seven percent of individuals possess the KIR2DL1 receptor for HLAC group 2. If they possess HLA-C group 2 allele(s) in their HLA type, these individuals have HLA-C2-specific NK cells which mediate alloreactions against cells from individuals who do not express HLA-C group 2 alleles. Finally, ~90% of individuals possess the KIR3DL1 receptor for HLA-Bw4 alleles. When they have HLABw4 allele(s) in their HLA type, these individuals may have HLA-Bw4-specific NK cells that are alloreactive against Bw4-negative cells. These KIR ligand mismatches often occur in haploidentical donor recipient transplant pairs.

3. Clinical effects of NK cell alloreactivity When exerted in the donor-versus-recipient direction, NK cell alloreactivity emerged as a crucial factor in improving outcomes of haploidentical transplantation (1-4). It reduced the risk of leukaemia relapse, did not cause graft versus host disease (GvHD) and markedly improved event-free survival (EFS) in a series of haploidentical transplants (57 acute myeloid leukaemia (AML) patients, 20 of whom were transplanted from NK alloreactive donors) (2). In an updated analysis (4), 112 highrisk AML patients received haploidentical transplants from NK alloreactive (n=51) or non-NK alloreactive donors (n=61). Transplantation from NK-alloreactive donors was associated with: - a significantly lower relapse rate in patients transplanted in any CR (3 vs. 47%) (p2 years of age) 2. Cy + Bu (+ MEL) in AML, CML and MDS.

5. Disease specific aspects 5.1. Inborn errors of the lymphohaematopoietic system In patients with profound deficiencies of the lymphoid immune system, such as SCID, the capacity for graft rejection is extremely low or absent. Preparative regimens are therefore unnecessary but HSCT results in an unusual form of lympho-haematopoietic chimerism. Donor cell engraftment after HLA identical sibling HSCT is usually restricted to the lymphocytes, whilst the myeloid system remains of host cell origin. The same approach in TCD HLA-mismatched HSCT has led to more variable results. Here, complete or partial graft failure, in particular failure of B-cell reconstitution, has been observed. This experience has resulted in the use of CT based preparative


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regimens in patients transplanted for SCID from HLA-mismatched donors. The regimen most frequently employed is that of Bu 2 mg/kg/day for 4 d + Cy 50 mg/kg/d for 4 d. In the presence of serious clinical conditions, e.g. pulmonary infections prior to the transplant, the use of chemotherapy may greatly increase the morbidity and mortality of the procedure. In these cases, HSCT may be performed without conditioning, as even partial immunological reconstitution may be of therapeutic benefit to the child. In less severe variants of lymphoid deficiencies and in all other inborn errors, conditioning is required for myelo-ablation and to prevent graft rejection and disease recurrence. Some young patients come to transplant with a number of complications related to their underlying disease, in particular poorly controlled chronic infections. The use of non-CT based conditioning protocols which allow stable engraftment remains a long-term goal. 5.2 Severe aplastic anaemia The marrow is empty in patients with SAA and the sole aim of the conditioning protocol is to provide immunosuppression is. Cy 50 mg/kg for 4 successive days is an appropriate conditioning regimen. It is very immunosuppressive and its use involves few serious long-term side-effects. Rejection remains a concern, especially in patients sensitised to their donors via blood products. The addition of ATG has reduced risk of rejection in one pilot study but prospective randomised and retrospective multicentre analyses have failed to confirm its value in nonsensitised recipients. Still, it is the most frequently used approach at the present time. The optimal conditioning regimen for patients with SAA and alternative donor transplants still needs defining. The addition of Fluda or low dose TBI (2 Gy) is under current investigation. All long-term follow up studies have shown a higher incidence of late malignancies in SAA patients given TBI or TAI during conditioning. 5.3. Fanconi’s anaemia Fanconi’s anaemia is a genetic disorder associated with diverse congenital abnormalities, progressive BM failure and increased risk of leukaemia and other cancers. HSCT is an effective treatment. The underlying molecular defects are heterogeneous but all result in defective DNA repair mechanisms. These patients are therefore extremely susceptible to the effects of CT/RT, in particular to irradiation and alkylating agents. Conditioning regimens must be reduced in intensity. Most regimens contain Cy 5–10 mg/kg/d for 4 days. Some centres add low dose TBI (400–450 cGy) or TAI or TLI (500 cGy) to overcome graft rejection. More recent approaches investigate the use of Bu or the addition of Fluda. 138

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CHAPTER 6 • Principles of conditioning

However, there is concern that the addition of TBI may increase the incidence of secondary malignancies in a group of patients already susceptible to further malignant disease. 5.4. Lymphoma The conditioning regimens for high dose therapy in lymphoma have been based on custom and practice rather than proof or confirmation by randomised trials. Most frequently used regimens avoid inclusion of TBI. Auto-HSCT: BEAM (BCNU, VP, ARA-C and MEL) has been the most commonly used regimen both for NHL and HD. Originally described jointly by groups in Lyon and London, it has become standard treatment both in Europe and, to a lesser extent, in the United States. The standard BEAM uses a total dose of VP of 800 mg/m2/d for 4 days. Several groups have attempted to change the individual components. None has proved superior and there is no evidence that any other regimen has more anti-tumour effect than BEAM. Allo-HSCT: BEAM and CBV have both been used in allografting but neither regimen is fully myeloablative. Fluda based RIC HSCT have been proposed. TBI plus CT is considered as the standard preparative regimen for allo-HSCT. The incidence of TRM is clearly associated with the status of the patients at the time of transplant. If transplant is performed for refractory HD or NHL, the TRM reaches 30–40%. Bu and Cy have also been used but Bu is not in common use in the treatment of lymphoma. There are no comparative data for Bu/Cy versus TBI containing regimens or for RIC versus standard conditioning HSCT. 5.5. Myeloma Relatively clear data are available concerning the best conditioning regimen for MM. Auto-HSCT: retrospective data on several thousand autologous HSCT for MM show clear results. Survival is best with a conditioning using MEL 200 mg/m2. No other regimen has shown better results. Allo-HSCT: standard conditioning with Cy TBI shows best long-term survival. RIC HSCT reduces early mortality but retrospective comparative data show inferior survival at +5 years. RIC HSCT may be used in planned double autologous allogeneic HSCT programs. The best approach is not known. 5.6. Solid tumours Dose intensification was the basic concept when auto-HSCT became an investigational tool for treatment of solid tumours. Initial results appeared promising and there was huge interest in the mid-nineties. Conditioning regimens were developed with "tumour specific" drugs in mind and a multitude of regimens appeared. None has


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proved to be superior to any other and HSCT in solid tumours with very few exceptions is still considered investigational. Conditioning regimens should follow those specified in the clinical protocols. They are discussed in the Chapter on solid tumours. Allo-HSCT is being investigated in more recent years as a tool for promoting graft vs. tumour effects. Hence, primarily RIC regimens are chosen with the aim of establishing donor type engraftment. Any RIC regimen can be used without a need for tumour specific therapy. RIC HSCT for solid tumours is still to be considered investigational.

6. Conditioning induced organ damage The desirable effects of conditioning regimens are offset by their highly predictable toxicities, which are responsible for considerable morbidity and mortality (see Table 3). These involve the GI, renal, hepatic, pulmonary and cardiac systems and include cardiac toxicity (Cy >1.5 g/m2/d; BCNU), pulmonary toxicity (TBI >8 Gy), mucositis, hepatic toxicity, bladder toxicity, renal toxicity and neurological toxicity. Prevention and treatment of early and late complications are discussed in detail in Chapters 9 and 12.

7. Quality management issues Application of high dose therapy in the context of HSCT is a complex and challenging therapy. It is prone to errors and all strategies should be undertaken to minimise such risks. This includes specifically the following points, as indicated in the JACIE standards and as outlined in Chapter 4. Each institution administering high dose CT in the context of HSCT must establish a quality management system for administration of this therapy. Such a system must be established in close cooperation with the institutional pharmacy and the nursing team. Conditioning has to be based on pre-printed orders. Regular check points at different levels and immediately prior to administration have to safeguard that the right patient is given the specified drugs at the correct dose and appropriate timing. All check points include the “4 eyes” principle. Similar considerations apply to the administration of TBI.

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Table 3: Factors influencing the outcome of HSCT* Disease factors Stage: increasing stage Chronic phase patients only: Basophil count (>3%) Sokal/Hasford score (higher) Leukocyte count (higher) Time interval (> 12 m) Patient-related factors Age (higher) Sex (male) Race Viral status (CMV positivity) Donor-related factors Histocompatibility (vs. HLA-id sibling) Identical twin Unrelated Mismatched Sex (female D for male R) Viral status (CMV positivity) Peri-transplant factors Conditioning (intensified) (reduced) GvHD prevention (intensified) (reduced) Cell content (CD34) high low Stem cell source: BM vs. PB Post-transplant factors Acute GvHD (increasing grade) Chronic GvHD (none vs. any)

RI

TRM

LFS

OS

≠

≠

Ø

Ø

≠ (≠) ≠ æ

æ (≠) æ ≠

Ø Ø Ø Ø

Ø Ø Ø Ø

? æ ?

≠ ≠ ≠ ?

Ø Ø ?

Ø Ø ?

æ

≠

Ø

Ø

≠ Ø Ø Ø ?

Ø ? ≠ ≠ ≠

Ø ? Ø Ø Ø

≠ ? Ø Øearly/≠late Ø

Ø ≠ ≠ Ø Ø æ (≠)

≠ Ø Ø ≠ Ø ≠ ≠

æ

æ

Ø æ æ ≠ Ø ?

? æ æ ≠ Ø ?

Ø Ø

≠ ≠

Ø Ø

Ø Ø

D: donor; R: recipient * These factors are confirmed for CML but apply in principle to most other situations

References 1. Thomas et al. A history of bone marrow transplantation. In: Thomas' Hematopoietic Cell Transplantation, 3rd Edition (Blume KG, Forman SJ, Appelbaum FR Eds.), Blackwell Publishing Oxford, Malden, Victoria, 2004, pp 4-8. 2. Storb RF, Lucarelli G, McSweeney, Childs PW. Hematopoietic stem cell transplantation for benign hematological disorders and solid tumors. Hematology 2003; 372-397. 3. Santos GW, Tutschka PJ, Brookmeyer R. Marrow transplantation for acute non-lymphocytic


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leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med 1983; 309: 1347-1353. 4. Ridell S, Appelbaum FR, Buckher CD, et al. High-dose cytarabine and total body irradiation with or without cyclophosphamide as a preparative regimen for bone marrow transplantation for acute leukemia. J Clin Oncol 1988; 6: 576-582. 5. Gore ME, Selby PJ, Viner C, et al. Intensive treatment of multiple myeloma and criteria for complete remission. Lancet 1989; ii: 879-882. 6. Philips G, Shepherd J, Bernett M, et al. Busulfan, cyclophosphamide and melphalan as a conditioning regimen for bone marrow transplantation in children with myelodysplastic syndrome. Leukemia 1994; 8: 1880-1888. 7. Slavin S, Nagler A, Naparstek E, et al. Non-myeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and non-malignant hematologic diseases. Blood 1998; 3: 756-763. 8. Giralt S, Estey E, Albitar M, et al. Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: Harnessing graft-versus-leukemia without myeloablative therapy. Blood 1997; 89: 4531-4536. 9. Copelan EA. Hematopoietic stem-cell transplantation. N Engl J Med 2006; 354: 1813-1826. 10.Appelbaum FR. Hematopoietic-cell transplantation at 50. N Engl J Med 2007; 357: 14721475. 11.Armand P, Antin JH. Allogeneic stem cell transplantation for aplastic anemia. Biol Blood Marrow Transplant 2007; 13: 505-516. 12.Blaise D, Vey N, Faucher C, Mohty M. Current status of reduced-intensity-conditioning allogeneic stem cell transplantation for acute myeloid leukemia. Haematologica 2007; 92: 533-541. 13.Attal M, Moreau P, Avet-Loiseau H, Harousseau JL. Stem cell transplantation in multiple myeloma. Hematology (Am Soc Hematol Educ Program) 2007; 311-316. 14.Hegenbart U, Niederwieser D, Sandmaier BM, et al. Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol 2006; 24: 444-453. 15.Secondino S, Carrabba MG, Pedrazzoli P, et al.; European Group for Blood and Marrow Transplantation Solid Tumors Working Party. Reduced intensity stem cell transplantation for advanced soft tissue sarcomas in adults: A retrospective analysis of the European Group for Blood and Marrow Transplantation. Haematologica 2007; 92: 418-420. 16.Satwani P, Cooper N, Rao K, et al. Reduced intensity conditioning and allogeneic stem cell transplantation in childhood malignant and nonmalignant diseases. Bone Marrow Transplant 2007; 26: in press. 17.Barrett AJ, Savani BN. Stem cell transplantation with reduced-intensity conditioning regimens: A review of ten years experience with new transplant concepts and new therapeutic agents. Leukemia 2006; 20: 1661-1672.

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Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Total body irradiation (TBI) is frequently used for conditioning in HSCT. Its biological effects depend primarily on: a) Radiation source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Total radiation dose applied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Combination of dose rate and fractions of doses . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Combination of total dose, dose rate and fractions of doses . . . . . . . . . . . . . . 2. The type of conditioning regimen is more important in autologous than in allogeneic HSCT, because: a) Graft versus tumour reaction is the only element in allogeneic HSCT for tumour control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Dose intensification of tumour specific therapy is the key element of autologous HSCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) There is no specific chemotherapy in allogeneic HSCT. . . . . . . . . . . . . . . . . . . . . . d) Monoclonal antibodies, such as CD20+ antibodies can only be integrated in the conditioning in autologous HSCT . . . . . . . . . . . . . . . . . . . . . . . . 3. Which of the following statements about reduced intensity conditioning (RIC) is correct? a) RIC has a specific advantage in patients with advanced malignancies . . . b) RIC has a specific advantage in patients with non-malignant conditions where no graft-versus-disease effect is required . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) RIC has a specific advantage in CMV positive patients with CMV negative donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Peripheral blood as a stem cell source is associated with less rejection than bone marrow in patients with RIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. There is an inherent dilemma in allogeneic HSCT: Increasing conditioning decreases risk of rejection and risk of relapse at the expense of increased toxicity; vice versa, decreased conditioning decreases direct regimen related toxicity at the expense of increased risk of rejection and relapse. Which of the following statements about this dilemma is true?


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a) Different regimens should be employed for patients with high-risk disease and low transplant risk compared to patients with low-risk disease but high transplant risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) T-cell depletion can abolish this dilemma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The statement does not hold true for unrelated cord blood transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The statement is not correct for non-malignant disorders . . . . . . . . . . . . . . . . . 5. The following conditioning regimens can be generally accepted as “established”; e.g., novel regimens need to be evaluated against this standard: a) Cyclophosphamide +/- ATG for severe aplastic anaemia . . . . . . . . . . . . . . . . . . . . b) Cyclophosphamide + busulfan for Hodgkin’s lymphoma . . . . . . . . . . . . . . . . . . . . c) Cyclophosphamide + TBI for multiple myeloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Fludarabine for myeloid malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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NOTES


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*

CHAPTER 7

Transfusion policy

D.H. Pamphilon

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*

CHAPTER 7

Transfusion policy

D.H. Pamphilon

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CHAPTER 7 • Transfusion policy

1. Introduction Haematopoietic stem cell transplant (HSCT) patients often require intensive blood component support. Transfusion may be complicated by transfusion transmitted infection (TTI) – both viral and bacterial, transfusion-associated (TA)-GvHD, febrile non-haemolytic transfusion reactions (FNHTR) and transfusion-related acute lung injury (TRALI). Alloimmunisation (AI) to red cell antigens may cause difficulties in selecting compatible blood whilst AI to the human leukocyte antigens (HLA) present on platelets may cause refractoriness to subsequent transfusions of randomly-selected platelets. It is therefore essential to define robust transfusion policies and procedures and these should be regularly audited. This Chapter describes the blood components available for transfusion including granulocytes and their clinical use in the setting of HSCT. The impact of reduced-intensity conditioning (RIC) transplantation on transfusion requirements is highlighted. Amongst infectious agents transmissible by blood components, cytomegalovirus (CMV) is particularly important in BMT patients and strategies to minimise CMV transmission in susceptible recipients are described.

2. General policies for the selection of high quality, appropriate transfusions The European Union Directive 2002/98/EC sets standards for the collection, testing, processing, storage and distribution of human blood and blood components (1). It requires that Blood Establishments should be licensed and this is of importance for both Blood Centres in EU countries that undertake these activities as well as hospitals that collect and issue e.g. granulocytes for transfusion. The most important aspects of the Directive are: • The fate of each unit of all blood components should be recorded and this record kept for 30 years, i.e. donor to recipient traceability • Robust Quality Systems should be in place • The processing of blood and blood components should be undertaken by licensed blood establishments (see above) • Training should be provided for hospital transfusion laboratory staff • Haemovigilance systems should be established to include the reporting of adverse events. Establishments are licensed by the Competent Authorities in EU Member States following inspection by a regulatory body – in the UK this is the Medicines and Healthcare Products Regulatory Authority (MHRA). Reports of compliance must be submitted.


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2.1. Testing of donated blood for infectious disease markers (IDM) A number of microbial agents may be transmitted by blood transfusion. These include hepatitis B and C, HIV-1 and -2, HTLV-1 and 2, CMV and syphilis. Blood Services routinely test blood for the following: • Hepatitis B: Hepatitis B surface antigen (HbsAg)* • Hepatitis C: Hepatitis C antibodies (anti-HCV)* • Human immunodeficiency virus 1 and 2: HIV-1 + 2 antibodies (anti-HIV 1+2)* • Human T-lymphotropic virus 1 and 2: HTLV-1 + 2 antibodies (anti-HTLV-1+2) • Syphilis. * These tests are mandated by the EU Blood Directive (2002/98/EC) (1) In addition, donations may be tested for: • anti-HBc, i.e. anti-hepatitis B core antigen • alanine aminotransferase (ALT): A surrogate marker of hepatitis C • HCV-RNA by PCR for hepatitis C • p 24 antigen for HIV-1. There is a variation from country to country in the number of tests performed on each blood donation. Testing for anti-CMV antibody to identify CMV seronegative donors is done on a proportion of blood donations, sufficient to identify enough CMV seronegative components for transfusion to those patients for whom it is appropriate. Bacterial contamination is a relatively common occurrence with an incidence estimated at 0.05–0.5% of components (2). The sources of bacteria are donor bacteraemia and contamination with bacteria present on the skin at the time of donation or present in blood packs. The organisms that most frequently contaminate red cell and platelet transfusions are shown in Table 1. Screening tests for bacteria in platelet concentrates (PCs) using automated blood culture systems e.g. BacT/ALERT have been evaluated (2) and are now used routinely by some transfusion services. PCs are not issued until at least 48 hours

Table 1: Bacteria that most frequently contaminate blood components Red cells Yersinia enterocolitica Pseudomonas fluorescens Other species

51% 27% 22%

Platelets Staph. epidermidis Staphylococcus aureus Salmonella choleraesuis Serratia marcescens Bacillus cereus Others

Figures are percentage of total contaminants for red cells and platelets respectively 148

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after collection but the storage period may be extended to 7 days once sterility has been evaluated. The risk of bacterial transmission is also minimised by careful donor selection, meticulous attention to sterility during venepuncture, diversion of the first 30 mL of blood collected (contains most of the bacteria) away from the primary collection pack and sterility during preparation of blood components. Bacterial contamination should be suspected in any patient who develops a febrile reaction characterised by fever, chills ± hypotension. Microbiological testing does not completely remove the risk of TTI, although the chance of infection in the UK after transfusion of screened blood components from known/previously tested donors is estimated to be less than 1 in 2 x 106 for HIV1, HBV and HCV. This risk will vary somewhat according to the donor selection and testing policies that are operative within a Blood Service. 2.2. Blood grouping and antibody testing The ABO and Rhesus D types of all donated blood are determined by standard techniques. This is a requirement of the EU Blood Directive (1). All donations are tested to exclude the presence of immune IgG antibodies that are reactive with common blood groups and which occur after an immunising stimulus such as pregnancy or transfusion. Selected units of red cells may be more extensively phenotyped (Kell, Duffy, Kidd, MNSs antigens) for patients who develop red cell alloantibodies. 2.3. Prevention of CMV transmission A proportion of HSCT patients are CMV seropositive pre-transplant or have seropositive donors. They require regular screening by PCR and antigenaemia testing together with ganciclovir therapy where appropriate to minimise the impact of virus reactivation and prevent clinical infection post-transplant. All CMV seronegative HSCT patients with CMV seronegative donors (neg/neg) and CMV seronegative patients with haematological and other disorders who are likely to proceed to a transplant should receive blood components that have a minimal risk of causing CMV acquisition (3). Studies show that the use of CMV seronegative components is associated with a less than 3% incidence of CMV infection and/or disease in neg/neg HSCT. CMV is transmitted via leukocytes, and leukodepletion also minimises the risk of CMV transmission. CMV seronegative and leukodepleted blood components are probably of equivalent efficacy but this view is not generally accepted (4, 5). Further evidence from prospective randomised controlled studies (PRCT) using pre-storage leukodepleted blood components is required. Centres must establish their own policies.


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2.4. Leukodepleted blood components Transfused leukocytes cause alloimmunisation (AI) to HLA Class 1 antigens (HLA AI) in a proportion of patients. This may be manifested clinically as FNHTRs, although these may also be caused by antibodies to neutrophils, platelets or plasma proteins and by cytokines such as interleukin (IL)-1, IL-6, IL-8 and tumour necrosis factor (TNF)-a which accumulate in stored blood components, especially PCs. HLA AI may cause accelerated destruction of transfused platelets that are HLA incompatible. This is clinically manifest as a failure to achieve a satisfactory increment after platelet transfusion (refractoriness). A summary of the adverse effects of transfused leukocytes is shown in Table 2. Donor dendritic cells (DC) which are present in red cell and platelet transfusions appear to be responsible for sensitisation to HLA. Studies show that removal of leukocytes to less than 5 x 106 per blood component prevents primary HLA AI in >97% of patients with haematological malignancies. The use of leukodepleted components also reduces secondary AI and refractoriness to platelet transfusion. Refractoriness is not always prevented since in >50% of cases it results from increased platelet destruction due to non-immune causes which include fever, splenomegaly, DIC and amphotericin therapy. AI is also associated with a higher incidence of graft failure in patients with severe aplastic anaemia. Filtration of blood or its components is best performed in Blood Centres and hospital blood banks. Data from studies where leukocytes were filtered from blood components at the bedside show that this may not be effective in preventing or reducing FNHTR, AI and refractoriness.

Table 2: Adverse effects of transfused leukocytes

150

HLA alloimmunisation causing

- FNHTR - Refractoriness to random donor platelets - Graft rejection - Shortened red cell survival

Transmission of microorganisms

- CMV - HTLV-1/11 - Toxoplasma gondii - Yersinia enterocolitica

Immunomodulation

- GvHD - Activation of viruses in host cells e.g. HIV-1 - Immune suppression of T- and NK-cell functions

Affecting the quality of stored blood

- Microaggregate formation - Metabolic deterioration during storage

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2.4.1. Indications for leukodepleted blood components (6) • Pre-HSCT in patients with SAA to reduce the likelihood of graft failure; • Pre- and post-HSCT to prevent recurrent FNHTR; • Pre-and post-HSCT to minimise HLA AI and platelet refractoriness. This is optional since there is no evidence of a significant impact on important clinical outcome measures such as survival post-HSCT except in patients with SAA. Nonetheless many Blood Services have implemented leukodepletion of a large proportion or, in some cases, all of their blood components. In the UK universal leukodepletion was implemented in 1999 with the aim of minimising the risk of transfusionassociated transmission of the causative agent of variant Creutzfeld-Jakob disease (vCJD); • As an alternative to CMV seronegative components. 2.5. Gamma-irradiation of blood components and TA-GvHD HLA incompatible third party leukocytes contained in donated blood components can engraft and initiate an alloreactive response after transfusion. This can cause TA-GvHD, manifest clinically by fever, rash, diarrhoea, jaundice and pancytopenia, and this is fatal in >90% of cases, so prevention is essential. Donor leukocytes are inactivated by gamma-irradiation of 2500 cGy and all components for HSCT recipients should be irradiated from the time that conditioning therapy is started and continued until 6 months post-transplant or until the lymphocyte count is 1 x 109/L in the absence of chronic GvHD. In addition, HLA matched PCs should be irradiated, as should those from family members, since HLA haplotype sharing may result in TA-GvHD even in immunocompetent patients. A summary of the indications for blood component irradiation is shown in Table 3. Platelets show normal functional characteristics through 5 days storage after irradiation with doses up to 5000 cGy. Red cells leak potassium during storage and

Table 3: Indications for irradiated blood components • • • • • • •

Allo-HSC recipients from time of conditioning therapy for 6 months or until the lymphocyte count is 1 x 109/L in the absence of cGvHD Allo-HSC donors Auto-HSC recipients (from 7 days before harvest until 3 months post transplant) All donations from HLA-matched donors or 1st or 2nd degree relatives All patients with Hodgkin disease at any stage of therapy All patients treated with purine analogues e.g. fludarabine All patients with congenital immunodeficiency states


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this is worsened by irradiation. Therefore, storage is limited to 14 days after irradiation with 2500 cGy. TA-GvHD has been shown to occur after 1500–2000 cGy and this dose range is not recommended (7).

3. Pre-transplant transfusions The following provisions apply: • Red cell transfusions for patients with sickle cell disease are initially matched for ABO, Rhesus D and Kell antigens but additional matching for the Rhesus CcEe and for Duffy (Fya Fyb), Kell (Kk), Kidd (Jka Jkb) and MNSs antigens may be required if the patient has developed alloantibodies; • Leukodepleted blood components should be transfused to all patients with aplastic anaemia (6); • Either CMV seronegative or leukodepleted blood components should be transfused to susceptible patients to prevent CMV acquisition (3); • Blood components should be gamma-irradiated for PBSC transplant patients during stem cell mobilisation and collection since transfused leukocytes might be captured in the PBSC harvest and subsequently induce TA-GvHD (7); • Blood components transfused to allogeneic marrow donors immediately pre- or intra-operatively should also be irradiated (7).

4. Blood component transfusions The following definitions for red cells, PCs, FFP, cryoprecipitate and granulocyte products were derived from the Guidelines for UK Blood Services. The requirement for red cell and platelet transfusions is decreased in the setting of RIC transplantation (8). 4.1. Red cells Red cells, usually suspended in an optimal additive solution (OAS) are transfused to correct anaemia due to marrow failure, haemorrhage or haemolysis, aiming to keep the haemoglobin or packed cell volume (PCV) above predefined levels to ensure good tissue oxygenation. Reduced intensity conditioning (RIC) transplants require fewer red cell transfusions. Transfusions may be: • Suspended in OAS, usually a combination of saline, adenine, glucose and mannitol (SAG-M): PCV 50–70%; volume 220–420 mL. This is the product of choice; • Derived from whole blood from which a proportion of the plasma has been removed – plasma reduced blood (PRB): PCV 50–60%; volume 200–450 mL; • Unmodified whole blood: volume 420–520 mL. This last term is misleading since platelets and labile coagulation factors deteriorate rapidly in stored blood. 152

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The storage period is 35–42 days at 4 ± 2ºC. Transfusion policy • Red cells should be matched for ABO and Rhesus D type (1). • Extended phenotyping may be necessary in patients, e.g. those with sickle cell disease, who have formed red cell alloantibodies after previous transfusions. • Red cells should be cross-matched against the patient’s serum by standard techniques prior to transfusion. • Thresholds should be defined for haemoglobin and PCV below which red cell transfusions are always given. Suggested arbitrary cut off points are Hb less than 8.0 g/dL and PCV less than 25%. • In adults 1 unit of red cells raises the Hb by 1.0 g/dL whereas in children the volume of blood to be transfused is derived from the formula: Volume = Increase in Hb (g/dL) required x 4 x weight (kg) 4.2. Platelet transfusions 4.2.1. Manufacture Platelet concentrates (PCs) are made: • From whole blood by centrifuging units in a “top – top” pack format to obtain platelet rich plasma (PRP), which is then further concentrated to give a PC. PRPPCs may be transfused individually or pooled in multiples – usually 6; • From whole blood by centrifuging units in a “bottom & top“ pack format to separate the buffy coat (BC), pooling 4 BCs and recentrifuging to separate PRP which is then expressed into a secondary storage container for PC preparation; • By collecting PCs directly on a cell separator. Dual arm, continuous flow apheresis is preferred and some cell separators collect PCs with an inherently low WBC content (Table 4) (9).

Table 4: Platelet content and WBC contamination of different types of platelet concentrates PRP-PC

BC-PC

Apheresis PC

Mean platelet Content x 1011/unit

3.4

3.2

3.15

Mean WBC Contamination x 106/unit

365

5.7

0.3

PRP-PC: platelet concentrate prepared from whole blood; BC-PC: platelet concentrate prepared from buffy coat; Apheresis PC: platelet concentrate obtained directly by apheresis. Data from the National Blood Service, Bristol Centre (9)


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The storage period is 5 days at 22 ± 2ºC unless bacterial screening has been carried out in which case it may be extended to 7 days. Transfusion policy Current practice, based on the results of randomised studies, is to transfuse PC prophylactically when the platelet count is less than 10 x 109/L. A recent Cochrane Systematic Review concluded that, whilst there is no reason to change current practice, blood products may become scarcer and further trials should be undertaken to compare prophylactic versus therapeutic platelet transfusion – i.e. PC given only when there is clinical bleeding (10). In autologous PB HSCT this has been found to be safe (11). Fewer PC transfusions are required in RIC allografted patients compared to those who receive full myeloablative conditioning (8). Best current practice is that: • PCs should be ABO and Rh compatible wherever possible since ABO incompatibility may reduce the expected count increment (CI) by 10–30%; • Group O PCs should be tested for high titre anti-A, B and if positive should only be transfused to group O recipients to avoid haemolysis caused by passive administration of antibody; • If Rh D positive platelets are given to an Rh D negative patient then give 250 IU polyclonal anti-Rh (D) immunoglobulin. Since the chance of Rh immunisation is probably less than 5% this may be omitted and the patients serum screened for immune red cell antibodies, or prior to a red cell transfusion; • Studies show that a threshold of 10 x 109/L in stable thrombocytopenic patients is optimal for prophylactic platelet transfusion; • A higher threshold of 20 x 109/L should be used in patients with fever, sepsis, splenomegaly and other well-established causes of increased platelet consumption; • If an invasive procedure is planned, e.g. central line insertion, the platelet count should be >50 x 109/L; • PCs should be transfused when there is significant clinical bleeding, irrespective of the platelet count; • PCs are contraindicated in patients with TTP; • In adults the usual dose of platelets is 3 x 1011 (an adult therapeutic dose – ATD) in a volume of 200–300 mL; • Children >30 kg receive one ATD. Children 2 mg/dL; hepatomegaly or pain in the right-upper quadrant; weight gain (>2% basal weight). Baltimore criteria: In first 21 days after HSCT, presence of bilirubin >2 mg/dL plus ≥2 of the following: painful hepatomegaly; ascites; weight gain (>5% basal weight). In both, other possible causes of these clinical features should be excluded before accepting the diagnosis of VOD (see differential diagnosis). Additionally, it is necessary to remember that some VOD cases can appear late after HSCT. Additional investigations Other complementary studies that can aid diagnosis are: Haemodynamic study of the liver carried out through the jugular or femoral veins (9): Despite its usefulness, this is only indicated to confirm the diagnosis of VOD before adopting a therapeutic approach that may be potentially hazardous for the patient. An hepatic venous gradient pressure (HVGP) ≥10 mmHg in a patient without previous liver disease allows a precise differential diagnosis with a high degree of specificity. However, a normal HVGP does not exclude the diagnosis of VOD. Liver biopsy: Thrombocytopenia usually present in this phase of HSCT precludes a transparietal liver biopsy; consequently hepatic tissue can only be obtained by means of a transvenous biopsy in the course of a haemodynamic study. In addition to the 184

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CHAPTER 9 • Early complications after HSCT

Table 1: Risk factors (3, 4, 6–8) Risk

Lower risk < Higher risk

Transplant type Donor type HLA compatibility Stem cell origin T-cell depletion Diagnosis Status of the disease

Syngeneic or autologous < allogeneic Sibling < another relative < unrelated HLA match < any mismatch Peripheral blood < bone marrow With TCD < without TCD non malignant disease < malignant disease Remission < relapse

Conditioning - Intensity - TBI

- Busulfan - Timing Age / Sex Karnofsky index ASAT/ALAT before HSCT Transplant number Previous hepatic irradiation Previous Mylotarg Status of the liver CMV serological status Fever in conditioning Hepatotoxic drugs Genetic predisposition

Cy alone < Cy + TBI < BVC (a) Fractionated TBI < single dose TBI Less than 12 Gy < more than 12 Gy Low dose rate < high dose rate IV BU < adjusted oral Bu < non adjusted oral Bu Interval Cy – TBI 36 hours < 12 hours Younger < older / men < women 100–90 < lower than 90 Normal < high First < second No < yes No < yes (b) Normal < fibrosis < cirrhosis or infiltration Negative < positive Absent < present Progestogens, ketoconazole, CsA, methotrexate, amphotericin B, vancomycin, acyclovir, IV Ig (c) GSTM1 positive < GSTM1 null genotype (d)

The most important risk factors are indicated in bold type. (a) BVC (BCNU, VP, Cy). (b) VOD incidence up to 64% (Wadleigh et al. Blood 2003). (c) Higher incidence of VOD with high-dose IVIg. (d) Srivastava et al., Blood 2004

classical histological changes of VOD (concentric non-thrombotic narrowing of the lumen of small intrahepatic veins) other less specific abnormalities can be observed in patients with a VOD syndrome, including eccentric narrowing of the venular lumen; phlebosclerosis; sinusoidal fibrosis and hepatocyte necrosis. Due to patchy nature of VOD a normal biopsy does not exclude the diagnosis. Ultrasound: A variety of abnormalities can be observed; gallbladder wall thickening, ascites, hepatomegaly and attenuate or reversed portal flow, but all of them are nonspecific. Biological markers: Although the serum of patients with VOD shows an increase in HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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levels of plasminogen activator inhibitor-1 (PAI-1) (marker with the highest specificity and sensitivity for VOD), aminopropeptides of type III collagen, and hyaluronic acid, all these measurements are of little utility in routine clinical practice. Differential diagnosis To accept the diagnosis of VOD all the following possible causes of similar clinical features should be excluded as far as possible, including: • Infections: Cholangitis lenta (sepsis of liver) / fungal infection / viral hepatitis • Immune dysfunctions: Acute GvHD of the liver • Drug toxicity: CsA, azoles, MTX, progestogens, trimethoprim-sulphamethoxazole, TPN, among others • Reduction of venous outflow / increased volume: Constrictive pericarditis / congestive heart failure / fluid overload / renal failure • Others: Pancreatic ascites / chylous ascites / infiltration of the liver. Prophylaxis of VOD (6, 8) (Table 2) Table 2: Prophylaxis of VOD Avoidance of risk factors • When possible delay HSCT if an acute hepatitis exists; adjust Bu dose or use IV Bu; fractionate TBI; avoid hepatotoxic drugs, etc. • In high risk patients, consider allo-RIC HSCT (lower incidence of VOD) Pharmacological The following drugs have been used to prevent VOD from the beginning of conditioning until day +21–30: • Sodium heparin: 100 U/kg/day by continuous infusion. Two randomised studies showed a beneficial effect but others have suggested that it is ineffective and dangerous • Prostaglandin E1: 0.3 µg/kg/h by continuous infusion. Evaluated in several clinical trials usually combined with heparin. When administered alone no beneficial effect was observed • Ursodeoxycholic acid: 600–900 mg/day p.o. Four randomised trials and 2 historically controlled studies have shown a reduction in incidence of VOD and in TRM • N-acetylcysteine. Very limited experience • Low molecular weight heparin: Enoxaparin 40 mg/day or fraxiparin 5000 U/day subcutaneously seem to be relatively safe and may have some effect but a large randomised study is needed to confirm these results • Pre-emptive ATIII replacement: Ineffective • Defibrotide. Several preliminary reports have shown encouraging results

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Treatment of established VOD (6, 8) (Table 3) Table 3: Treatment of established VOD First line therapy Symptomatic (a)

-

Specific (b)

-

Restriction of salt and water intake ± diuretics Maintain intravascular volume and renal perfusion by means of albumin, plasma expanders and transfusions (haematocrit >30%) Defibrotide 6.25 mg/kg IV in 2 h infusion q 6 h IV during 14 days (1) (c) (d) rt-PA 0.05 mg/kg/h during 4 hours (maximum 10 mg/day) for 2–4 days ± sodium heparin 20 U/kg as a bolus (maximum 1000 U) followed by 150 U/kg/day by continuous infusion for 10 days (e)

Other measures Symptomatic (a)

-

Specific

-

Low dose dopamine (effectiveness not demonstrated) Analgesia Paracentesis / thoracocentesis Haemodialysis / haemofiltration Mechanical ventilation TIPS (transvenous intrahepatic portosystemic shunt) (f) Surgical shunt Liver transplantation

rt-PA: recombinant tissue plasminogen activator. (a) Symptomatic treatment should be established first, reserving specifies measures for most severe cases. (b) Although other agents have been used (antithrombin III, prostaglandin, corticosteroids, glutamine/vitamin E, N-acetylcysteine, etc.) the only ones occasionally effective are those mentioned. (c) Defibrotide permits the resolution of 50–55% of severe VOD with multiorgan dysfunction and a 47–60% of survival at day +100 with no secondary effects in adults and children (8, 10). (d) In a randomised study defibrotide at 25 mg/kg/day has shown similar effectiveness to the classical dose of 40 mg/kg /d (8). (e) rt-PA has been shown to be effective only in patients with a non-advanced VOD. Its use is contraindicated in patients with multi-organ dysfunction syndrome (MODS), haemorrhages or severe hypertension. (f) Despite improvement in portal hypertension and ascites, long term efficacy and survival are extremely poor


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VOD evolution (3, 4, 6 ,7) (Table 4) Table 4: VOD evolution Complete resolution on day +100 w/o treatment Complete resolution on day +100 with treatment Non resolution before death (c) or on day +100 Mortality attributable to VOD by day +100 (e)

Classification (a) Frequency (b) Mild VOD 8–23% Moderate VOD 48–64% Severe VOD (d) 23–28% 1–3% of all HSCT 18–28% of all VOD 75–95% of severe VOD (f)

(a) Classification described by Seattle group for retrospective evaluation of VOD. (b) Values observed in two large series (3, 6); (c) In many cases VOD is not the direct cause of death but contributes to it. (d) The severity of VOD can be predicted by means of a mathematical model (Bearman et al., JCO 1993). (e) Data from pre-defibrotide era. (f) The equivalent predicted mortality in non-severe VOD cases ranges between 10 and 20%

3.2. Capillary leak syndrome (CLS) (11) Pathogenesis The injury to the capillary endothelium produces a loss of intravascular fluids into interstitial spaces and the clinical manifestations. Incidence The absence of well-established clinical criteria for its diagnosis precludes an accurate estimation of its incidence. Additionally, the differential diagnosis with VOD, ES or IPS can be very difficult. Clinical features Development, in the first 15 days after HSCT, of: • Weight gain (>3% in 24 hours), and • Generalised oedemas (ascites, pleural effusion, pericarditis) that characteristically does not respond to frusemide treatment. Other features occasionally observed are: tachycardia, hypotension, renal insufficiency of pre-renal origin and hypoalbuminaemia. Differential diagnosis From engraftment syndrome (ES): Its earlier development, the absence of skin rash and the poor response to corticosteroids. From VOD: The absence of jaundice and painful hepatomegaly, and the poor response to furosemide. From IPS: The presence of generalised oedema. Risk factors The use of G-CSG, GM-CSF or K-CSF; high cumulative dose of CT in the pre-HSCT phase; 188

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unrelated or HLA mismatched donor grafts. Treatment To withdraw growth factors. Despite being systematically used the response to corticosteroids is poor. There is no other specific treatment. Evolution There is a high mortality if it progresses to MODS. 3.3. Engraftment syndrome (ES) (12–14) Pathogenesis Massive release of pro-inflammatory cytokines by tissues injured by intensive conditioning and by recovering neutrophils has been hypothesised to play a role. Incidence Variable depending on the diagnostic criteria used. After auto-HSCT: from 5 to up to 25% in patients with breast cancer or autoimmune diseases. After conventional allo-HSCT: only occasionally described (possibly because of the difficult differential diagnosis from GvHD). After allo-RIC: 10% in a recent series (14). Clinical features Development, within 72 hours of the start of neutrophil recovery, of the following major clinical criteria: • High fever of a non-infectious origin (unresponsive to antibiotics and negative cultures); • Skin rash affecting >25% body surface and not attributable to an allergic reaction; • Lung infiltrates or hypoxia not attributable to fluid overload, lung embolism, or congestive heart failure. Other symptoms occasionally observed are diarrhoea, weight gain and liver, kidney or CNS dysfunction (minor criteria). Diagnosis There are no well-established criteria for its diagnosis. Spitzer (12): 3 major criteria or 2 major and one or more minor criteria; Majolino (13): fever with either skin rash, pulmonary infiltrates or diarrhoea; Gorack (14): ≥2 major criteria plus weight gain. Risk factors Most cases of ES have been described since the introduction of growth factors and use of PBSCT. For this reason, a high number of CD34+ cells, faster engraftment and use of growth factors (especially GM-CSF) are considered to be the main risk factors as well as the underlying disease (breast cancer, multiple sclerosis, POEMS syndrome). Treatment MethylPDN 1 mg/kg q 12 h (3 days) with progressive tapering over one week. An appropriate empiric antibiotic treatment should always be maintained due to the difficulty of excluding an infectious origin of the fever.


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Evolution Complete resolution in 1–5 days in >80% of cases if steroids are introduced early. 3.4. Diffuse alveolar haemorrhage (DAH) (15, 16) Pathogenesis Very similar to VOD pathogenesis but affecting the lungs. Incidence The reported incidence ranges from 1 to 5% in auto-HSCT and from 3 to 7% in alloHSCT. Some authors consider that underlying undetected infections can play a role in DAH pathogenesis and postulate that infection-associated alveolar haemorrhage and DAH should be considered as equivalents. Clinical features Despite some cases of late-onset DAH, it is usually diagnosed within the first 30 days after HSCT. The main manifestations are: • Dyspnoea, non productive cough, tachypnoea • Hypoxaemia that can require oxygen-therapy • Chest X-ray or CT with focal or diffuse interstitial or alveolar infiltrates located in middle and inferior lung fields • Bronchoalveolar lavage (BAL) progressively bloodier, and not attributable to infection (absence of pathogens in BAL), thrombocytopenia, fluid overload or heart failure. Successive aliquot of 20 mL, in at least three segmentary bronchi, become pro-gressively more bloodstained (indicating blood in the alveoli). Risk factors DAH is not related to low platelet counts. Factors that favour this complication are older age, previous thoracic radiation, allogeneic donor, myeloablative conditioning, and severe acute GvHD. Treatment After publication of some small retrospective series high-dose methylPDN (250–500 mg q 6 h, 4–5 days and tapering in 2–4 weeks) was considered the treatment of choice. However, many other authors have not observed that corticosteroids modify the poor outcome associated with DAH. Recombinant FVIIa has been used with success in some cases. The possible role of cytokine antagonists and antiinflammatory agents should be evaluated. Evolution The overall mortality rate at 60 days from the onset of the haemorrhage is around 75% despite in many patients the death is not directly related to the haemorrhage. 3.5. Thrombotic microangiopathy (TMA) (17–20) TMA is the term used to describe haemolytic uraemic syndrome (HUS) and thrombotic 190

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thrombocytopenic purpura (TTP) associated with HSCT. Pathogenesis Conditioning regimen related toxicity, together with other triggering factors that are not clearly understood, produces a generalised endothelial dysfunction with intravascular platelet activation and formation of platelet-rich thrombi within the microcirculation. In contrast to classical TTP, ADAMTS13 activity very rarely falls below 10%. Incidence Less than 4% in auto-HSCT. Up to 15% in allo-HSCT (7% in an EBMT survey). Clinical manifestations Usually develop around day +60 but early (day +4) and late (2 years) episodes have been described. Characterised by: • Microangiopathic haemolytic anaemia (MHA) (anaemia, >2–5% schistocytes, LDH and other markers of haemolysis) • Thrombocytopenia or increase in transfusional requirement • Fever of non-infectious origin • Renal dysfunction and/or neurological abnormalities (cortical blindness, seizures, typical images in CNS CT-scan). Diagnostic criteria for HSCT-associated TMA (Table 5) Table 5: Diagnostic criteria for HSCT-associated TMA Blood & Marrow Transplant Clinical Trials Network consensus (18) 1) RBC fragmentation and 2 schistocytes per high-power field on PB smear 2) Concurrent increased serum LDH 3) Concurrent renal (a) and/or neurologic dysfunction w/o other explanations 4) Negative direct and indirect Coombs test International Working Group (19) 1) Increased percentage (>4%) of schistocytes in the blood 2) De novo, prolonged or progressive thrombocytopenia 3) Sudden and persistent increase in LDH 4) Decrease in Hb concentration or increased RBC transfusion requirement 5) Decrease in serum haptoglobin concentration (a) Doubling of serum creatinine from baseline

Risk factors A higher incidence has been observed in patients receiving TBI, calcineurin inhibitors (CNI), sirolimus, urelated or HLA-mismatched donor grafts, or developing GvHD or CMV/fungal infections.


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Clinical forms Two main forms of TMA can be observed: 1. CNI-associated nephrotoxicity (or neurotoxicity) with MHA: Classically develops early after HSCT, is related to toxic levels of CNI, and is reversible after stopping its administration. Usually has a favourable evolution if it improves quickly after stopping CNI. 2. Not associated with CNI toxicity, with two clinical forms: a. Conditioning associated HUS: TMA primarily affecting the kidney, often causing oliguric or anuric renal failure with hypertension, MHA and thrombocytopenia, and b. Fulminating multifactorial TMA: Early after HSCT, renal failure, CNS disturbances, hypertension, MHA and thrombocytopenia associated with GvHD, viral or fungal infection. Most cases have a fatal evolution and do not respond to CNI suppression, plasma exchange or other treatments (see below). Prevention The only reasonable measure is to have a close control (2–3 times per week) of CNI, LDH and creatinine levels. If any of them increase peripheral blood smear, haptoglobin and CNI metabolites should be tested. Treatment The only effective measure in some cases is to inmediately stop CNI, adding another agent for GvHD prophylaxis/treatment (corticosteroids, mycophenolate, azathioprine). Plasma exchange cannot be currently considered the standard of care despite some success (less than 50% of responses and 70–90% of mortality in published series possibly due to selection bias). Some authors have reported successful results with anti-TNF MoAb (etanercept/infliximab), defibrotide, daclizumab, rituximab, and eicosapentaenoic acid. 3.6. Idiopathic pneumonia syndrome (IPS) (21) Pathogenesis The formerly used term of interstitial pneumonia has been progressively abandoned because it does not correspond to the real pathologic findings. Apparently, this syndrome is the result of a diversity of lung insults, including the toxic effects of conditioning, immunologic cell-mediated injury, inflammatory cytokines and, probably, occult pulmonary infections. Incidence As a consequence of the improvement of diagnostic methods the incidence of IPS has reduced from more than 20% in earlier series of allo-HSCT to less than 10% at present time (8.4 and 2.2% after conventional and allo-RIC, respectively, in a recent series). It is uncommon in auto-HSCT setting. 192

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Clinical features Development around day +21 of: • Fever, non-productive cough • Tachypnoea, hypoxaemia • Diffuse alveolar or interstitial infiltrates on X-ray or scan. Diagnosis The diagnosis is confirmed when the previous clinical manifestations are associated with: • Absence of infectious pathology or DAH in FBS, BAL or lung biopsy, and • Absence of other possible pathogens (lung oedema, lung haemorrhage, fat embolism, leukaemic infiltration, or lung toxicity due to leucoagglutinins). Risk factors Myeloablative conditioning, age older than 40, and grade III–IV acute GvHD. With myeloablative HSCT, the use of TBI, especially in patients older than 40 years is an additional risk factor. Treatment Supportive care combined with prophylaxis and treatment of infections. Some patients improve with methylPDN and some successes have been described with antiTNF MoAb (etanercept/infliximab). Evolution Up to 50–70% of patients will die due to a progressive impairment of respiratory function. This percentage reaches 97% if mechanical ventilation is required. 3.7. Multiple-organ dysfunction syndrome (MODS) (22) Pathogenesis All mechanisms previously mentioned. Incidence Unknown, due to the difficulty in differentiating this from the syndromes already described. Clinical features This diagnosis should be considered when early after HSCT a patient presents two or more of the following: • CNS dysfunction (>4 points on the scale of Folstein) • Lung dysfunction (O2sat < 90% in two occasions separated by more than 2 hr in the same day) • Renal dysfunction (creatinine >1.5 mg/dL [>133 mmol/L]) • Hepatic dysfunction (VOD criteria, see 3.1.). Treatment There is no effective treatment and this syndrome is irreversible.


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References 1. Sencer SF, Haake RJ, Weisdorf DJ. Hemorrhagic cystitis after bone marrow transplantation. Risk factors and complications. Transplantation 1993; 56: 875-879. 2. Gine E, Rovira M, Real I, et al. Successful treatment of severe hemorrhagic cystitis after hemopoietic celltransplantation by selective embolization of the vesical arteries. Bone Marrow Transplant 2003; 31: 923-925. 3. McDonald GB, Hinds MS, Fisher LD et al. Veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: A cohort study of 355 patients. Ann Intern Med 1993; 118: 255-267. 4. Carreras E, Bertz H, Arcese W, et al. Incidence and outcome of hepatic veno-occlusive disease (VOD) after blood or marrow transplantation (BMT): A prospective cohort study of the European group for Blood and Marrow Transplantation (EBMT). Blood 1998; 92: 3599-3604. 5. Lee JL, Gooley T, Bensinger W, et al. Veno-occlusive disease of the liver after busulfan, melphalan, and thiotepa conditioning therapy: Incidence, risk factors, and outcome. Biol Blood & Marrow Transplant 1999; 5: 306-315. 6. Carreras E. Veno-occlusive disease of the liver after hematopoietic cell transplantation. Eu J Haematol 2000; 64: 281-291. 7. DeLeve LD, Shulman HM, McDonald GB. Toxic injury to hepatic sinusoids: Sinusoidal obstruction syndrome (veno-occlusive disease). Semin Liver Dis 2002; 22: 27-42. 8. Ho VT, Linden E, Revta C, Richardson PG. Hepatic veno-occlusive disease after hematopoietic stem cell transplantation: Review and update on the use of defibrotide. Semin Thromb Hemost 2007; 33: 373-388. 9. Carreras E, Gra_ena A, Navasa M, et al. Transjugular liver biopsy in BMT. Bone Marrow Transplant 1993; 11: 21-26. 10.Richardson PG, Murakami C, Jin Z et al. Multi-institutional use of defibrotide in 88 patients after stem cell transplantation with severe veno-occlusive disease and multisystem organ failure: response without significant toxicity in a high-risk population and factors predictive of outcome. Blood 2002; 100: 4337-4343. 11.Nürnberger W, Willers R, Burdach S, Göbel U. Risk factors for capillary leak-age syndrome after bone marrow transplantation. Ann Hematol 1997; 74: 221-224. 12.Speizer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001; 27: 893-898. 13.Maiolino A, Biasoli I, Lima J, et al. Engraftment syndrome following autologous hematopoietic stem cell transplantation: Definition of diagnostic criteria. Bone Marrow Transplant 2003; 31: 393-397. 14.Gorak E, Geller N, Srinivasan R, et al. Engraftment syndrome after nonmyeloablative allogeneic hematopoietic stem cell transplantation: Incidence and effects on survival. Biol Blood Marrow Transplant 2005; 11: 542-550. 15.Afessa B, Tefferi A, Litzow MR et al. Diffuse alveolar hemorrhage in hematopoietic stem cell transplant recipients. Am J Respir Crit Care Med 2001; 166: 641-645. 16.Majhail NS, Parks K, Defor TE, Weisdorf DJ. Diffuse alveolar hemorrhage and infectionassociated alveolar hemorrhage following hematopoietic stem cell transplantation: 194

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Related and high-risk clinical syndromes. Biol Blood Marrow Transplant 2006;12: 10381046. 17.Daly AS, Xenocostas A, Lipton JH. Transplantation-associated thrombotic microangiopathy: Twenty-two years later. Bone Marrow Transplant 2002; 30: 709-715. 18.Ho VT, Cutler C, Carter S, et al. Blood and marrow transplant clinical trials network toxicity committee consensus summary: Thrombotic microangiopathy after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005;11: 571-575. 19.Ruutu T, Barosi G, Benjamin RJ, et al. European Group for Blood and Marrow Transplantation; European LeukemiaNet. Diagnostic criteria for hematopoietic stem cell transplantassociated microangiopathy: Results of a consensus process by an International Working Group. Haematologica. 2007; 92: 95-100. 20.Batts ED, Lazarus HM. Diagnosis and treatment of transplantation-associated thrombotic microangiopathy: Real progress or are we still waiting? Bone Marrow Transplant 2007 Jul 2; [Epub ahead of print]. 21.Fukuda T, Hackman RC, Guthrie KA, et al. Risks and outcomes of idiopathic pneumonia syndrome after nonmyeloablative and conventional conditioning regimens for allogeneic hematopoietic stem cell transplantation. Blood 2003; 102: 2777-2785. 22.Gordon B, Lyden E, Lynch J, et al. Central nervous system dysfunction as the first manifestation of multiorgan dysunction syndrome in stem cell transplant patients. Bone Marrow Transplant 2000; 25: 78-83.

Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Late-onset haemorrhagic cystitis usually is produced by: a) The direct action of cyclophosphamide on the bladder. . . . . . . . . . . . . . . . . . . . . b) The sum of several toxic factors that produce a bladder damage . . . . . . . . . . c) A polyomavirus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) A bacterial infection of the urinary tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) The neutropenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Which of the following complications could not be attributed to an endothelial dysfunction? a) Engraftment syndrome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Veno-occlusive disease of the liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Haemorrhagic cystitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Leak capillary syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) Thrombotic microangiopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


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3. Which of the following is not a clinical manifestation of VOD? a) Weight gain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Painful hepatomegaly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Ascites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Platelet refractoriness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) Diarrhoea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. All but one of the following are classical manifestations of engraftment syndrome, which one? a) Skin rash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Back pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Hypoxaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) Diarrhoea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Which is the main cause of thrombotic microangiopathy after HSCT? a) Bacterial infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Graft allo-reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Immunological phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Cyclosporin toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e) Renal failure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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*

CHAPTER 10

Infections after HSCT

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CHAPTER 10 • Infections after HSCT

1. Introduction Despite considerable progress in the management of the complications of HSCT, infection remains an important cause of post-transplant morbidity and mortality, mainly after allogeneic HSCT. The major advances in the management of infectious complications have come from better understanding of the mechanisms of the complex depression of immunity observed during the first months after transplant and their role in the predisposition to given infections, and also from well-designed therapeutic trials. Although the proportion of infectious deaths after allogeneic HSCT has decreased over the last two decades (1), much remains to be done to further decrease this risk and implement more efficient preventive and prophylactic strategies adapted to this high-risk population. Even though the risk of infectious deaths is much lower after autologous transplant, the risks of the procedure are greater than those of conventional chemotherapy, and preventive policies should be implemented in any transplant program.

2. The timing of immune reconstitution determines the timing of infections Stem cell transplantation offers a unique model of gradual immune reconstitution, and illustrates perfectly the relationship between the type of immune deficiency and the occurrence of infection due to special pathogens. Immune reconstitution after HSCT is the topic of another Chapter of this book, and will not be detailed here. However, some basic observations deserve to be emphasised. After allogeneic HSCT following conventional (i.e. myeloablative) conditioning regimens, the sequence of infections can be divided into three periods: 1. The first is the aplastic phase following the conditioning regimen until neutrophil recovery from the donated marrow. During this phase, the infectious complications of HSCT patients are not very different from those encountered in other profoundly neutropenic patients such as acute leukaemia patients, except, in most cases, for more severe mucosal damage, especially after total body irradiation. This is also the beginning of the at-risk period for fungal infections, mainly aspergillosis. Viral infections, especially HSV, are also common. Infection-related mortality at this time is mainly due to severe bacterial sepsis, pneumonia, and fungal infections. 2. The second phase corresponds to the period from initial marrow engraftment to at least the third or fourth month, and is characterised by cell-mediated immune deficiency with decreased number and function of specific and non-specific


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cytotoxic cells. For many years CMV infection, which is mainly due to reactivation, was the greatest problem during this phase. However, the routine practice of early diagnosis through PCR or antigenaemia and prophylactic or pre-emptive therapy have both been shown to decrease mortality due to CMV disease. Other viral diseases, less frequent than CMV, have also been described during this phase, especially those due to adenovirus and enteric and respiratory viruses. The occurrence and severity of GvHD is the main factor delaying immune recovery and favouring infections. 3. The third phase, beginning at the fourth month, is considered to be the late posttransplantation period. Here again, immune reconstitution is mainly influenced by the presence and severity of chronic GvHD. Most patients have immunoglobulin deficiency, particularly of IgG2, which is responsible for a decrease in the response to polysaccharide antigens. Allogeneic HSCT recipients are particularly vulnerable to encapsulated bacteria (e.g. S. pneumoniae and H. influenzae). In the absence of chronic GvHD, this deficiency will often be transient and will resolve over time. In other cases, it may persist indefinitely. However, even in these cases, active immunisation may be beneficial, especially with conjugate vaccines. Although most infectious mortality is observed during the first 6 months after transplant, late infections may be life-threatening (particularly S. pneumoniae) and should not be neglected, especially in patients with chronic GvHD. The development of non-myeloablative approaches has modified the timing and features of the main infectious complications after transplant, although their overall incidence is not greatly changed. Neutropenia almost disappears or is significantly reduced, as is mucositis, and therefore the risk for early bacterial or fungal infections decreases (2). However, patients who undergo reduced intensity conditioning (RIC) regimens are usually older than those who receive conventional conditionings. As the main risk factor for severe infections is GvHD, and as, until now, the risk of GvHD is roughly comparable whatever the conditioning regimen, patients who receive reduced intensity conditioning remain at risk of infections, especially viral and fungal infections. Actually, although we may agree that RIC regimens decrease the overall transplant-related mortality, there is no clear evidence from the literature that RIC decreases infection-related mortality (3, 4). After autologous HSCT, morbidity and mortality related to infection is much lower. Since more than 95% of autologous HSCT are performed with peripheral blood stem cells, neutropenia generally lasts no more than 14 days, and the risk of bacterial sepsis is close to that observed in similar patients after chemotherapy. Fungal risk is extremely low and one may consider that these patients are not at risk for 200

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Aspergillus, and in standard situations they do not need rooms with filtered air. However, in addition to the risk of the autologous HSCT procedure itself, these patients may also be at risk due to previous immunosuppressive factors, such as prolonged steroid therapy, or hypogammaglobulinemia, which may favour other, non neutropenia-related, infections. Late infections may also occur after autologous transplant, but are exceptional without total body irradiation.

3. Clinical management of fever and infectious complications Because allogeneic transplant patients are those at higher risk for opportunistic infections and severe outcome, most of the recommendations given below mainly apply to these patients, but autologous transplant recipients should benefit from similar approaches, at least for the first 2 months after transplant. Two special clinical settings are taken as examples of the management of these patients when infection is probable, or even just possible, because they require special attention with essential diagnostic and therapeutic procedures. 3.1. Fever Febrile neutropenia is a special situation where bacterial infection must be the main target of empirical anti-infective therapy. There are not many differences between febrile neutropenia in patients at the neutropenic phase of stem cell transplant, and febrile neutropenia following chemotherapy, except that patients conditioned with total body irradiation are more likely to suffer from mucositis which is one of the main factors for streptococcal bacteraemia. Only 30% of febrile neutropenic episodes are microbiologically documented, with 20% due to Gram-positive cocci - mainly coagulase negative staphylococci - and 10% due to Gram-negative bacteria. As in any febrile neutropenia, broad-spectrum antibiotics should be administered promptly after blood cultures and sampling of any clinical site of infection whenever possible. Guidelines from the recent ECIL 1 (European Conference on Infections in Leukaemia) have clarified the need for aminoglycosides (5) and glycopeptides (6) in febrile neutropenia: the group considers that in standard situations betalactam monotherapy is as efficacious as betalactam plus aminoglycoside combination, both as initial empirical therapy and in case of persistent fever. Therefore, considering their potential toxicity, the use of aminoglycosides is not recommended, except in case of severe sepsis or septic shock where aminoglycosides are an initial option for first line antibiotic therapy, but where they should be quickly reconsidered according to the clinical outcome (5). Similarly, the use of glycopeptides as initial antibacterial therapy - combined with a betalactam - should be restricted to patients with hypotension or shock, skin or soft tissue infections (including high suspicion of central catheter infection) or in patients


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with known colonisation with methicillin - resistant S. aureus (6). If fever persists after 3 to 5 days of empirical antibacterials, fungal and viral infection must be considered, and an empirical antifungal treatment must be administered (7). Several antifungals may be used in this indication, but liposomal amphotericin B and caspofungin are recommended (8). Routine screening for fungal infection by galactomannan antigenaemia and lung CT-scan offers new hope of moving from the classical empirical strategy to a more risk-targeted, so-called “pre-emptive”, strategy (9, 10), but this strategy is, until now, not standardised for allogeneic HSCT recipients. When a transplant patient is febrile but not neutropenic, any infection should be considered and possible diagnoses should be made according to the clinical presentation and timing of fever after transplant. Although fever can be a feature of GvHD, most febrile episodes in transplant patients are due to infection, and diagnostic methods should be used to identify the responsible pathogen. The main difference between neutropenic and non-neutropenic patients is that in nonneutropenic patients, it is usually possible to investigate the patient properly before starting anti-infective drugs. For example, when an episode of pneumonia occurs within the first 6 months of transplant, and has developed over several days or weeks, it is usually possible to wait for fibre-optic bronchoscopy and BAL, whenever possible within 24h, before giving antibiotics. However, it is not always advisable to wait even in non-neutropenic patients, e.g. those with hypoxemic pneumonia, or severe sepsis. 3.2. Pneumonia Pneumonia complicates the course of half of allogeneic HSCT recipients treated with conventional conditioning regimens. There are many possible causes of pulmonary infiltrates observed after transplant. However, two thirds are due to infection. Pulmonary oedema, pulmonary embolism, alveolar haemorrhage, and alveolar proteinosis, may also occur, especially within the first 3 months after transplant. Fibre-optic bronchoscopy with BAL is the main diagnostic tool. It must be performed early in the course of pneumonia, before administration of antibacterials whenever possible, and a protected sample should be obtained for quantitative microbiological assessment, by either brushing or aspiration. BAL has a limited diagnostic yield for fungal infections. However, despite this limitation, it is the procedure with the highest risk/benefit ratio for first-line investigation. Since the use of prophylactic or preemptive treatment for CMV infection, which makes the occurrence of CMV pneumonia very rare, special attention must be paid to the possibility of respiratory virus pneumonia. Transbronchial lung biopsy is associated with a greater risk of pneumothorax and bleeding, and no greater diagnostic benefit for most causes of pneumonia in the transplant setting, except for mycobacterial and fungal infections. Open lung biopsy should be considered in sub-acute pneumonia when BAL does not 202

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provide a clear diagnosis. Although there is not much available data about the value of indirect non-invasive procedures in such patients (i.e., legionella, pneumococcal, cryptococcal antigens in blood and/or urine), such procedures deserve to be used since they may easily provide indirect evidence of the cause of pneumonia.

4. Bacterial infections Two important points should be remembered: - Timing and immune reconstitution are key points in making a possible diagnosis at the bedside of a transplant patient suspected of bacterial infection. - Additionally, HSCT patients are among the most immunodepressed patients in the hospital. Therefore, they are at risk for nosocomial infections, depending on the environmental measures and the epidemiology of the centre. Bacterial infections occurring during the neutropenic phase are usually the same as those observed during any chemotherapy-induced neutropenia, i.e. streptococci and Gram-negative bacteria. Neutrophil recovery usually marks the end of the bacterial risk for most autologous HSCT patients, but not for allogeneic HSCT recipients who remain at high risk for nosocomial infection for many weeks after neutrophil recovery, especially when they stay in the hospital for the treatment of severe GvHD with multiple complications, and keep their central IV line. After 2 months, the risk for encapsulated bacteria, mainly for S. pneumoniae and H. influenzae, appears and is strongly - but not exclusively - linked to chronic GvHD. Specific deficiencies in anti-S. pneumoniae and anti-PRP antibodies - the main H. influenzae type B capsular polysaccharide - have been noted, which may predispose the patient to these infections. S. pneumoniae may cause bacteraemia and/or pneumonia or sinusitis. Fulminant fatal outcomes may be observed, even years after transplant. S. pneumoniae infections are mainly observed in patients with GvHD and in those who have received TBI (11). Although it rarely causes fulminant disease, H. influenzae may be responsible for upper and lower respiratory tract infection, and bacteraemia. Other rare bacterial infections have been reported after transplant, i.e., mycobacterial or legionella infection. However, due to the rarity of these cases, the timing and risk factors are not well identified. 4.1. Prevention of bacterial infections During the early phase of transplant, three preventive measures must be considered: a) Because of the hospital environment and its resistant bacteria, the physical environment of transplant patients should aim to decrease the risk of nosocomial infection, as in any immunocompromised patient. Different measures can be implemented. The easiest is simple protection including mask, gloves, and


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gowns, which is usually enough for autologous transplant patients. Although there is no clear demonstration in the literature that such measures are superior to hand washing alone in preventing cross-transmission, this kind of isolation is usually easier to control enforce than hand washing alone, and such visible protection usually discourages the staff from making unnecessary approaches to the patient during the neutropenic phase. The control of room air quality through filtration (HEPA) is the main measure that can be taken to decrease the risk of aspergillosis but there is no demonstration that such air treatment has any impact on the incidence of bacterial pneumonia. Water control is also necessary, in any units caring for high-risk patients, to avoid contamination of the patients with Legionella sp. or P. aeruginosa. b) The second measure is the prevention of bacterial translocation by oral antibacterials - absorbable such as quinolones, or non-absorbable such as nonabsorbable gut decontamination - and a diet low in microbes. The gut is the main reservoir for Gram-negative bacteria, and a source of subsequent entry into the body. A diet low in microbes is generally recommended for these patients, but the measures vary from one centre to another. The minimal measures are to avoid fresh vegetables and fruits, and any food suspected to content high quantities of bacteria. This is a logical recommendation, although its benefit alone has never been clearly shown. Gut decontamination is differently approached in different centres and countries. Quinolones have been shown, in comparative trials, to decrease the risk for Gram-negative bacteraemia and the number of days of fever in patients with prolonged neutropenia. They are widely used for allogeneic HSCT patients in Europe, especially ciprofloxacin. In acute leukaemia patients and autologous HSCT recipients, a large trial comparing levofloxacin to placebo has shown that the use of levofloxacin during the neutropenic phase significantly reduces the risk of fever, bacterial infection and bacteraemia, and especially of Gram-negative infections, reducing the cost of intravenous antibacterials for febrile neutropenia (12). So far, there are no data on levofloxacin in allogeneic HSCT patients. Once started, quinolones should be given until the first febrile episode, or until the recovery of neutropenia in the absence of fever. However, quinolones may select Gram-negative quinolone-resistant bacteria. Therefore, their use is not recommended in units with a high level of quinolone resistance among Gramnegative bacteria, and should be associated with periodic monitoring of the local epidemiology (13). The other option, widely used in some European countries, is the use of non-absorbable antibiotics (i.e. colimycine and aminoglycosides). They similarly reduce the risk for Gram-negative bacteraemia, but historical trials failed to show any survival benefit. Non-absorbable gut decontamination 204

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has constraints, especially the need to be combined with sterile food and with frequent microbiological controls of the stools to look for the possible selection of resistant strains. Whatever the option (quinolones, or non-absorbable gut decontamination), an antifungal prophylaxis must also be given to decrease the risk of overgrowth of yeasts in the gut. c) The third measure is the management of central IV lines. Catheters may be the source of bacteraemia, with significant morbidity and potential mortality. During neutropenia, it is controversial whether a catheter may be left in situ when blood cultures have documented a pathogen, except in the case of methicillin resistant S. aureus, Candida sp., Bacillus sp. and Corynebacterium JK, and any hospitalacquired resistant pathogen, such as P. aeruginosa or Acinetobacter sp, for which there are clear recommendations to take the catheter out (2). Central catheter management should be formalised in any transplant unit in written procedures. From the third month after transplant, transplant patients may be profoundly hypogammaglobulinaemic (immune globulin levels below 3 g/L). These patients should receive IV immunoglobulin replacement to maintain immunoglobulin serum levels over 3 g/L, especially when they have chronic GvHD and are receiving immunosuppressive therapy. The most logical and cost-effective approach to prevent S. pneumoniae and H. influenzae infections is active immunisation. For S. pneumoniae, the historical trials with polysaccharide vaccines have been disappointing since they show that patients with GvHD, or receiving steroids, those with the higher risk of infection, have a poor antibody response, especially before 6 months post-transplant. Therefore, long-term antibiotic prophylaxis with penicillin is still the practice in most centres for allogeneic transplant recipients, but the development of penicillin resistance makes this policy unsatisfactory. The recent availability of a conjugate pneumococcal vaccine offers new hopes of improving the protection of HSCT patients since the conjugation of the pneumococcal polysaccharides to proteins elicits a T-cell dependent immune response. A first study shows that 3 doses of the heptavalent pneumococcal conjugate vaccine, given at 3, 6 and 12 months after allogeneic HSCT induces protective IgG concentrations in 60% of the patients regardless of donor immunisation (14). Another study in children showed that 3 doses of the heptavalent vaccine given from 6 months allows protective antibody levels in more than two thirds of the patients (15). A prospective EBMT trial recently completed shows that the administration can be given as soon as 3 months post-transplant. Similarly, the H. influenzae type-B tetanus conjugate vaccine has been shown to be highly effective, giving a satisfactory response in 85% of immunised HSCT


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patients, and should be administered from 4 months. Probably because of the high immunogenicity of such conjugate vaccines, the response does not seem to be influenced by the presence of GvHD. Therefore, these vaccines are recommended in all patients, and even more in those with GvHD who are at higher risk of infection. The main recommendations of the IDWP for active immunisation in transplant patients are summarised in Table 1 (16).

5. Fungal infections Aspergillus is the most serious fungal infection after allogeneic stem cell transplant (17), and is the main cause of infectious death (18). Reported incidences vary from 0 to 20% of transplants. The most common site is the lung and GvHD is the main risk factor. A first peak of incidence occurs during the neutropenic period, particularly in leukaemic patients who may have been previously colonised. A second wave of aspergillosis occurs during the 2nd and 3rd months after transplant and is strongly linked to GvHD; this is now more important than the first peak and late cases are more and more frequent. In patients previously infected by Aspergillus during induction or consolidation treatment phases, fungal recurrence may occur in roughly one third of the patients (19) and the use of a secondary antifungal Aspergillus prophylaxis is widely admitted, although the optimal choice of the drug is not fixed. Despite major improvements in the treatment of fungal infections (20), the mortality of Aspergillus in allogeneic HSCT patients remains over 50% in recent series. PCR and galactomannan antigenaemia may help in the early detection of Aspergillus infections. Candida infection is more rare and has no special clinical presentation in transplant patients when compared to other haematology patients. More and more nonAspergillus, non-Candida infections are reported in HSCT patients. Pneumonias due to endemic fungi, such as histoplasmosis or coccidioidomycosis, particularly in North America, must be considered in these patients, as well as the emerging fungi, including Trichosporon, Alternaria, Fusarium, and the Mucorales. Any of these fungi can cause pneumonia in transplant patients, as in similarly immunocompromised hosts, and their management needs a close collaboration between the mycologist and the clinician. 5.1. Prevention of fungal infections The most efficient protection from fungal air-born infections during the neutropenic phase of allogeneic transplant is the use of air filtration with positive pressure. This kind of isolation has been shown to decrease the early risk of Aspergillus in 206

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Table 1: EBMT recommendations for immunisations of long-term survivors after allogeneic and autologous stem cell transplantation (16) Available forms

Available data in HSCT patients

Recommended after HSCT

Strength No of of recommen- doses dation+

Time Improved after HSCT by donor (months) vaccination

BACTERIAL VACCINES S. pneumoniae

PS Conjugate PS

Yes Yes

Yes AII Yes (subgroups) CII

1 3

12 Unclear

No Yes

H. influenzae type B

Conjugate PS

Yes

Yes

BII

3

6

Yes

N. meningitidis types A and C N. meningitidis type C

PS

Yes

Individual assessment

CII

1

6-12

Unknown

Conjugate PS

No

1

6

Unknown

BCG

Live

No

Contra-indicated EII

NA

NA

Unknown

Tetanus

Toxoid

Yes

Yes

BII

3

6-12

Yes

Diphtheria

Toxoid

Yes

Yes

BII

3

6-12

Likely

B. pertussis#

Acellular, toxoid +/- other antigens

Yes

See text

CIII

3

6-12

Unknown

Inactivated

Yes

Yes, yearly

AII

1

4-6

Unknown

Inactivated polio Inactivated

Yes

Yes

BII

3

6-12

Unknown

VIRAL VACCINES Influenza

Hepatitis B

Inactivated plasma Yes or recombinant DNA derived

See text

BII

3

6-12

Yes

Hepatitis A virus

Inactivated

No

In endemic areas CIII and in travellers

3

6-12

Unknown

Measles§

Live

Yes


BII

1

24*

Unknown

Rubella§

Live

Yes


BIII

1

24*

Unknown

Mumps§

Live

Yes


CIII

1

24*

Unknown

Varicella

Live

Limited


CIII

Unclear Before HSCT Unknown or at 24*

Yellow fever

Live

Limited

Individual asses- CIII sment, travellers

1

24*

Unknown

+The recommendations are graded according to the CDC system; *Not in patients with chronic GvHD or ongoing immunosuppression; #Combination vaccines including also tetanus, diphtheria, and pertussis with or without HIB and poliovirus components are available; §Usually combined in a MMR vaccine; BCG: Bacille Calmette-Guérin; HIB: Haemophilus influenzae type b; PS: Polysaccharide


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allogeneic HSCT. Good management of air filters, and effective collaboration with the infection control unit of the hospital are of paramount importance for allogeneic transplant units, and for haematology departments in general. Epidemics of mould infections have been reported in stem cell transplant units and have led to interruption of transplant programs until the infections can be controlled (21). However, air filtration, of course, does not provide any protection from yeast infections. In two randomised trials, fluconazole prophylaxis at the dose of 400 mg/day has been shown to reduce the risk for invasive fungal infections, especially Candida infections, and is associated with a long-term survival benefit. However, because non-albicans Candida are more and more frequent in haematology patients, the benefit of fluconazole prophylaxis is probably lower than ten years ago, and anyway unsatisfactory considering the main concern of the transplant community is mould infections. Recently, Ullmann et al. have shown that posaconazole (600 mg/d), when given from the onset of GvHD, significantly decreases the incidence of proven and probable invasive fungal infection, especially Aspergillus (22). In autologous HSCT, except in centres with a high incidence of candidaemia, the risk of invasive Candida infection is low, and therefore the use of fluconazole in this setting is optional (23). 5.2. Treatment of fungal infections Voriconazole, a triazole antifungal agent, is fungicidal active against Aspergillus and Candida species, including non-albicans Candida, and other rare fungal infections such as Fusarium or Scedosporium species which occur in stem cell transplant patients. In the first-line treatment of aspergillosis, it has been shown to be more effective and better tolerated than amphotericin B (20). Despite a substantial number of side effects (including visual disturbances, confusion, skin reactions, and liverfunction abnormalities), and potential drug interactions, especially with cyclosporin, voriconazole is widely used after stem cell transplant. A recent, large prospective study comparing 3 vs. 10 mg/kg/d of liposomal amphotericin B in first line treatment of Aspergillus infections did not find any advantage to the 10 mg dose, but showed comparable results of the 3 mg/kg/d arm with the voriconazole study (24). Echinocandins have been studied in refractory aspergillosis with encouraging results but no study of first-line therapy has been published so far. Echinocandins are excellent candidates for combinations with either azoles, or polyenes. However, there are, until now, no prospective data published on first line therapy of fungal infections with echinocandin combinations. These combinations will be extremely expensive, and there is no evidence as yet that they will provide better results than 208

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single drug regimens. Prospective, comparative trials need to be done before any conclusion can be drawn. The ECIL 1 has recently published treatment guidelines for Candida and Aspergillus infections (25).

6. Viral infections Viral infections are frequent after HSCT. They may be life threatening, especially when affecting lung, liver, or central nervous system in allogeneic HSCT recipients. The availability of new antiviral agents in the last 2–3 years, together with data from comparative trials, have allowed a better control of herpes virus infections. However, due to the subsequent decrease in CMV infection and CMV disease, new viral infections have emerged, especially due to respiratory viruses and adenovirus. Herpes simplex virus (HSV) infections are extremely common, due to reactivation in seropositive patients. The main early manifestation is mucous lesions, difficult to distinguish from chemotherapy-induced mucositis in the absence of viral documentation. These lesions are painful and maybe the portal of entry of bacteria from the gut. In seropositive patients, prophylaxis with acyclovir or valaciclovir is recommended to decrease the risk of reactivation during the early phase of transplant. In case of HSV infection, IV acyclovir is usually effective. Acyclovir resistance is rare in HSCT patients, but this possibility must be considered in case of HSV disease documented during prophylaxis. Due to common mechanisms at the cellular level (thymidine-kinase dependent), resistance to acyclovir is associated with resistance to ganciclovir and famciclovir. The best choice in case of acyclovir resistance of HSV is foscavir. Cytomegalovirus (CMV) disease has historically been a main cause of death in allogeneic transplant patients, except where both donor and recipient are seronegative. Since the demonstration that CMV infection usually precedes CMV disease, and considering the poor prognosis of CMV disease even when treated, two strategies - prophylactic and pre-emptive - have been developed in order to reduce the risk of CMV disease. First, prophylactic trials comparing IV ganciclovir versus placebo showed that ganciclovir prevents the risk of CMV infection and disease, but does not improve survival and additionally favours the delay of specific immune reconstitution, and consequently, the occurrence of late CMV infection. This prophylactic strategy was finally not more effective than a pre-emptive strategy. Therefore, prophylactic strategies are usually reserved for patients with high-risk of CMV disease, such as mismatched transplant recipients. For other patients, a preemptive strategy is more cost-effective. Controlled trials are needed to know


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whether the oral pro-drug of ganciclovir, valganciclovir, will be as effective and no more toxic than the IV ganciclovir. First experiences are encouraging but suggest caution is needed because of the different pharmacokinetics of the two drugs (26). The clinical impact of theses differences should be explored further. Although most first line pre-emptive strategies have used ganciclovir, foscavir has been shown, in an EBMT comparative trial, as effective and no more toxic than ganciclovir (27). Both can be used as first-line treatment of CMV infection, for an initial duration of 2 weeks. If CMV is still detected after 2 weeks of therapy, an additional course of 2 weeks should be given. Cidofovir has been studied only in uncontrolled trials and because of its toxicity profile; its use should be reserved for second line preemptive therapy. These strategies must be based on sensitive diagnostic methods of CMV infection. Allogeneic HSCT recipients must be monitored for CMV in peripheral blood at least weekly with a sensitive method, until day 100 after transplant, and longer in case of prolonged GvHD or previous CMV reactivation. Antigenaemia testing detects CMV antigen pp65 in leukocytes by immune staining with monoclonal antibodies. This test is semiquantitative and rapid. PCR is also widely used. The quantification of viral load seems to be important since higher levels of CMV DNA are indicators for a higher risk of CMV disease. The availability of Real Time or Light Cycler technologies has improved the quantitative evaluation of the viral load. Detection of mRNA by nucleic acid sequence-based amplification (NASBA) is also available and has been shown to be similarly effective as pp65 antigenaemia or detection of DNA by PCR for the early detection of CMV infection. Because the risk of CMV disease is extremely low after autologous transplant, systematic CMV screening is not recommended in this population, except in high-risk patients, like those receiving CD34-selected grafts, or patients who have recently received fludarabine, 2-CDA, or alemtuzumab. In additionally to antiviral strategies, CMV prophylaxis must include transfusion policies to avoid acquisition of CMV through blood products, especially for CMV seronegative recipients of seronegative donors. These patients must receive blood products either from CMV seronegative donors exclusively, or leukocyte-depleted products. Although less established by prospective trials, the same policy is logical for CMV seronegative recipients of autologous transplants. Varicella-zoster virus (VZV) infections occur both after allogeneic and autologous transplants. Primary varicella may be severe in both populations. IV acyclovir is the therapy of choice. Valaciclovir can also be used, especially in late infections, and in patients who do not receive high-dose immunosuppressive drugs. These patients, when given valaciclovir, must be correctly managed in case of rash extension or 210

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systemic manifestations, and informed of the possible need for switching to the IV form of the antiviral. Human herpes virus 6 (HHV6) infection after allogeneic HSCT has been associated with pneumonia, delayed marrow engraftment, and particularly with prolonged thrombocytopenia and encephalitis. HHV6-DNA is frequently detected in blood during the first months after transplant in asymptomatic patients, so that its implication in clinical symptoms is usually difficult to establish, except in encephalitis when HHV6-DNA is detected in CSF. Epstein-Barr virus associated lymphoproliferative disease (EBV-LPD) is a lifethreatening complication occurring after allogeneic HSCT. Monitoring of the EBV viral load by quantitative PCR permits the early detection of EBV reactivation that may lead to EBV-LPD. Recipients of a T-cell-depleted HSCT, or patients conditioned with antithymocyte globulins are at higher risk of EBV-LPD. In these patients, preemptive therapy of EBV reactivation with rituximab has been shown to improve outcome. Infusion of EBV-specific cytotoxic T-cells has also been studied in highrisk patients with elevated EBV-DNA levels. However, the exact indications for preemptive therapy based on EBV-viral load for preventing EBV-LPD are not yet clear and should highly depend on the transplant population. Prospective trials are needed. 6.1. Respiratory virus infections Respiratory viruses, including respiratory syncytial virus (RSV), parainfluenza virus, rhinovirus, and influenza virus, appear to be now more frequent than CMV pneumonia. A prospective study from the EBMT showed an incidence of respiratory virus pneumonia of 2.1% in allogeneic, and 0.2% in autologous transplant patients (28). Most cases in this series were due to RSV or influenza A. The mortality of these infections also varies among series, and with the time after transplant and the degree of immunosuppression, but it may be as high as 80% in RSV pneumonia. Few data are available in the literature on the efficacy of antivirals in RSV pneumonia. Due to the rarity of the disease, and the poor prognosis of the lower tract infections, prospective trials are extremely difficult to perform. In the European experience, there was no clear advantage or disadvantage of adding IV ribavirin to either aerosolised ribavirin or to IV immune globulins. The best therapeutic option for RSV pneumonia is not established. Due to the risk of spread in the transplant unit, it is important to diagnose these patients very early, and to prevent transmission in the ward. Oseltamivir has been used in an open study to control an influenza A outbreak and appears to be safe in allogeneic transplant recipients (29).


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6.2. Adenovirus infections Adenovirus infections can be a cause of severe disseminated infections in allogeneic HSCT recipients. Patients receiving mismatched or unrelated donor transplants, patients with severe acute GvHD, patients showing isolation from multiple sites or from blood are at high risk of developing organ involvement due to adenovirus. There are currently no established strategies for prophylaxis or treatment of adenovirus disease. Spontaneous recovery may be observed. However, in high-risk patients such as children receiving mismatched transplants, adenovirus infection usually develops into adenovirus disease, which has a high mortality rate, and pre-emptive treatment should be logical but has not been studied extensively. A recent retrospective study by the IDWP of the EBMT has showed that cidofovir was effective in 10/16 patients with invasive adenovirus disease (30). Ribavirin has been used in case reports with varying outcome. Both drugs - cidofovir and ribavirin - may be effective in preemptive therapy. Prospective controlled studies are needed to define the best strategy.

7. Other infections Toxoplasmosis occurring after HSCT has been mainly investigated in Europe, due to a higher seroprevalence of the disease when compared to US. Patients at risk are those who are seropositive for toxoplasmosis before transplant, irrespectively of the serology of the donor. Blood PCR allows early detection of toxoplasma reactivation. A prospective study from the IDWP on 106 allogeneic toxoplasma seropositive recipients, screened weekly by blood PCR, showed an incidence of PCR-documented infection of 16%, and an incidence of toxoplasma disease of 6% up to 6 months post-transplant. All patients developing disease were previously or simultaneously PCR-positive in blood (31). Most of these reactivations occur in patients with GvHD, while trimethoprim-sulfamethoxazole has been stopped for side effects, and replaced by aerosolised pentamidine for P. jiroveci prophylaxis. Whether asymptomatic toxoplasma infection documented by blood PCR should be treated is not yet clear. However, there are reports in the literature where high-risk patients, with severe GvHD, developed toxoplasma infection which evolved, in the lack of specific therapy, to toxoplasma disease with a high mortality rate. P. jiroveci pneumonia must be prevented in allogeneic stem cell transplant recipients from engraftment to at least 6 months, even longer in case of prolonged immunosuppression. The best option is trimethoprim-sulfamethoxazole. In case of intolerance, alternatives are dapsone or aerosolised pentamidine, but the latter does not provide optimal protection (32). 212

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8. Other preventive strategies Many issues regarding infectious complications in HSCT patients remain to be explored, especially in allogeneic HSCT recipients. Active immunisation against tetanus, poliovirus, and diphtheria is highly recommended in all transplant populations, due to the usual loss of specific immunity both after autologous and allogeneic stem cell transplantation. This is the only way to allow leukaemic patients to have similar levels of immunity to these pathogens - even rarely encountered - as the normal population. Immunisation with live vaccines is classically prohibited in immunocompromised patients. However, transplant patients may require active immunisation with vaccines which only exist in a live form, i.e. measles, mumps, or yellow fever. Ljungman et al. have showed that live vaccine to measles, mumps and rubella may be safely administered to transplant children, as far as they are at more than 2 years after transplant, have no GvHD, and are not receiving immunosuppressive drugs. Immune globulins have been administered for years to allogeneic HSCT recipients to try to prevent the occurrence of GvHD and to reduce infectious mortality. A French double-blind study comparing 3 doses of IVIg to placebo in recipients of HLA-identical sibling donors shows that IVIg administration did not affect the incidence of infections over the first 6 months after transplant, the occurrence of GvHD, or survival. On the other hand, severe veno-occlusive disease was significantly more frequent in patients receiving high doses of IVIG (250 mg/kg and 500 mg/kg/weekly) (18). Similar findings have been reported in another randomised trial in unrelated transplant patients. Consequently, considering the availability of other strategies now available for infection prophylaxis, and considering the high cost of IVIg, prophylactic administration of IVIg is not recommended in allogeneic transplant recipients.

References 1. Gratwohl A, Brand R, Frassoni F, et al. Cause of death after allogeneic haematopoietic stem cell transplantation in early leukaemias: An EBMT analysis of lethal infectious complications and changes over calendar time. Bone Marrow Transplant 2005; 36: 757769. 2. Junghanss C, Marr KA, Carter RA, et al. Incidence and outcome of bacterial and fungal infections following nonmyeloablative compared with myeloablative allogeneic hematopoietic stem cell transplantation: A matched control study. Biol Blood Marrow Transplant 2002; 8: 512-520. 3. Diaconescu R, Flowers C, Storer B, et al. Morbidity and mortality with nonmyeloablative compared to myeloablative conditioning before hematopoeitic cell transplantation from HLA matched related donors. Blood 2004; 104: 1550-1558. 4. Scott BL, Sandmaier BM, Storer B, et al. Myeloablative vs nonmyeloablative allogeneic


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transplantation for patients with myelodysplastic syndrome or acute myelogenous leukemia with multilineage dysplasia: A retrospective analysis. Leukemia 2006; 20: 128-135. 5. Drgona L, Paul M, Bucaneve G, et al. The need for aminoglycosides in combination with betalactams for high-risk, febrile neutropenic patients with leukaemia. Eur J Cancer 2007; Suppl. 5: 13-22. 6. Cometta O, Marchetti O, Calandra T. Empirical use of anti-Gram positive antibiotics in febrile neutropenic cancer patients with acute leukaemia. Eur J Cancer 2007; Suppl. 5: 23-31. 7. Hughes WT, Armstrong D, Bodey GP, et al. 2002 guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Clin Infect Dis 2002; 34: 730-751. 8. Marchetti O, Cordonnier C, Calandra T. Empirical antifungal therapy in neutropenic cancer patients with persistent fever. Eur J Cancer 2007; Suppl. 5: 32-42. 9. Maertens J, Theunissen K, Verhoef G, et al. Galactomannan and computed tomographybased pre-emptive antifungal therapy in neutropenic patients at high risk for invasive fungal infection: A prospective feasibility study. Clin Infect Dis 2005; 41: 1242-1250. 10.Cordonnier C, Pautas C, Maury S, et al. Empirical versus pre-emptive antifungal strategy in hig-risk febrile neutropenic patients: A prospective randomized study. 48 th ASH Annual meeting, Orlando, Florida, 9-12 December 2006. 11.Engelhard D, Cordonnier C, Shaw PJ, et al. Early and late invasive pneumococcal infection following bone marrow and stem cell transplantation. Br J Haematol 2002; 117: 444-450. 12.Bucaneve G, Micozzi A, Menicheti F, et al. Levofloxacin to prevent bacterial infection in patients with cancer and neutropenia. N Engl J Med 2005; 353: 977-987. 13.Bucaneve G, Castagnola E, Viscoli C, et al. Quinolone prophylaxis for bacterial infections in afebrile high risk neutropenic patients. Eur J Cancer 2007; Suppl. 5: 5-12. 14.Molrine DC, Antin JH, Guinan EC, et al. Donor immunization with pneumococcal conjugate vaccine and early protective antibody responses following allogeneic hematopoietic cell transplantation. Blood 2003; 101: 831-836. 15.Meisel R, Kuypers L, Dirksen U, et al. Pneumococcal conjugate vaccine provides early protective antibody responses in children after related and unrelated allogeneic hematopoietic stem cell transplantation. Blood 2007; 109: 2322-2326. 16.Ljungman P, Engelhard D, Locasciulli A, et al. Vaccination of stem cell transplant recipients. Recommendations of the Infectious Diseases Working Party of the EBMT. Bone Marrow Transplant 2005; 35:737-746. 17.Marr KA. Epidemiology and outcome of mold infections in hematopoietic stem cell transplant recipients. Clin Infect Dis 2002; 34: 909-917. 18.Cordonnier C, Chevret S, Legrand M, et al. Should immunoglobulin therapy be used in allogeneic transplantation? A randomized, double-blind, dose-effect, placebo-controlled, multicenter trial. Ann Intern Med 2003; 139: 8-18. 19.Offner F, Cordonnier C, Ljungman P, et al. Impact of previous aspergillosis on the outcome of bone marrow transplantation. Clin Infect Dis 1998; 26: 1098-1103. 20.Herbrecht R, Denning DW, Patterson TF, et al. Voriconazole versus Amphotericin B for primary therapy of invasive aspergillosis. New Engl J Med 2002; 347: 408-415. 21.Gratwohl A, McCann S, Byrne JL, et al. Outbreaks of infectious disease in stem cell transplant units: A silent cause of death for patients and transplant programmes. Bone Marrow 214

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Transplant 2002; 29: S 53. 22.Ullmann A, Lipton J, Vesole DH, et al. Posaconazole or Fluconazole for prophylaxis in severe graft-versus-host disease. N Engl J Med 2007; 356: 335-347 23.Maertens J, Frere P, Lass-Flörl C, et al. Primary antifungal prophylaxis in leukaemia patients. Eur J Cancer 2007; Suppl. 5: 43-48. 24.Cornely OA, Maertens J, Bresnik M, et al. Liposomal amphotericin B as initial therapy for invasive mold infection: A randomized trial comparing a high-loading dose regimen with standard dosing. Clin Infect Dis 2007; 44: 1298-1306. 25.Herbrecht R, Flückiger U, Gachot B, et al. Treatment of invasive Candida and Aspergillus infections in adult haematological patients. Eur J Cancer 2007; Suppl. 5: 49-59. 26.Einsele H, Reusser P, Bornhaüser, et al. Oral valganciclovir leads to higher exposure to ganciclovir than intravenous ganciclovir in patients following allogeneic stem cell transplantation. Blood 2006; 107: 3002-3008. 27.Reusser P, Einsele H, Lee J, et al. Randomized multicenter trial of foscarnet versus ganciclovir for preemptive therapy of cytomegalovirus infection after allogeneic stem cell transplantation. Blood 2002; 99: 1159-1164. 28.Ljungman P, Ward KN, Crooks BNA, et al. Respiratory virus infections after stem cell transplantation. A prospective study from the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 2001; 28: 479-484. 29.Vu D, Peck AJ, Nichols WG, et al. Safety and tolerability of oseltamivir prophylaxis in hematopoietic stem cell transplant recipients: A retrospective case-control study. Clin Infect Dis 2007; 45: 187-193. 30.Ljungman P, Ribaud P, Eyrich M, et al. Cidofovir for adenovirus infection after allogeneic stem cell transplantion (HSCT). A retrospective survey of the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 2003; 31: 481-486. 31.Martino R, Bretagne S, Einsele H, et al. Early Detection of Toxoplasma Infection by Molecular Monitoring of Toxoplasma gondii in Peripheral Blood after Allogeneic Stem Cell Transplantation. Clin Infect Dis 2005; 40: 67-78. 32.Center for Disease Control. Guidelines for preventing opportunistic infections among hematopoeitic stem cell transplant recipients. Recommendations of CDC, the Infectious Diseases Society of America, and the American Society of Blood and Marrow Transplantation. October 20, 2000 / 49 (RR10); 1-128. (www.cdc.gov).

Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Among these statements comparing RIC regimens and conventional regimens, one is true. Which one? a) RIC decreases the incidence of encapsulated bacterial infections . . . . . . . . . HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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b) RIC increases the incidence of early bacterial infections . . . . . . . . . . . . . . . . . . . c) RIC decreases the incidence of fungal infections . . . . . . . . . . . . . . . . . . . . . . . . . . . d) RIC delays the occurrence of CMV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Among these statements about S. pneumoniae vaccination, one is true. Which one? a) The conjugate pneumococcal vaccine is a live vaccine . . . . . . . . . . . . . . . . . . . . . b) The conjugate pneumococcal vaccine includes more antigens than the olysaccharide vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The polysaccharide vaccine is recommended in allogeneic SCT recipients from 3 months after transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The conjugate pneumococcal vaccine is more immunogenic because it induces a T-cell response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Among these statements about the use of antibacterials in febrile neutropenic patients, one is true. Which one? a) The use of aminoglycosides is not recommended in febrile neutropenic patients without severe sepsis or septic shock. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) The use of glycopeptides in combination with a betalactam is recommended as first line treatment of febrile neutropenic patients in standard situations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) In neutropenic patients, the risk of S. aureus bacteraemia is increased in the presence of severe mucositis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) In neutropenic patients, the risk of streptococcal bacteraemia is increased in the presence of catheter infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Among these statements about antifungal strategies in allo-HSCT, one is true. Which one? a) Posaconazole prophylaxis decreases the incidence of invasive fungal infection when given from transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Posaconazole prophylaxis decreases the incidence of invasive fungal infection when given from the onset of GvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Increasing the dose of liposomal Amphotericin B from 3 to 10 mg/kg in first line treatment of aspergillosis improves the clinical response . . . . d) It has been shown in a prospective study that the combination of 216

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caspofungin with voriconazole is more efficient than voriconazole alone in first line treatment of aspergillosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Among these statements about toxoplasmosis after allo-HSCT, one is true. Which one? a) Toxoplasmosis after allo-HSCT occurs almost exclusively in patients who were seropositive for toxoplasma before transplant . . . . . . . . . . . . . . . . . . . . . . . . b) Reactivation documented by blood PCR is observed in approximately 30–40% of seropositive allogeneic transplant recipients. . . . . . . . . . . . . . . . . . . c) Asymptomatic toxoplasma infection never develops into toxoplasma disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Aerosolised pentamidine prophylaxis is effective against both P. jiroveci and toxoplasma infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


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*

CHAPTER 11

Graft versus host disease

A. Devergie

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CHAPTER 11 • Graft vs. host disease

Graft versus host disease (GvHD) is the most frequent complication after allogeneic haematopoietic stem cell transplantation (HSCT). GvHD can occur despite aggressive immunosuppressive prophylaxis even when the donor is a “perfectly” matched (HLA-identical) sibling. It is a consequence of interactions between antigen (Ag)presenting cells of the recipient and mature T-cells of the donor.

1. History When the first allografts were performed, it was shown that patients given marrow from donors other than monozygotic twins were likely to develop what was then called “secondary disease”. Clinical manifestations involved the skin, intestinal tract and liver. They were similar to those observed in mice neonatally transplanted with allogeneic spleen cells (runt disease) and in some immunodeficient children who had received blood transfusions. Patients given allo-HSCT fulfilled the three conditions necessary for the development of GvHD, as defined by Billingham in 1966 (1): • Administration of immunocompetent cells • Histo-incompatibility between donor and recipient • Inability of the recipient to destroy or inactivate the transfused or transplanted cells.

2. Pathophysiology A 3-step process reflects the current view of the development of acute GvHD (Table 1) (2). Phase 1: Effect of conditioning: Underlying malignancy, effects of previous therapies and conditioning-related tissue damage lead to generation of large amounts of inflammatory cytokines such as TNF-a and IL-1 that enhance early activation of host Ag-presenting cells (APCs). Translocation of lipopolysaccharide (LPS) across damaged intestinal mucosa activates the innate immune system and promotes the inflammatory cytokine cascade.

Table 1: Immunobiology of aGvHD as a 3-step process Phase

Cells

Cytokines, chemokines

1

Effect of conditioning

Host APCs (dendritic cells) Epithelial cell damage

TNF-a, IL-1 Adhesion molecules, LPS

2

T-cell activation

Donor T-cells (mainly CD4+) Host APCs

IL-2 IFN-g

3

Cellular and inflammatory effector phase

CTLs NK cells

“Cytokine storm” TNF-a / IL-1 / nitric oxide


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Phase 2: T-cell activation: In this milieu, transplanted naïve donor T lymphocytes (and other cellular compartments) interact with host APCs, are activated and express cytokines such as IFN-g, IL-2 and TNF-a (among others), leading to T-cell expansion. Phase 3: Cellular and inflammatory effector phase: This phase is a complex cascade of multiple effectors including cytotoxic effector cells (CTLs), using Fas- and perforin-mediated mechanisms, natural killer (NK) cells and large granular lymphocytes (LGLs), followed by the generation of inflammatory cytokines, such as TNF-a, IL-1 and macrophage-derived nitric oxide. Interactions of innate (LGL) and adaptive (alloreactive T-cells) immune responses lead to organ damage. Additional complexity has been added by the recent description of regulatory cell populations including regulatory T-cells (Treg), regulatory APC populations and perhaps mesenchymal stem cells (3).

3. Classification of GvHD The traditional definition of acute (aGvHD) or chronic GvHD (cGvHD) was based on the time of onset after transplantation (less or more than 100 days after HSCT). This distinction is no longer tenable. For instance, aGvHD may present beyond 3 months in patients who have received reduced-intensity conditioning (RIC), and symptoms characteristic of cGvHD may occur before D 100. Furthermore, some signs and symptoms are common to both cGvHD and aGvHD. The recent National Institutes of Health (NIH) Consensus Conference proposed a definition of aGvHD or cGvHD, each with 2 subcategories, based on the specificity of signs and symptoms rather than the criterion of time of onset (Table 2) (4).

4. Incidence and risk factors The median incidence of clinically significant (grade II-IV) aGvHD is about 40% but

Table 2: Definition of acute and chronic GvHD Category

Time of manifestation

aGvHD features

cGvHD features

Classic

£ 100 days

Yes

No

Persistent, recurrent, late onset

> 100 days

Yes

No

Classic

No time limit

No

Yes

Overlap syndrome

No time limit

Yes

Yes

aGvHD

cGvHD

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ranges from 10 to 80% according to a number of risk factors (Table 3). The risk of aGvHD increases with the use of unrelated donors, multiparous female donor, older age of the recipient, graft type (cord blood has a lower rate and peripheral blood stem cells have a higher rate than marrow), and certain conditioning regimens. Although the central role of HLA matching in HSCT is well established, it has become apparent that other genetic systems affect the development of GvHD. Minor histocompatibility antigens (mHA) are peptides from polymorphic cellular proteins, encoded by regions outside the major histocompatibility complex (MHC). Interestingly, some mHA are expressed mainly by malignant cells and represent attractive target for the induction of Graft-versus-Leukaemia (GvL) effect without promoting GvHD. Genes controlling inflammatory processes, such as cytokines, chemokines and their receptors, can modulate GvHD. Gene polymorphisms affecting IL-1, IL-6, IL-10, TNF, TGF-b, and IFN-g have all been implicated in the incidence and severity of GvHD, both in experimental models and immunogenetic analysis of retrospective clinical data (5). These findings suggest that, in the future, the genotyping of patients and/or donors with respect to a panel of cytokines, chemokines, pharmacogenes, etc. will possibly complement histocompatibility typing and increase our ability to predict the risk of transplant-related toxicity (6).

Table 3: Risk factors for the development of GvHD Donor

Recipient

HLA compatibility (related/unrelated) Sex mismatched (FÆM) Alloimmunisation (parity, transfusions) Source of SC (PBSC > BM > CB)

Age Conditioning regimen Prevention of GvHD

5. Histopathology Apoptosis of cells in the tissue layer responsible for proliferation and regeneration is a typical histologic feature of GvHD and is the final event of the allogeneic reaction (Phase 3: Cellular and inflammatory effector phase). The targets are epithelial cells, including basal and suprabasal cells of the epidermis, the intestinal epithelium and the biliary duct epithelium. In the three target organs, the characteristic lesion of GvHD is the same, with infiltrating immune cells in the vicinity of the apoptotic cell giving a feature classically termed “satellite cell necrosis”. 5.1. Skin biopsies Skin biopsies are widely used in the diagnosis of a GvHD. The dermoepidermal junction


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is most severely affected: There is epidermal and basal cell vacuolar degeneration, disorganisation of epidermal cell maturation, eosinophilic body formation and melanocyte incontinence. In the first 3 weeks after transplant, the microscopic lesions induced by the conditioning regimen, i.e. lymphocytic infiltrates and epithelial damage, can be very similar to early lesions of GvHD. When the histological lesions are mild, it can be useful to look for satellite cell necrosis by electron microscopy, or to use an anti-TNF antibody to stain activated intraepithelial lymphocytes. In very severe forms of aGvHD with epidermal necrolysis (Lyell syndrome) clinical diagnosis is obvious. Skin biopsy would show satellite cell necrosis of all the keratinocytes of the basal layer leading to the dissociation of the upper part of the epidermis from the basal layer. 5.2. Gut biopsies Gut biopsies are performed as a diagnostic procedure to explore gastro-intestinal (GI) tract symptoms, mainly nausea or diarrhoea. Rectal or colon biopsies have been generally replaced by gastro-duodenal biopsies. The target cells for GvHD are basal epithelial cells localised in the epithelium of the crypts, which undergo apoptosis in the close vicinity of intraepithelial lymphocytes. In the acute phase of GvHD, it is important to distinguish lesions of GvHD from those induced by virus, particularly CMV. Classically, epithelial cells infected by CMV have voluminous clear inclusions and become necrotic. However, in GI lesions of recent onset, viral inclusions can be difficult to see and, in some cases, CMV and GvHD can be associated. Therefore, the possibility of CMV infection should be excluded using antibodies directed against CMV early proteins. 5.3. Liver biopsies Liver biopsies are performed only to rule out viral infection or drug toxicity when isolated hepatic GvHD is suspected. The target is the epithelium of the biliary canal and the lymphocytic infiltrates are mainly localised in portal tracts. Foci of necrotic hepatocytes can be observed, but there is no sclerosis in the acute phase of GvHD. Hepatic small bile ducts show segmental disruption, injury to the periductular epithelium, bile duct atypia and cellular degeneration. Cholestasis may be present.

6. Acute GvHD 6.1. Clinical manifestations and grading 6.1.1. Skin A maculo-papular rash, often involving the palms and soles usually marks the onset 222

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of aGvHD. Lesions may be pruritic and/or painful. The rash then spreads and can involve the entire body surface. In more severe cases, bullae can form and surface areas can desquamate, leading to extremely painful denudation associated with protein loss. 6.1.2. Liver Liver involvement results in cholestatic hepatopathy, with or without jaundice, in which the cholestatic enzymes are substantially raised whilst the transaminases show only non-specific changes. The clinical diagnosis of aGvHD of the liver is difficult since distinguishing liver impairment due to therapy associated hepatotoxicity, infection, veno-occlusive disease or GvHD is not always possible. 6.1.3. Gastro-intestinal Involvement of the GI tract primarily manifests as nausea and green watery diarrhoea. The enteral fluid loss is used as a measure of gut involvement. Severe abdominal pain, bloody diarrhoea and massive enteral fluid losses accompany advanced disease. A variant of mild enteric GvHD involving only upper GI tract has been described. Symptoms include anorexia and nausea without diarrhoea and this usually responds well to immunosuppressive therapy. 6.1.4. Concomitant signs of acute GvHD These include fever, decrease in performance status and weight loss. Other tissues such as lymphoid organs, mucous membranes, conjunctivae, exocrine glands and the bronchi may also be involved, but these are not included in the clinical staging and grading established by Glucksberg and more recently by the International Bone Marrow Transplant Registry (IBMTR). 6.1.5. Grading In 1974, Glucksberg published the first aGvHD classification (7). Each organ is staged from 0 to 4 (Table 4). These stages are combined to calculate an overall grade including objective assessment of organ function and subjective assessment of performance status (Table 5). It is routine practice to dichotomise GvHD severity into clinically insignificant grades 0–I and clinically significant grades II–IV. In recognition of the system’s limitations, a Consensus Workshop was held in 1995 and a modified grading was system proposed (Table 6) (8). The latter system retained the objective organ staging criteria of the Glucksberg system but excluded the subjective criteria of clinical performance. Thus, a revised system was developed by the IBMTR (Table 7) (9). To compare prospectively the Glucksberg and IBMTR classifications, a prospective


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multicentre study was conducted in 607 patients receiving T-cell replete allografts in 18 transplantation centres (10). Patients were scored weekly for aGvHD. The 2 classifications performed similarly in explaining variability in survival by aGvHD score, although the Glucksberg classification predicted early survival better. There was less physician bias or error in assigning grades with the IBMTR scoring system. With either system, only the maximum observed grade had prognostic significance for survival.

Table 4: Grading system: Stage for each organ Stage

Skin / Maculo-papular rash

Liver / Bilirubin

GI / Diarrhoea

+

500 mL

++

25-50% of body surface

51-102 mmol/L

> 1000 mL

+++

Generalised erythroderma

103-255 mmol/L

> 1500 mL

++++

Generalised erythroderma with bullae formation and desquamation

> 255 mmol/L

Severe abdominal pain with or without ileus

Table 5: Overall grading system (Glucksberg) Grade of aGvHD

Degree of organ involvement

I

Skin: + to ++

II

Skin: + to +++ Gut and/or liver: + Mild decrease in clinical performance

III

Skin: ++ to +++ Gut and/or liver: ++ to +++ Marked decrease in clinical performance

IV

Skin: ++ to ++++ Gut and/or liver: ++ to ++++ Extreme decrease in clinical performance

Table 6: Consensus Conference on aGvHD grading

224

Grade

Skin

Liver

Gut

I

Stage 1–2

0

0

II

Stage 3 or

Stage 1 or

Stage 1

III

–

Stage 2–3 or

Stage 2–4

IV

Stage 4 or

Stage 4

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Table 7: IBMTR Severity Index for aGvHD Skin

Liver

GI

INDEX

Stage (max)

Extent of rash

Stage (max)

Bilirubin (µmol/L)

Stage (max)

Diarrhoea (mL/d)

A

1

255 or

4

Pain, ileus

Thus, the authors failed to demonstrate a clear advantage of one system for grading aGvHD. 6.2. Prevention and treatment Acute GvHD is the major cause of early TRM. Mortality is due not only to GvHD itself but also to treatment related complications, both being responsible for a profound immune deficiency with frequent opportunistic infections. A major goal is prevention of GvHD. However, to date, it has not been possible to separate the deleterious effect of GvHD from the beneficial GvL effect. This explains why it is difficult to define the best strategy to prevent GvHD. Once GvHD occurs, the most important predictor of long-term survival is the primary response to therapy, as results with secondary treatments have been disappointing. 6.2.1. Prevention of GvHD a) Post transplant immunosuppressive treatment of the recipient (Table 8) Currently, most centres use a combination of a calcineurin inhibitor (cyclosporin (CsA) or tacrolimus) with “short course” methotrexate (MTX). Although other regimens are being explored, this standard regimen, described twenty years ago (11), has been shown repeatedly to result in a reasonable balance of GvHD and GvL in matched sibling transplants after ablative conditioning regimen. For higher risk groups or groups receiving non-conventional grafts (such as mismatched donors, older patients, reduced intensity regimen etc.), the best prophylaxis is less clearly established and other immunosuppressive drugs have been used: Sirolimus has been used in combination with tacrolimus and low dose (5 mg/m2) short MTX or with tacrolimus alone with prompt engraftment and minimal TRM (12). The efficacy of mycophenolate mofetil (MMF) associated with CsA has been studied mainly after RIC regimens (13). MMF may substitute MTX in the standard CsA combination because of less mucositis and overall good tolerance.


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Table 8: GvHD prophylaxis agents and mechanisms of action Mechanism of action

Dose

Cyclosporin (CsA)

Calcineurin inhibitor: blockade of T-cell activation

3 mg/kg IV

Tacrolimus

Calcineurin inhibitor: blockade of T-cell activation

0,02 mg/kg IV

Methotrexate (MTX)

Antimetabolite, folic acid analogue

15 mg/m2 D+1, 10 mg/m2 D+3, +6 and +11

Methylprednisolone (MP)

Receptor-mediated lympholysis + additional mechanisms

0,5–1 mg/kg

Mycophenolate mofetil (MMF)

Inhibition of DNA synthesis: lymphocyte apoptosis

1.5 to 3 g/d

Sirolimus (Rapamycine)

Macrolide antibiotic: blockade of T- and B-cell activation

12 mg D–3 then 4 mg/d

Antithymocyte globulin (ATG)

Rabbit or equine antibodies against human T-cells

2,5 mg/kg/d x 4

Alemtuzumab (Campath-1H)

Humanised monoclonal antibody to CD52

MethylPDN is the first line treatment for aGvHD, but its role in the prevention of GvHD is controversial. Several combination using prednisone in combination with MTX, CsA or both have been reported without substantial benefit. b) In vitro or in vivo T-cell depletion (TCD) of the transplant For patients receiving mismatched grafts, more intensive immunosuppression is usually needed. Methods of ex vivo TCD as well as pharmacologic in vivo TCD (anti-thymocyte globulin, alemtuzumab) have been used. In general, these methods reduce aGvHD but increase the incidence of infection (due to delayed reconstitution of the immune system) and the incidence of relapse (due to a decreased GvL effect). Several randomised or comparative studies have been performed comparing in vitro TCD to CsA + MTX, but so far, it has not been conclusively established whether TCD can improve LFS (14). 6.2.2. Treatment of aGvHD a) Primary treatment The most important predictor of long-term survival is the primary response of GvHD to therapy. Methyl-prednisolone (MP) at a dose of 2 mg/kg/d is the best initial therapy for aGvHD. This treatment, associated with a calcineurin inhibitor, is given for 7 to 14 days, and then tapered slowly if there is a complete response to therapy. 226

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Complete responses occur in 25 to 40% of patients with grade II to IV aGvHD. However, the likelihood of response decreases with increasing severity of the disease and the dynamic of responses may differ between target organs and between patients. Failure of therapy is usually defined as: • Progression after 3 days, or • No change after 7 days, or • Incomplete response after 14 days. b) Secondary treatment Patients in whom initial therapy has failed will receive a secondary treatment. Table 9 provides a summary of available agents. Numerous strategies have been used but only very few controlled studies have been conducted and there are no criteria to identify patients who are likely to respond to any second line treatment. Overall, results have been disappointing. The rate of partial and complete response to second line therapy varies from 35 to 70%, but the 6–12 months survival is low, in the order of 30%, in most large trials, because of high incidence of infectious complications or recurrence of GvHD. Treatment of steroid-refractory aGvHD has not made major advances in the last decade, as shown in several reviews recently published (15–17). In patients with predominant GvHD of the skin, which is refractory to steroids, the use of phototherapy may be an attractive and non-toxic strategy. In case of visceral

Table 9: Second line treatment of steroid-refractory aGvHD Methylprednisolone (2–5 mg/kg) Immunosuppressive drugs: - Tacrolimus, MMF, sirolimus (if not used for prophylaxis) Oral non-absorbable steroids (in case of GI involvement) Anti-thymocyte globulin Monoclonal antibodies: - Anti-IL-2 receptor (CD25) antibody: Inolinomab, basiliximab, daclizumab, denileukin difitox - Anti-TNFa antibody: Infliximab, etanercept - Anti-CD52 antibody (broad specificity T-cell antibody): Alemtuzumab (Campath 1H) - Anti-CD147 antibody (anti activated T- and B-cells): ABX/CBL - Anti-CD3 (broad specificity T-cell antibody): Visilizumab, OKT3 Pentostatin: - Inhibitor of adenosine-deaminase Extracorporeal photopheresis: - Suppression of T-cell reactivity and cytokine release; induction of regulatory T-cells Mesenchymal stem cells: - Immunomodulatory and tissue repairing effect


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GvHD, despite concerns about toxicity, second line treatment can be either polyclonal antibodies (i.e. ATG) or monoclonal antibodies. Other approaches, such as the use of mesenchymal stem cells, need to be explored in prospective randomised trials. There is agreement that efforts must be aimed at reducing steroid exposure and protecting the patient against infectious complications. c) Supportive care Supportive measures are very important. They include GI rest, parenteral hyperalimentation, replacement therapy of enteral losses of fluids, pain control, and infectious prophylaxis. Nevertheless, despite all preventive measures, viral, bacterial and mycotic infections are common and are the most frequent causes of death in patients with severe aGvHD. Ultimately, the outcome of aGvHD depends on two main factors: the overall grade of aGvHD and the response to treatment.

7. Chronic GvHD 7.1. Clinical manifestations and grading Chronic GvHD is the primary cause of late morbidity and non-relapse mortality in transplant survivors. Chronic GvHD has features resembling autoimmune and other immunologic disorders such as scleroderma, Sjögren syndrome, primary biliary cirrhosis, wasting syndrome, bronchiolitis obliterans (BO), immune cytopenias and chronic immunodeficiency. Symptoms usually present within 3 years after allogeneic HCT and are often preceded by a history of aGvHD. Manifestations of cGvHD may be restricted to a single organ or tissue or may be widespread. Chronic GvHD can lead to debilitating consequences, e.g. joint contractures, loss of sight, end-stage lung disease and profound chronic immunosuppression. Risk factors for cGvHD have been identified, including prior aGvHD, older patient age, the use of female donors for male recipients, use of donor lymphocyte infusion, use of unrelated or HLA-mismatched donors, and, more recently, the use of PBSC as a source of stem cells. 7.1. Diagnosis and staging The list of signs and symptoms of cGvHD, as established by the NIH working group report on diagnosis and staging (4), is shown in Table 10, with a distinction between diagnostic signs (that establish the diagnosis of cGvHD without the need for further testing or symptom), distinctive signs (not found in aGvHD but not sufficient to establish the diagnosis of cGvHD), other features of cGvHD (non-specific) 228

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Table 10: Signs and symptoms of cGvHD Organ/site

“Diagnostic”

Skin

Poikiloderma Depigmentation Lichen planuslike features

Nails

Other

Both acute and chronic Erythema, maculopapular rash, pruritus

Dystrophy

Scalp Mouth

“Distinctive”

Alopecia Lichen planus

Eyes

Xerostomia

Mucositis

Keratoconjunctivitis Photophobia, sicca blepharitis

Genitalia

Lichen planus

GI tract

Strictures of oesophagus

Exocrine pancreatic insufficiency

Liver

Anorexia, weight loss Bilirubin or alkaline phosphatase > 2 x upper limit of normal

Lung

Bronchiolitis obliterans

Muscles, fascia, joints

Fasciitis, joint contractures

Myositis

Cramps, arthralgias

Haematopoietic and immune

Thrombocytopenia eosinophilia, lymphopenia

Other

Ascites, pericardial or pleural effusions

and common signs or symptoms (found in both aGvHD and cGvHD). The diagnosis of cGvHD requires at least one diagnostic manifestation of cGvHD or at least one distinctive manifestation, with the diagnosis confirmed by pertinent biopsy or laboratory test. Historically, cGvHD was classified as limited or extensive on the basis of a small retrospective study (18). The NIH working group proposed a new global scoring system, which includes both the number of organs or sites involved and the severity within each affected organ: score 0 = no symptoms, score 1 = mild symptoms, score 2 = moderate symptoms and score 3 = severe symptoms. Mild cGvHD involves only 1 or 2 organs (except lung) with a maximum score of 1. Moderate cGvHD involves at least 1 organ with score 2, or 3 or more organs with score 1 (or lung score 1). Severe cGvHD indicates a score of 3 in any organ (or score 2 in the lung) (Table 11). HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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Table 11: Global scoring of cGvHD, according to the number of sites and the severity for each organ or site Number of organs

Mild cGvHD

Moderate cGvHD

Severe cGvHD

1 organ or site

Score 1

Score 2

Score 3

2 organs or sites

Score 1

Score 2

Score 3

3 or more organs

Score 1

Score 3

Lung involvement

Score 1

Score 2

7.3. Prevention and treatment Despite the fact that significant aGvHD is one of the strongest predictors for the development of cGvHD, successful efforts to reduce aGvHD with combinations of immunosuppressive agents, such as tacrolimus or MMF, have not resulted in a reduced incidence of cGvHD (19). Other methods, such as in vivo or in vitro TCD have been studied but the benefits due to a decreased incidence of acute and cGvHD need to be balanced against the consequences of delayed immune reconstitution. The role of prolonged administration of CsA for cGvHD prophylaxis has been studied and has shown only a non-significant reduction in the risk of extensive cGvHD with no difference in overall survival or disease free survival (20). 7.3.1. Primary treatment A combination of CsA and prednisolone has been the standard frontline therapy for cGvHD for almost 20 years (21). The initial dose of steroid ranges from 1 to 1.5 mg/kg/day for at least 2 weeks then the dose is slowly tapered, according to response. Duration of therapy is also determined by response, but is prolonged, usually continuing for close to 12 months, even in patients achieving complete resolution. The morbidity associated with steroid therapy is significant, including avascular necrosis, glucose intolerance requiring administration of insulin, infections, hypertension, changes in body habitus, cutaneous atrophy, cataracts, osteoporosis, emotional lability, interference with sleep, and growth retardation in children. So far, the addition of other immunosuppressive drug to standard upfront therapy, such as thalidomide, has not led to any significant difference in cGvHD response rate or survival (22). There is a need for randomised prospective trials to investigate the addition of other immunosuppressive drugs to upfront therapy in an effort to improve outcomes and ameliorate steroid-related side-effects. Such a trial, sponsored by the EBMT, has been activated in 2007 (double-blind placebo-controlled trial comparing CsA plus steroids with or without myfortic (the biologically active component of MMF) as primary treatment for extensive cGvHD). 230

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7.3.2. Salvage therapy There is no standard second-line therapy for patients who have failed primary treatment. Various approaches have been developed, including low dose total lymphoid irradiation, PUVA therapy, extracorporeal photochemotherapy, MMF, tacrolimus, and thalidomide. All these treatments have been reported to improve clinical manifestations (23). Recent publications have described encouraging responses following therapy with rituximab (anti-CD20 monoclonal antibody) (24). The efficacy of sirolimus (25) or of pentostatin (26) has also been investigated and some objective responses were described. 7.3.3. Antimicrobial prophylaxis and supportive care Infection is the leading cause of death among patients with cGvHD. Antimicrobial prophylaxis is a very important aspect of the treatment of these patients. Infections with Streptococcus pneumoniae and Haemophilus influenzae are frequent and must be prevented with prophylactic antibiotics and/or vaccination. Pneumocystis pneumonia occurring late after HSCT is strongly associated with cGvHD and prophylaxis must be given to patients receiving long-term immunosuppressive treatment. Additional treatments include ursodeoxycholic acid in patients with hepatic GvHD, anti-osteoporosis agents, and artificial tears in patients with eye involvement. A multidisciplinary approach to the management of patients with cGvHD is essential, including dermatologists, ophthalmologists, stomatologists, gynaecologists and others. In retrospective series of patients with extensive cGvHD, only 10 to 30% became long-term survivors. Late mortality is related not only to opportunistic infections but also to an increased incidence of secondary cancers.

8. Conclusion In conclusion, early institution of effective immunosuppressive therapy has changed the outcome for patients with GvHD, but larger studies demonstrating safety and efficacy of second line treatments for patients non-responsive to standard therapy with steroids are mandatory.

References 1. Billingham RE. The biology of graft versus host reactions. Harvey lectures 1966-1967; 62: 71-78. 2. Reddy P, Ferrara JLM. Immunobiology of acute graft-versus-host disease. Blood Reviews 2003; 17: 187-194. HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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3. Tse WT, Pendleton JD, Beyer Wm, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: Implication in transplantation. Transplantation 2003; 75: 389-397. 4. Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic Graft-versus-host Disease: I. Diagosis and Staging Working group report. Biol Blood Marrow Transplant 2005; 11: 945956. 5. Dickinson AM, Charron D. Non-HLA immunogenetics in hematopoietic stem cell transplantation. Curr opin Immunol 2005; 17: 517-525. 6. Baron C, Somogyi R, Greller LD, et al. Prediction of graft-versus-host disease in humans by donor gene-expression profiling. PLoS Med 2007; 4: e23. 7. Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graft versus host disease in human recipients of marrow from HLA-matched sibling donors. Transplantation 1974; 18: 295-304. 8. Przepiorka D, Weisdorf D, Martin P, et al. Consensus conference on acute GvHD grading. Bone Marrow Transplant 1995; 15: 825-828. 9. Rowlings PA, Przepiorka D, Klein JP, et al. IBMTR severity index for grading acute GvHD: retrospective comparison with Glucksberg grade. Br J Haematol 1997; 97: 855-864. 10.Cahn JY, Klein JP, Lee SJ, et al. Prospective evaluation of 2 acute GvHD grading system: A joint Société française de Greffe de Moelle et Thérapie Cellulaire (SFGM-TC), Dana Farber Cancer institute (DFCI), and International Bone Marrow Transplant Registry (IBMTR) prospective study. Blood 2005; 106: 1495-1500. 11.Storb R, Deeg HJ, Whitehead J, et al. Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukaemia. N Engl J Med 1986; 314: 729-735. 12.Cutler C. Li S, Ho VT, et al. Extended follow-up of methotrexate-free immunosuppression using sirolimus and tacrolimus in related and unrelated donor peripheral blood stem cell transplantation. Blood 2007; 109: 3108-3114. 13.Niederwieser D, Maris M, Shizuru JA, et al. Low-dose total body irradiation and fludarabine followed by hematopoietic cell transplantation from HLA-matched or mismatched unrelated donors and post grafting immunosuppression with cyclosporine and mycophenolate mofetil can induce durable complete chimerism and sustained remission in patients with haematological diseases. Blood 2003; 101: 1620-1629. 14.Wagner JE, Thompson JS, Carter S, et al. Effect of GvHD prophylaxis on 3-year DFS in recipients of unrelated donor bone marrow (T-cell Depletion Trial): A multi-centre, randomised phase II-III trial Lancet 2005; 366: 733-741. 15.Kim SS. Treatment options in steroid-refractory acute GvHD following hematopoietic stem cell transplantation. The Annals of Pharmacotherapy 2007; 41:1436-1444. 16.Bacigalupo A. Management of acute GVHD. Br J Haematol 2007; 137: 87-98. 17.Deeg JH. How I treat refractory acute GVHD. Blood 2007; 109: 4119-4126. 18.Shulmann H, Sullivan KM, Weiden PL, et al. Chronic graft-versus-host disease in man: A clinic pathologic study of 20 long term Seattle patients. Am J Med 1980; 69: 204-217. 19.Fraser CJ, Baker KS. The management and outcome of chronic graft-versus-host disease. Br J Haematol 2007; 138: 131-145. 232

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20.Kansu E, Gooley T, Flowers ME, et al. Administration of cyclosporine for 24 months compared with 6 months for prevention of chronic graft-versus-host disease: A prospective randomized clinical trial. Blood 2001; 98: 3868-3870. 21.Sullivan KM, Witherspoon RP, Storb R, et al. Alternating day cyclosporine and prednisone for treatment of high-risk chronic graft-versus-host disease. Blood 1988; 72: 555-561. 22.Arora M, Wagner JE, Davies SM, et al. Randomized clinical trial of thalidomide, cyclosporine and prednisone versus cyclosporine and prednisone as initial therapy for chronic GvHD. Biol Blood Marrow Transplant 2001; 7: 265-273. 23.Vogelsang GB. How I treat chronic graft-versus-host disease. Blood 2001; 97: 1196-1201. 24.Cutler C, Miklos D, Kim HT, et al. Rituximab for steroid-refractory chronic graft-versushost disease. Blood 2006; 108: 756-762. 25.Jurado M, Vallejo C, Perez-Simon JA, et al. Sirolimus as part of immunosuppressive therapy for refractory chronic graft-versus-host disease. Biol Blood Marrow Transplant 2007; 13: 701-706. 26.Jacobsohn DA, Chen AR, Zahurak M, et al. Phase II study of pentostatin in patients with corticosteroid-refractory chronic graft-versus-host disease. J Clin Oncol 2007; 25: 42554261.

Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. The prognosis of aGvHD is mainly related to: a) The diagnosis of the initial disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) The age of the donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The % of body surface involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The response of GvHD to initial therapy with 2 mg/kg methyl prednisolone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. One factor is not significantly associated with an increased risk of cGvHD: a) The use of a male donor for a female recipient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) The previous occurrence of an aGvHD grade ≥ I . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The use of PBSC as a source of stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) The age of the recipient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. T-cell depletion of the transplant is: a) Associated with a better long term survival in all patients when compared with a non T-cell depleted transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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b) Associated with an increased risk of cGvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) An effective prophylaxis of aGvHD but is associated with an increased risk of graft failure/relapse/opportunistic infections . . . . . . . . . . . . . . . . . . . . . . . d) Associated with an increased risk of secondary solid tumour . . . . . . . . . . . . . . 4. The “classical” prevention of GvHD is: a) A combination of methylprednisolone 1 mg/kg/d and a calcineurin inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) A combination of a calcineurin inhibitor and short course methotrexate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Mycophenolate mofetil alone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) A course of ATG during 10 days . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Chronic GvHD is: a) Associated with an increased risk of relapse of the leukaemia . . . . . . . . . . . . b) Associated with an increased risk of secondary cancer . . . . . . . . . . . . . . . . . . . . . c) Associated with an increased risk of veno-occlusive disease . . . . . . . . . . . . . . d) Observed only after a grade II to IV aGvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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*

CHAPTER 12

Long term survivorship, general health status, quality of life and late complications after HSCT

A. Tichelli, C-P. Schwarze, G. Socié

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CHAPTER 12 • Late effects

1. Introduction Large numbers of patients now survive long term following haematopoietic stem cell transplantation (HSCT). The aim of HSCT is not only to cure the patients from their primary disease, but also to allow the long-term survivor to obtain a normal health status, to return to work or to school and to have a normal social life. Immediate survival is therefore no longer the sole concern. Still, HSCT remains associated with considerable morbidity and mortality. Long-term health status and the development of late events related to HSCT have therefore gained increasing interest. Secondary malignant diseases are of particular clinical concern as more patients survive the early phase after transplantation and remain free of their original disease. Nonmalignant late effects are heterogeneous, and although often non-life threatening they significantly impair the quality of life of long-term survivors. This Chapter presents an overview of these malignant and non-malignant late complications, describes the consequences of these late events on the general health status, social integration and quality of life in long-term survivors, and provides recommendations regarding their prevention and early treatment (1, 2). Readers will find extensive literature summary and references in recently published reviews (3–5).

2. Non malignant late complications (Table 1) (2) 2.1. Late ocular effects 2.1.1. Ocular complications of the posterior segment These can be divided into microvascular retinopathy, optic disk oedema, haemorrhagic complications and infectious retinitis. Fungal infections typically occur within 120 days of HSCT, while Herpes zoster, CMV and Toxoplasma retinitis occur later. Ischaemic retinopathy with cotton-wool spots and optic-disk oedema has been described in 10% of patients following HSCT. Microvascular retinopathy occurs mainly after TBI conditioned allogeneic HSCT in patients receiving cyclosporin as GvHD prophylaxis. In most cases, the retinal lesions resolve with withdrawal or reduction of immunosuppressive therapy. 2.1.2. Ocular complications of the anterior segment The two most common late complications affecting the anterior segment are cataract formation and kerato-conjunctivitis sicca syndrome. Posterior subcapsular cataracts have long been recognised in recipients of HSCT as one of most frequent late complications of TBI. After single dose TBI almost all patients develop cataracts within 3 to 4 years and most if not all require surgical repair. Although the probability of developing cataracts after fractionated TBI is


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around 30% at 3 years the incidence may be more than 80% 6 to 10 years post HSCT (6, 7). Use of TBI in general and, single fractionations in particular and the use of

Table 1: Non-malignant late tissue and organ toxicity in long-term survivors Organ

Clinical manifestation

Risk factors

Monitoring

Intervention

Eye

Cataracts

Radiation Steroids

Split lamp examination

Fractionation of TBI Surgical repair

Keratoconjunctivitis Radiation GvHD

Schirmer test

Treatment of GvHD Topical lubricants Topical steroids

Restrictive or dilated Anthracyclines cardiomyopathy Mediastinal Arrhythmia radiotherapy Autonomic neuropathy

LVEF 24-hour ECG

Treatment of cardiac insufficiency Pace maker

Infection GvHD Smoking

Pulmonary function testing Chest radiographic testing if clinically indicated

Treatment of infection Immune globulin replacement Treatment of GvHD

Restrictive lung disease

Radiation Chemotherapy Infection

Pulmonary function testing Chest radiographic testing if clinically indicated

Fractionation of radiation Lung shielding Treatment of infection Steroids

Chronic hepatitis C

HVC infection Iron overload

Liver tests Hepatitis serologies Viral load if positive

Treatment of hepatitis C Treatment of iron overload (phlebotomy or chelation therapy)

Liver cirrhosis

HCV infection Iron overload

Ferritin

Heart

Respira- Chronic obstructive tory tract lung disease Bronchiolitis obliterans

Liver

Chronic GvHD of the liver Kidney

Nephropathy

TBI Chemotherapy (platinum) Cyclosporin

Liver biopsy if indicated

Treatment of GvHD

Renal function tests

Control of hypertension

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Organ

Clinical manifestation

Skeletal

Monitoring

Intervention

Avascular necrosis Steroids of the bone Radiation

Radiographic testing

Avoidance of long term treatment with steroids Symptomatic relief of pain Orthopoedic measures Surgical repair

Osteoporosis

Dual photon densitometry

Sex hormone replacement Treatment of osteoporosis (bisphosphonates, calcium, vitamin D)

Chronic stomatitis Chronic GvHD Radiation

Oral inspection

Oral hygiene Treatment of GvHD

Dental late effects

Radiation Chronic GvHD

Dental inspection

Prophylaxis: oral hygiene, instruction for dental care, brushing teeth, application of fluoride Treatment of caries

Thyroid gland

Hypothyroidism

Radiation

TSH, T4 annually

Thyroid hormone replacement

Gonadal function

Gonadal failure

Radiation Chemotherapy

FSH, LH, testosterone (males), oestradiol (females)

Hormone replacement Sperm banking in males

Fertility

Infertility

Radiation Chemotherapy

FSH, LH, testosterone (males), oestradiol (females) Sperm analysis

Cryopreservation of sperm fluid before HSCT

Nervous system

Leukoencephalopathy

Cranial radiation Intrathecal chemotherapy

Evaluation according to symptoms

Peripheral neuropathy

Chemotherapy GvHD

Atherosclerosis Cerebrovascular events Cardiovascular events Peripheral vascular events

Established Cardiovascular risk cardiovascular risk factors factors GvHD (?) Radiation (?)

Oral

Vascular compartment

Risk factors

Steroids, cyclosporin, tacrolimus Hypogonadism, TBI, chemotherapy Immobility

Correction of cardiovascular risk factors


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steroid treatment for longer than 3 months are associated with a significant risk of cataract development. The development of cataracts is also more likely if the TBI is delivered at a high dose rate. Finally, in prospective studies of the incidence of cataracts according to various risk factors, patients who receive cyclophosphamide and TBI (Cy/TBI) have a higher incidence of cataract formation than those treated with busulfan and cyclophosphamide (BuCy). The only treatment for cataract is to surgically remove the opacified lens from the eye to restore transparency of the visual axis. Today, cataract surgery is a low risk procedure and improves visual acuity in 95% of eyes that have no other pathology. Keratoconjunctivitis sicca syndrome is usually part of a more general syndrome with xerostomia, vaginitis and dryness of the skin. All these manifestations are closely related to cGvHD, which may lead in its most extensive forms to a Sjögren like syndrome. The ocular manifestations include reduced tear flow, keratoconjunctivitis sicca, sterile conjunctivitis, corneal epithelial defects, and corneal ulceration. The incidence of late-onset keratoconjunctivitis sicca syndrome may reach 20% fifteen years after HSCT, but reaches nearly 40% in patient with cGvHD, compared to less 10% in those without GvHD. Risk factors for late-onset keratoconjunctivitis include cGvHD, female sex, age >20 years at HSCT, single dose TBI, and the use of methotrexate for GvHD prophylaxis. Treatment is based on the management of cGvHD with regular use of topical lubricants. Topical corticosteroids may improve symptoms but can cause sight-threatening complications if used inappropriately in Herpes simplex virus or bacterial keratitis. 2.2. Pulmonary late effects 2.2.1. Restrictive lung disease Restrictive lung disease is frequently observed 3 to 6 months after HSCT in patients conditioned with TBI and/or receiving an allogeneic HSCT but in most cases it is not symptomatic. Restrictive disease is often stable and may recover, partially or completely, within 2 years. However, some patients do develop severe late restrictive defects and may eventually die from respiratory failure. 2.2.2. Chronic obstructive lung disease Chronic obstructive pulmonary disease can be detected in up to 20% of long-term survivors after HSCT. It has been mainly associated with cGvHD, but other potential risk factors including TBI, hypogammaglobulinemia, GvHD prophylaxis with methotrexate, and infections have been described. Mortality is high among these patients, particularly in those with an earlier onset and rapid decline of FEV1. Symptoms consist of nonproductive cough, wheezing and dyspnoea; chest radiography 240

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is normal in most cases. High-resolution CT scanning may reveal non-specific abnormalities. Symptomatic relief can be obtained in some patients with bronchodilators; however in most cases obstructive abnormalities are not improved by this treatment. Patients with low IgG and IgA levels should receive immunoglobulins to prevent infections, which may further damage the airways. Immunosuppressive therapy may be of benefit but typically improvements occur in less than 50% of cases. Asymptomatic patients with abnormal pulmonary function tests (PFTs) should be closely monitored for the development of respiratory symptoms; an early recognition of airflow obstruction allows the initiation of treatment at a potentially reversible stage. Obliterative bronchiolitis (OB), the best characterised obstructive syndrome, has been reported in 2 to 14% of allogeneic HSCT recipients and carries a mortality rate of 50%. OB is strongly associated with cGvHD and low levels of immunoglobulins. Initial symptoms often resemble those of recurrent upper respiratory tract infections, and then persistent cough, wheezing, inspiratory rales and dyspnoea appear. PFTs gradually deteriorate with severe and non-reversible obstructive abnormalities. Chest radiographs and CT scanning may reveal hyperinflation with or without infiltrates and vascular attenuation; however radiological findings do not correlate with lung function changes. Bronchoscopy with transbronchial biopsy can help to rule out infection and may reveal obliteration of bronchioles with granulation tissue, mononuclear cell infiltration or fibrosis. It is not clear to what extent combined immunosuppressive treatment can be effective in the treatment of this disease, which typically does not respond to treatment with steroids. Azathioprine and mycophenolate mofetil may lead to improved symptoms in some cases. Prophylaxis and prompt treatment of infections are the most important elements of clinical management and may help to alter the clinical course of the disease. 2.3. Late complications of bones and joints 2.3.1. Avascular necrosis of bone (AVN) The incidence of AVN varies from 4% to more than 10%. The mean time from transplant to AVN is 18 months and the first clinical manifestation is usually pain. Early diagnosis can rarely be made using standard radiography alone and magnetic resonance imaging is the investigation of choice. The hip is the affected site in over 80% of cases with bilateral involvement occurring in more than 60% cases. Other sites include the knee (10% of patients with AVN), the wrist and the ankle. Symptomatic relief of pain and orthopoedic measures to decrease the pressure on the affected joints are of value, but most adult patients with advanced damage will require surgery. The probability of total hip replacement following a diagnosis of


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AVN is approximately 80% at 5 years. While short-term results of joint surgery are excellent in the majority (>85%) of cases, it is clear that long term follow-up of the prostheses are needed in young patients who have a long life expectancy. Studies evaluating risk factors for AVN have clearly identified the use of steroids (both total dose and duration) as the strongest risk factor. Thus, unnecessary long-term low dose steroids for non-active chronic GvHD should be avoided. The second major risk factor for AVN is TBI, the highest risks being associated with receipt of single doses of 10 Gy or higher or >12 Gray in fractionated doses. 2.3.2. Osteoporosis (8) The degree of reduction in bone mass can be quantified on dual photon densitometry. The cumulative dose and number of days of glucocorticoid therapy and the number of days of cyclosporine or tacrolimus therapy showed significant associations with loss of bone mass. Non-traumatic fractures may occur in 10% of patients. Using WHO criteria, nearly 50% of the patients have low bone density, a third have osteopenia and roughly 10% have osteoporosis, 12–18 months post transplant. Preventative measures of osteoporosis must include sex-hormone replacement in patients with gonadal failure; the efficacy of new treatments for osteoporosis in long-term survivors of HSCT requires further evaluation. 2.4. Endocrine function after HSCT 2.4.1. Thyroid dysfunction Seven to 15.5% of patients will develop sub-clinical hypothyroidism (slightly-high serum TSH and normal free-T4 levels) in the first year post HSCT. It is not yet clear if patients who develop sub-clinical hypothyroidism should be treated with Lthyroxine since the majority of these cases are mild, compensated and may resolve spontaneously. One possible approach is to monitor TSH and free-T4 levels twice yearly and to consider L-thyroxine treatment only if the TSH concentration remains high or is increasing. The frequency of hypothyroidism requiring L-thyroxine replacement therapy is highly variable depending to a large extent on the type of pre-transplant conditioning applied: nearly 90% in patients who have received 10 Gy single-dose TBI, 14–15% of patients following fractionated TBI, and smaller numbers after conditioning with BuCy. Treatment with L-thyroxine is indicated in all cases of frank hypothyroidism (elevated TSH with low free-T4 blood levels). Thyroid hormone levels should be measured 4–6 weeks after commencement of replacement therapy, and dosage should be tailored thereafter to the individual patient and adjusted according to thyroid function evaluation every 6 months. Elderly patients 242

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should have an ECG prior to commencing treatment to exclude associated ischaemic heart disease and/or arrhythmias. Autoimmune thyroiditis, presumably transferred via donor cells, has also been reported. 2.4.2. Gonadal failure Gonadal failure (both testicular and ovarian) is a common long-term consequence of the chemotherapy given prior to HSCT, and of the pre-transplant conditioning. The major cause of gonadal damage leading to hypergonadotrophic-hypogonadism is irradiation. Similar damage can also be caused by busulfan. In males, the testicular germinal epithelium (Sertoli-cells), the site of spermatogenesis, is more vulnerable to radiation and chemotherapy than the testicular Leydig-cell component, which is involved testosterone secretion. Therefore, testosterone levels are usually normal even where spermatogenesis is reduced or absent. The serum FSH is typically elevated while LH levels may remain in the normal range. The great majority of patients will not, therefore, require testosterone replacement to ensure sexual activity, libido, erection and ejaculation. Sex-hormone replacement therapy (SHRT) with testosterone derivatives in males is indicated in patients with severe uncompensated hypogonadism. In females, hypergonadotrophic-hypogonadism is almost inevitable as the ovaries are more vulnerable to irradiation and chemotherapy than the testes. Busulfan is one of the most gonadotoxic agents while cyclophosphamide is usually associated with only minor effects on gonadal function. The majority of adult females will need SHRT in order to maintain menstruation and bone turnover/mineralisation. In prepubertal girls who do not undergo puberty spontaneously post-HSCT, oestrogen treatment should be started at the age of 12–13 years to promote breast and uterine development and the pubertal growth spurt. The dose of oestrogen treatment will need to be gradually increased and a combination of cyclical oestro-progesterone treatment introduced after 1–2 years to initiate menstruation and to reduce the risk of future osteoporosis. SHRT can be interrupted once every 2–3 years, for a period of six months, to evaluate possible spontaneous recovery of ovarian activity, which occurs in the minority of women. Due to the high incidence of gonadal dysfunction and early menopause in patients after HSCT, an annual clinical and biological gynaecological assessment is recommended. 2.4.3. Special considerations in puberty Hypogonadism occurs in up to 70% of paediatric patients after HSCT. Male patients are more likely than females to enter and progress through puberty. During spontaneous puberty, measurement of testosterone is recommended if the pubertal growth spurt is blunted. A high percentage of female patients will need SHRT. The


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probability of ovarian recovery after fractionated TBI is higher in younger patients but after busulfan hypogonadism prevails. Baseline measurements of LH and FSH are recommended at 8 yrs in females and 9 yrs in males. The timing and progression of puberty have a major influence on growth and final height. This must be considered when initiating replacement therapy. Delaying puberty beyond the ages of 13 and 14 yrs is usually not advisable. Temporary cessation of replacement therapy after completion of puberty and growth should be considered in order to evaluate spontaneous recovery. Precocious puberty occurs in a few cases and necessitates gonadotrophin releasing hormone (GnRH)-agonist treatment. Central/hypogonadotrophic hypogonadism can be diagnosed by a GnRH test. 2.4.4. Fertility following stem cell transplantation The overall incidence of pregnancy is low (6 colour flow to be performed will ensure high quality, sensitive and specific monitoring of MRD. Critical to the success of multiparameter flow cytometry is the requirement for inter-laboratory standardisation and this is currently being addressed by the Euroflow Consortium, a European Commission funded study to standardise fast and sensitive diagnosis and follow up of haematological malignancies (1). 2.1.4. Polymerase chain reaction analysis PCR based approaches have particular relevance in the detection of MRD due to their high specificity and sensitivity. In patients where a defined chromosomal lesion is present, primers are designed to bind to the nucleic acid of each of the gene partners in the translocation, thus allowing specific detection of the chromosomal lesion. In many cases, RNA will be the primary target but as PCR does not amplify RNA, the RNA must first be converted into cDNA using the enzyme reverse transcriptase (RT). When a chromosomal lesion is not present, MRD analysis relies on detection of clone-specific rearrangements such as the rearranged immunoglobulin (Ig) or Tcell receptor (TCR) genes (Ig-TCR-gene-rearrangements). These are particularly


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relevant for MRD detection in ALL, as many cases, even those without chromosomal aberrations, have rearranged IgH or TCR genes. Sensitivities of RT-PCR range from (10-4) to (10-6) depending on the type of leukaemia. Standardisation of detection of clonal IgH and TCR fingerprints and of the most frequent fusion transcripts in ALL (E2A-PBXI, MLL-AF4, BCR-ABL, TEL-AML) and the intrachromosomal microdeletion generating the SIL-TALI fusion gene, by the European Biomed and Europe Against Cancer Networks has allowed robust MRD detection to be available for single centre and multicentre studies (2). While semi-quantitative MRD studies yielded interesting results, emphasising the relevance of MRD detection, the introduction of real time quantitative PCR approaches (RQ-PCR) has empowered the clinical application of the results of MRD studies (3, 4). The European Study Group of MRD detection in ALL (ESG-MRD-ALL) has produced excellent standards and guidelines, harnessing the work of 30 European laboratories in concert to establish reproducible approaches for detecting, quantifying and assessing the relevance of MRD detection (4). To avoid contamination of PCR assays with old PCR products, a number of suggestions have been made, including use of dUTP and uracil N glycosylase (UNG), UV irradiation of surfaces, materials and reagents. However, physical separation of the individual part of the PCR assay into sample preparation, pre-PCR and post-PCR areas should be the central part of any contamination control strategy. Although carryover of amplified sequences contributes to the majority of the false positives, sampleto-sample contamination can also be a factor. Consequently, precautions must be taken in all aspects of sample handling, including use of plugged pipettes and tips at all stages of the process.

3. MRD detection in acute leukaemia Immunophenotyping and PCR based systems are currently the most relevant methods for detecting MRD, as they are highly sensitive and can be employed in the analysis of virtually all cases of acute leukaemia. With respect to immunophenotyping the most frequently expressed aberrant phenotypes in B-cell precursor ALL involve TdTdim/CD10+ and CD38dim/CD34+. CD19 is a useful additional gating marker. A panel of markers is employed at diagnosis, allowing selection of the most appropriate LAPs for MRD analysis in individual patients (Figure 2). In T-ALL, the employment of Tcell markers, particularly TdT and cytoplasmic CD3 with CD7 as an additional gating marker allows the majority of T-ALL to be detected. Judicious choice of the marker panel and its evaluation in individual patients allows the selection of robust MRD LAPs.

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CHAPTER 13 • Molecular monitoring after HSCT

Figure 2: MRD detection by immunophenotyping

MRD analysis was performed using the combination of CD66 and CD10 as a potential LAP. CD10 is labelled with PE and CD 66 with FITC. This combination does not appear on normal B-cells. By day 28 post therapy, leukaemia cell load has been reduced but MRD can still be detected

3.1. Methods 3.1.1. PCR of chromosomal aberrations RT-PCR can be applied in 40-45% of childhood precursor B-cell ALLs where 5 main targets for PCR analysis are used (Figure 3) and in 35–40% of adult precursor B-ALL, but is only relevant in 15–25% of T-ALLs. An example of an RT-PCR screen is shown in Figure 4. While standard RT-PCR is useful in establishing the appropriate target for MRD detection, RQ-PCR is the “gold standard” for MRD detection and evaluation. TaqMan probes designed for each of the 5 translocations most commonly seen in childhood ALL allow the kinetics of MRD to be determined by sequential RQ-PCR (Figure 5). 3.1.2. RQ-PCR of junctional regions of Ig and TCR gene rearrangements The variable (V), diversity (D) and junctional (J) regions of both the Ig and TCR genes undergo rearrangement to generate the primary antigen specific repertoire of the immune system. In normal lymphoid cells, there is a huge diversity of possible rearrangements whereas in lymphoid malignancies, the clonal nature of the disease means that cells derived from the precursor leukaemic clone will carry the same Ig or TCR rearrangement. Thus the junction of either region can be used as a target


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Figure 3: Frequency of chromosomal translocations observed in childhood B-cell ALL

The chromosomal translocation and genes involved in each translocation are indicated in the key legend

Figure 4: Molecular screening for translocations in childhood B-ALL

At diagnosis the sample is screened to ensure that the correct translocation as judged by karyotyping is detected. Two primer sets (A, B) which flank the fusion breakpoint are used to amplify material from the diagnostic sample (in the example shown the sample is positive for the E2A-PBX1 fusion gene). In order to confirm positivity, a confirmatory PCR is performed using a nested set of primers (C) located internal to the original PCR primers. Once the confirmatory screen has been performed, samples can be requested for subsequent MRD analysis 260

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Figure 5: Methodology of real-time PCR

Real time quantitative PCR (RQ-PCR) for MRD analysis for TEL-AML1. Amplification of different dilutions of fusion transcript (A) and calculation of the threshold cycle (Ct) for each dilution allows a standard curve to be generated (B) which will permit quantification of MRD levels in the follow-up samples. Equal amounts of RNA are examined at each time point allowing changes in leukaemia burden to be determined (C). Control gene amplification of a house-keeping gene ensures that equal amounts of RNA are being analysed (D)

for PCR based detection systems. Use of consensus PCR primers and subsequent DNA sequencing identifies patient specific sequences, allowing the design of TaqMan probes which can be employed in RQ-PCR assays to assess MRD quantitatively during treatment (Figure 6). As clonal evolution of Ig/TCR gene rearrangements between diagnosis and relapse has been described in 5–30% of cases in different studies, it is essential to employ at least two Ig/TCR targets for each patient being monitored by MRD. The employment of rigorous controls and the use of standardisation protocols (ESG-MRD-ALL) ensure that correct interpretation of MRD data will be performed in different laboratories and allows appropriate comparison


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Figure 6: Design of patient specific clonality marker

g

d

Schema for MRD analysis using clonality makers. The example shown is screening for an IgH clonality marker

of MRD data from different clinical studies, thus allowing robust incorporation of MRD based stratification into clinical protocols. 3.2. MRD detection in ALL Following the initial seminal papers on MRD in childhood leukaemia (5, 6), MRD analysis has been demonstrated to have clinical utility in a number of studies, both in de novo and relapsed ALL as well as in ALL patients undergoing allogeneic HSCT. This has led to the incorporation of MRD analysis into risk stratification in a number of childhood ALL protocols (7). MRD detected at early stages of treatment (< 3 months) reflect the patient’s response to the treatment regimen. A rapid clearance of residual disease is considered predictive of a more favourable outcome. Stratification based on MRD kinetics may allow (i) identification of high-risk disease and patients may require more intensive treatment (ii) identification of patients at low risk of relapse who might benefit from a reduction in treatment, thus reducing toxicity. 262

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3.3. MRD detection in acute myeloid leukaemia (AML) Protocols involving all trans-retinoic acid (ATRA) combined with anthracycline-based chemotherapy lead to cure in approximately 70% patients with PML-RARa-associated acute promyelocytic leukaemia (APL). PCR of the PML-RARa rearrangement can help identify patients who may require additional therapy or patients at low risk who may be spared unnecessary toxicity. As a consequence, molecular monitoring at 3 monthly intervals has been incorporated into a number of treatment algorithms. Based on samples acquired from patients entered in the Spanish PETHEMA protocols RQPCR has also been used to refine the significance of MRD positivity by stratifying patients into those at high, intermediate and low risk of relapse (8). Although the data is not as mature, MRD analysis in patients with t(8;21) and inv16 rearrangements employing both flow cytometry and RQ-PCR in tandem has demonstrated that mean differences in MRD levels between relapsing and non-relapsing patients are statistically significant and cut off levels could be assigned to risk of relapse (9). A number of other potential markers including WTI and nucleophosmin have been identified in acute leukaemia but their clinical relevance requires further study.

4. MRD detection in chronic leukaemias 4.1. MRD detection in chronic myeloid leukaemia (CML) Over 90% of cases of CML are associated with the chromosomal translocation t(9;22), and virtually all patients with CML express BCR-ABL fusion transcripts (e13a2 or e14a2) In initial studies, BCR-ABL fusion transcripts were detected by RTPCR based assays. Nested PCR (where a second round of PCR is performed using primers internal to the primer set used in the first round of PCR) can allow detection of a single BCR-ABL expressing leukaemia cell in 106 normal cells. The quality of RNA is assessed by amplification of a housekeeping gene transcript (e.g. ABL, BCR, Gus, etc). 4.1.1. Predictive value of early and late RT-PCR positivity Until the year 2000, allo-HSCT was the standard method of treating many patients with CML. Early RT-PCR positivity can be detected up to 9 months post allo-HSCT in patients receiving unmanipulated stem cell products in first chronic phase but over time patients usually revert to RT-PCR negativity. Early RT-PCR positivity is not associated with an increased risk of relapse. However early positive RT-PCR results may be relevant for patients who received a T-cell depleted allo-HSCT, and may be used to initiate donor lymphocyte infusions and/or tyrosine kinase inhibitors to prevent frank relapse. Late RT-PCR positivity (≥1 year post HSCT) in both unmanipulated and T-cell depleted allo-HSCT identifies patients at high risk of relapse.


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Serial testing is a prerequisite for using the results of RT-PCR to help guide therapy, as the kinetics of MRD evolution are more relevant than individual positive or negative results. Because of this, quantitative PCR approaches have now become the gold standard in assessing BCR-ABL transcripts in CML (10). Although originally designed to detect early relapse after allo-HSCT these techniques are now being used to assess the depth of response to the tyrosine kinase inhibitors. Quantitation of mRNA by RQ-PCR has been rigorously evaluated in a number of laboratories using either fluorescent-based Taqman or Light Cycler approaches. These approaches, which monitor product accumulation during the extension phase of PCR, are now being used for molecular quantification of MRD in many laboratories (Figure 7). Rising numbers of BCR-ABL transcripts following allo-HSCT precede cytogenetic and haematological relapse and are used as indicators for intervention with approaches such as DLI, which result in long-term disease free and RT-PCR negative remissions. Recent attempts to standardise mRNA quantitation by RQ-PCR for BCR-ABL have involved a multi-centre study which employed a lyophilised preparation of a K562 cell line as a potential quality control reagent (11). Vials were sent to 22 laboratories in 4 continents and results indicated that this QC control could be successfully employed in different laboratories with different PCR instruments, a first step in providing a universal QC reagent for BCR-ABL, RQ-PCR analysis. More recently there has been a worldwide effort to harmonise the methodology and reporting of RT–PCR for BCR-ABL transcripts (12).

Figure 7: Real-time quantification of BCR-ABL expression

Real time (RQ) PCR utilises a DNA hybridisation probe (e.g. a TaqMan probe) in addition to a set of fusion gene specific primers. The probe is constructed such that a fluorescent and a quencher moiety are linked together. In this configuration, no fluorescence is generated. During PCR, as the Taq polymerase extends the annealed primers during the extension phase of PCR, its exonuclease activity cleaves the bond between the reporter and the quencher, thus allowing the fluorescent moiety to be released into solution. During the linear phase of PCR, there is a direct correlation between the amount of target and the amount of fluorescence generated. Collection of this real time PCR data allows accurate quantification of the target, allowing MRD levels to be accurately quantified 264

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4.2. MRD detection in chronic lymphocytic leukaemia (CLL) Until relatively recently research into cellular abnormalities in CLL was hampered by the inability to make human B cells divide. Newer techniques such as FISH molecular analysis and 3–4 colour flow cytometry have allowed the establishment of a number of potential prognostic markers (CD38, Zap 70, somatic hypermutation) and to begin to look for residual disease following treatment. Treatment, with the exception of the few patients receiving allo HSCT, was until recently confined to palliation. With the advent of newer therapeutic regimens including purine analogues (fludarabine), cyclophosphamide and the monoclonal antibody Rituximab, complete response (CR) has become a possibility. The absence of identifiable CLL cells in the blood or bone marrow by flow cytometry confirms a CR and serial examinations for the re-emergence of MRD in the guise of CD5/CD19 + CD20/CD79b neg cells may serve as an indication for further therapy or a change in strategy (Figure 8). Figure 8: B-CLL MRD analysis by flow cytometry


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5. Chimerism following allogeneic HSCT The term chimerism following HSCT refers to the proportion of donor and recipient cells found in the host at any given time following the transport procedure. Proportions of each may change over time and a schema for definitions is given in Table 1.

Table 1: The four distinct categories of Chimerism in the human following allogeneic HSCT Donor Chimerism (DC)

All cells are of donor origin following allogeneic HSCT

Transient Mixed Chimerism (TMC)

A mixed profile with a proportion of cells (typically 1–5%) post HSCT of recipient origin is detected in the first 6 months post transplant; subsequently the patient reverts to donor chimerism

Stable Mixed Chimerism (SMC)

A mixed profile with a proportion of cells (usually 1–20% recipient cells) detected post allogeneic HSCT and remain at a constant level over time

Progressive Mixed Chimerism (PMC)

A mixed profile with a proportion of recipient cells. These recipient cells increase to >10% recipient cells over time

5.1. Techniques to measure chimerism 5.1.1. Cytogenetics Cytogenetic analysis can be performed, provided there is a marker which distinguishes between donor and recipient cells, such as the presence of the Y chromosome in sex mismatched transplants. In addition the presence of a karyotypic abnormality can also serve as a marker. However chimerism studies using cytogenetic analyses are compromised by low sensitivity and the need for dividing cells. Use of fluorescent in situ hybridisation (FISH) analysis can increase sensitivity and eliminates the need for dividing cells but is probably only of real benefit in the sex mismatched transplant setting. 5.1.2. DNA based techniques Southern blotting techniques using either single copy or minisatellite probes were initially used to detect post-transplant chimerism. While these techniques allow some degree of assessment of virtually all allo-HSCT, the relative lack of sensitivity and the laborious nature of the techniques has prevented their widespread use. Most 266

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laboratories now use PCR based techniques, exploiting polymorphisms in DNA to distinguish between donor and recipient cells. Variable number tandem repeats (VNTRs) can be amplified to allow size differences between donor and recipient cells to be used in PCR-based post-transplant chimerism analysis but the most widely used approach involves PCR of short tandem repeats (STR) (Figure 9). The Eurochimerism Concerted Action, a consortium of leading laboratories in this field from 10 European Countries, has developed a Eurochimerism marker panel (EUR-STR) and

Figure 9: STR DNA polymorphisms to evaluate chimerism after allo-HSCT -HSCT

Select informative polymorphism(s)

Study post-HSCT pattern

Short tandem repeat PCR (STR-PCR) exploits the presence of small repeated sequences of DNA which can vary in the number of repeats between donor and recipient. An example of 2 types of STR is shown (A) a dinucleotide CA repeat and (B) a tetranucleotide TTTA repeat Variation in repeat length between donor and recipients allows post transplant chimerism to be assessed. A panel of STR markers is first used to identify informative polymorphisms (C) which are then used to monitor samples from the recipient post HSCT (D). In this example an initial period of donor chimerism is followed by re-emergence of recipient cells and disease relapse. Samples can be electrophoresed through a gel matrix as indicated or by capillary electrophoresis. Activation of the fluorescent primers by an argon laser using for example a 310 or 3100 DNA sequence detector (Applied Biosystems) allows quantitation of donor and recipient profiles. D: Donor Profile; R: Recipient profile; 1-5: Serial chimerism profiles post-HSCT; O: Operator profile (to control for contamination)


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has shown its application to quantitative chimerism analysis. Furthermore it has established the allele frequencies of the EUR-STR panel in different European populations, has standardised the nomenclature and has developed DNA extraction methodologies for chimerism analysis (www.eurochimerism.org). Chimerism techniques employ a fluorescent based PCR methodology providing a suitable format for application in clinical laboratories. 5.2. Significance of chimerism analysis following allo-HSCT “Classic” allo-HSCT is preceded by myeloablative conditioning regimens, the aim of which is the complete destruction of host haematopoiesis. In patients with leukaemia, the re-emergence of cells of host origin frequently heralded leukaemia relapse, although rarely normal host cells may partially repopulate host haematopoiesis. Transient mixed chimerism (TMC) may occur in the first 6–9 months post transplant, particularly following matched unrelated donor HSCT (MUD-HSCT) and may be associated with a lower risk of GvHD. Since the advent of reduced intensity stem cell transplantation (RIC-HSCT) the relevance of serial chimerism analysis has been re-stabilised. The philosophy of RICHSCT is to reduce the potency of the conditioning therapy to reduce toxicity and not to attempt myeloablation. This theoretically results in a period of mixed haematopoietic chimerism in the host following infusion of donor stem cells. Over time, donor cells will predominate and exercise their graft versus leukaemia effect (GvL) (Figure 10). In the presence of persistent or progressive MC, DLI is performed in an attempt to establish donor chimerism, albeit at the risk of precipitating GvHD. Chimerism in these recipients is best evaluated by the use of lineage specific analysis. Selection of appropriate cell populations (T-cells, myeloid cells etc) is first performed and the individual populations are subsequently subjected to chimerism analysis. Achievement of complete donor chimerism in the T-cell lineage may be of particular relevance to the long-term success of this approach. Chimaeric analysis therefore is useful to follow the success of donor lymphocyte infusions (DLI) for relapsed leukaemia (especially CML) or when DLI is used to convert mixed chimaeras into donor chimaeras following RIC-HSCT (13). 5.2.1. Chimerism analysis in severe aplastic anaemia (SAA) Although an extremely successful therapy for SAA, allo HSCT in this setting is significantly different from acute or chronic leukaemia. Current conditioning regimens are not myeloablative (cyclophosphamide alone or with ATG). The presence of mixed chimerism therefore is common and stable mixed chimerism (SMC) is associated with a good outcome and absence of GvHD. Emergence of progressive mixed chimerism (PMC) usually precedes disease relapse/graft rejection and carries 268

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Figure 10: Chimerism after a non-myeloablative stem cell transplant

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Shows recipient (a) and donor (b) STR profiles prior to NST. Following reduced intensity conditioning a mixed chimaeric state is common (c). Overtime, this may progress to full donor chimerism (d). Persistent mixed chimerism is an indication for DLI (e) to convert the patient to a full donor chimaera (d)


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a poor prognosis. In contrast to the leukaemias, allo HSCT for SAA may be followed by graft rejection. This may be early, when there is no evidence of donor engraftment, or may occur later following transient donor engraftment with a subsequent rise in host cells and graft rejection which may be interpreted as disease relapse. Reduction in exposure to multiple red cell and platelet transfusions and newer treatment regimens have reduced the graft rejection rate to 2 yrs) even better values than after BM grafts (2). Post-HSCT events

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May in turn have a worsening effect on IR, especially aGvHD and cGvHD, relapse and infectious complications (EBV or CMV viruses, fungal infections, toxoplasmosis) either directly or through drugrelated side effects.


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CHAPTER 15 • Immune reconstitution and allo-HSCT

4. Assessment of B-cell reconstitution 4.1. B-lymphocyte phenotyping with B-lineage markers (CD19, CD20, CD21) and activation or differentiation markers (CD5, CD27) CD19+ B-cells normalise by one year after transplant. B-cell regeneration may be associated with transient appearance of monoclonal B-cell expansions. 4.2. Quantification of serum total IgG, IgM and IgA and of IgG subclasses After a decline in the first few months after HSCT, levels of specific antibodies to protein Ag frequently encountered after transplantation (e.g. CMV) return to pretransplantation levels within 1 yr. In contrast, antibodies to protein Ag that are unlikely to be encountered after HSCT (e.g. tetanus, measles, polio) continue to decline. This supports the recommendation of post-HSCT vaccination. Antibody levels in the first year, are affected primarily by pre-HSCT antibody levels in the recipient (3). A persistent defect in IgA, especially in patients with cGvHD explains mucosal infections of the respiratory and digestive tracts. IgG2 and IgG4 subclasses are also deficient in the case of GvHD, accounting for the increased susceptibility to infections, primarily those due to encapsulated bacteria (e.g. Streptococcus pneumoniae or Haemophilus influenzae). PBSC recipients do not have higher antibody levels than BM recipients. 4.3. Vaccinations Vaccinations with inactivated or conjugated vaccines (see Chapter 10) should be initiated when CD4 and B-lymphocyte counts are sufficient to expect efficacy, usually from 6 months post-transplant onwards.

5. NK-cell reconstitution NK-cells are lymphocytes that act early in the immune response against infection and tumour-transformed cells. Based on phenotyping (CD16 and CD56), they are the first lymphocyte subpopulation to be reconstituted in all graft settings, usually within 3 months. The genetic organisation and function of NK receptors, either inhibitory or activating, has been unravelled in the past few years. NK-cell receptors are encoded by 2 structurally distinct families of molecules: The killer immunoglobulin-like receptors (KIR) and the lectin-like CD94:NKG2 heterodimers. Every NK-cell expresses at least one inhibitory receptor specific for autologous HLA Class I, thereby ensuring selftolerance. As KIR and HLA segregate independently and as unrelated individuals almost always have different KIR genotypes, we predict that approximately 25% of


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transplants between HLA-identical siblings involve KIR identity and approximately 75% KIR disparity. For transplantation with an HLA-MUD, the frequency of KIR incompatibility approaches 100%. In the haploidentical TCD graft setting, a beneficial effect of KIR disparity on GvL has been evidenced (4). This is an important finding which needs to be extended to other types of grafts and which may change our current criteria of donor/recipient matching based on HLA compatibility (see Chapter 3). There are still few studies directly assessing NK reconstitution at the level of KIR and lectin-like NK receptors expression level and function (5). This study and others showed that CD94:NKG2A may be expressed earlier than KIR and that most patients reconstitute a donor-type NK repertoire depending on their KIR genotype. Different NK subsets are now more precisely defined and especially the CD56brightCD16and CD56dimCD16+, respectively prone to cytokine production or cytotoxicity. The rapid recovery of NK-cells after graft is due to an expansion of CD56brightCD16-. The precise role of this NK subset in GvHD and GvL is a key issue in HSCT.

6. T-cell reconstitution 6.1. Naïve and memory T-cells Memory T-cells are the first to expand after HSCT; they may be either of donor origin in the case of a non-TCD BM or, in the case of a TCD, originate from host T-cells that have survived the conditioning regimen (6). They respond quickly to previously encountered pathogens, are easier to trigger, faster to respond and enter tissues more readily than naïve T-cells. They are frequently directed towards periodically reactivated herpes viruses, CMV or EBV, which they keep under control. They constitute the majority of oligoclonal T-cell expansions found in healthy adults, especially in the CD8+ population. They are also less dependent than naive T-cells upon recognition of self MHC-peptide complexes in their survival and expansion in the periphery. In the long term, broad immune responses need the reconstitution of a naïve T-cell repertoire able to respond to a broad range of pathogens encountered by the host and to tumour antigens. Reconstitution of this compartment is an ongoing process which requires a functional thymus for the recovery of a complete T-cell ontogeny. The thymus itself may be a target of the alloreactive immune attack with possible consequences on thymic selection, escape of self-reactive T-cell clones and perpetuation of GvHD. This has been well documented in animal models (7) and deserves more insight in humans. 6.2. How to evaluate naïve and memory T-cell populations? The current immunological tests assess naïve and memory lymphocyte populations: 300

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CD45RO for memory T-cells and CD45RA or CD62L for naïve T-cells are the most usual markers. However, naïve T-cells may undergo expansion without phenotypic changes and have a long lifespan, up to 20 yrs. In addition, memory CD45RO+ T-cells may revert to a naive CD45RA+ phenotype, especially in case of persistent infection with herpes viruses. Therefore, other markers should be added to definitely assess naïve T-cells and the different categories of memory T-cells. CCR7, a molecule involved in the homing of T-cells to lymph nodes is especially valuable. A combination of these markers allows the definition of: - Naïve T-cells: CD45RAhighCD45RO-CCR7+CD28+ - 2 populations of CD8+CD45RA- memory cells: - CCR7+ “central memory”, expressing L-selectin (CD62L) - CCR7- “effector memory”, L-selectin-, IL-2 dependent, which migrate to inflammatory sites and secrete IFN-g. T-cell diversity (“T-cell repertoire”) and thymic function can be directly evaluated. The size of the T-cell repertoire and the extent of T-cell diversity has been measured only recently, the value of about 25 x 106 different TCR ab complexes being lower than that was previously estimated. T-cell diversity is contributed by the naïve population and in healthy adults memory T-cells, which account for approximately 1/3 of the total T-cells, contribute to less than 1% of the abT-cell diversity. A practical consequence for HSCT is that evaluation of T-cell repertoire diversity reflects the extent of the naïve T-cell compartment. Various approaches known as “Immunoscope” or “spectratyping” may be used. They are based on the size diversity analysis of the CDR3 b-chain region as an index of the diversity of the whole abT-cell population. T-cell repertoire diversity in allo-HSCT recipients is a function of: - Number and diversity of infused T-cells with the graft (TCD, age of the donor, source of graft) - Residual T-cells present in the recipient - Thymic pathway of regeneration, for which the age of the recipient is the main factor - Immunosuppressive treatment and complications (GvHD, viral infections). Overall, early after HSCT (within 6 months after graft) many abnormalities of the T-cell repertoire are demonstrable but are difficult to correlate with the clinical status of the patient. Conversely, later after the graft (after 1 yr at least) and ongoing for at least 2 to 3 yrs post-transplant, it is possible to correlate repertoire disturbance with the occurrence of GvHD, severe infectious complications or relapse. T-cell repertoire reconstitution is delayed in case of TCD or in CD34+ purified grafts and is improved where there is full donor haematopoiesis. Techniques of TCR b-chain sequencing have clearly separated T-cell clones mediating GvHD and GvL and could be used in the future to monitor GvHD-causing clones in HSCT recipients (8). HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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The reconstitution and maintenance of a diverse repertoire in the peripheral lymphocyte pool is dependent on the generation of functional thymocytes throughout adult life. These recent thymic emigrants can now be evaluated by measuring the episomal DNA excision circles of the TCR d locus deleted during recombination of the a locus in all functional ab T-cells (known as “TREC” for T-cell receptor rearrangement excision DNA circles). This ex vivo marker of thymic function has been used in allo-HSCT monitoring: - TREC levels are low until 3–6 months after allo-BMT. Low TREC values are associated with increasing patient age and TCD but mainly with GvHD (9), leukaemia relapse or opportunistic infections - High TREC levels and a broad T-cell repertoire have been associated with an efficient IR after CB transplantation in the long term (2) although there is some delay in IR in that setting (10). The thymic function of the recipient before graft could be associated with a more favourable outcome in terms of survival, GvHD and bacterial or viral infections (11). It could be a valuable prognostic factor predicting IR after transplant. 6.3. How is the Ag-specific immune response reconstituted after allo-HSCT? Naïve T-cell reconstitution is a key issue for the long-term recovery of immune responses but memory T-cells are also needed for an efficient and timely response towards pathogens. Therefore, especially in some graft settings (TCD, CBT, HLA mismatch UD) adoptive immunotherapy can be used in an attempt to compensate for the lack of Ag specific immunocompetent T-cells. In order to do this, it is necessary to evaluate patients at risk and to be able to monitor Ag specific immune responses towards pathogens. Herpes viruses (CMV, EBV) are of primary importance in HSCT because reactivation of EBV can result in potentially fatal EBV-associated lymphoproliferative disease and because of the frequency of late CMV reactivation in the host even under pre-emptive therapy. It is possible: - To monitor EBV and CMV-specific cytotoxic responses by Elispot functional assays or intracellular cytokine staining which are easier to perform than the conventional 51Cr release cytotoxic assay in a routine laboratory - To use the tetramer technology to stain directly ex vivo CD8+ T-cells reactive with peptide/HLA complexes and to characterise these cells in terms of phenotype and function. This is a very sensitive method which can stain 1/5000 CD8+ T-cells (or 1/5 x 104 PBMC). It may also be used to isolate Ag-specific T-cells by cell sorting and to expand them in vitro. It is becoming a routine laboratory analysis for CMV (12) and EBV (13) specific CD8+ T-cell responses.

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7. From monitoring to immune intervention The combination of these various structural (Immunoscope for TCR diversity analysis, TREC for thymic function) and functional approaches (Elispot, tetramer staining) should enable more precise immune monitoring of patients at risk of relapse or persistent severe infectious complications, particularly in the context of adoptive immunotherapy. The tetramer approach has direct benefits for the identification of patients with impaired CD8+ specific cytotoxic responses who may be eligible for cellular adoptive immunotherapy against CMV (14) or for CD20-specific MoAb treatment (rituximab) to prevent post-HSCT lymphoproliferative disorder during EBV reactivation (13). The transfer of viral-specific CTL is possible with tetramer-sorted CTL without culture (15). Based on experimental models, other attempts could be pursued to improve IR and graft outcome: - Improve thymic function recovery, by growth factors such as KGF, Flt3l or androgen blockade - Selective depletion of alloreactive T-cells from donor lymphocytes (16) - Use of minor histocompatibility Ag as tumour Ag to mediate GvL effect, as described for HA-1 minor antigen (17) - Genetic modification of T-lymphocytes with TK suicide gene (18) - Haploidentical NK immunotherapy (19) - Immune modulation through alternative stem cell sources (mesenchymal stem cells), dendritic cells or Treg manipulation. Manipulation of immune system homeostasis to facilitate the emergence of regulatory CD4+CD25high T-cells (or Treg) which have been shown in animal models to control GvHD without impairing GvL (20). In humans, although evidence for the role of Treg has been less clear, it appears that an in situ defect in these populations could be associated with aGvHD (21). Importantly, Treg could be expanded in vitro and keep their functional properties, thus being an approach of choice in the future for controlling GvHD.

Acknowledgments This work was supported by research grants from the Cancéropôle Ile-de-France, EC programs EUROBANK, EUROCORD and FP6 ALLOSTEM (#503319).

References 1. Storek J, Dawson MA, Storer B, et al. Immune reconstitution after allogeneic marrow transplantation compared with blood stem cell transplantation. Blood 2001; 97: 3380-3389.


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2. Talvensaari K, Clave E, Douay C, et al. A broad T-cell repertoire diversity and an efficient thymic function indicate a favorable long-term immune reconstitution after cord blood stem cell transplantation. Blood 2002; 99: 1458-1464. 3. Storek J, Viganego F, Dawson MA, et al. Factors affecting antibody levels after allogeneic hematopoietic cell transplantation. Blood 2003; 1001: 3319-3324. 4. Farag SS, Fehniger TA, Ruggeri L, et al. Natural killer cell receptors: New biology and insights into the graft-versus-leukemia effect. Blood 2002; 100: 1935-1947. 5. Shilling HG, McQueen KL, Cheng NW, et al. Reconstitution of NK cell receptor repertoire following HLA-matched hematopoietic cell transplantation. Blood 2003; 101: 3730-3740. 6. Roux E, Dumont-Girard F, Starobinski M, et al. Recovery of immune reactivity after T-celldepleted bone marrow transplantation depends on thymic activity. Blood 2000; 96: 2299-2303. 7. Hauri-Hohl MM, Keller MP, Gill J, et al. Donor T-cell alloreactivity against host thymic epithelium limits T-cell development after bone marrow transplantation. Blood 2007; 109: 4080-4088. 8. Michalek J, Collins RH, Hill BJ, et al. Identification and monitoring of graft-versus-host specific T-cell clone in stem cell transplantation. The Lancet 2003; 361: 1183-1185. 9. Weinberg K, Blazar BR, Wagner JE, et al. Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood 2001; 97: 1458-1466. 10.Komanduri KV, St John LS, de Lima M, et al. Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T cell skewing. Blood 2007; 110: 4543-4551. 11.Clave E, Rocha V, Talvensaari K, et al. Prognostic value of pretransplantation host thymic function in HLA-identical sibling hematopoietic stem cell transplantation. Blood 2005; 105: 2608-2613. 12. Aubert G, Hassan-Walker AF, Madrigal JA, et al. Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogenic stem cell transplants. J Infect Dis 2001; 184: 955-963. 13.Clave E, Agbalika F, Bajzik V, et al. Epstein-Barr virus (EBV) reactivation in allogeneic stemcell transplantation: Relationship between viral load, EBV-specific T-cell reconstitution and rituximab therapy. Transplantation 2004; 77: 76-84. 14.Peggs KS, Verfuerth S, Pizzey A, et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines. The Lancet 2003; 362: 1375-1377. 15.Cobbold M, Khan N, Pourgheysari B, et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. J Exp Med 2005; 202: 379-386. 16.Amrolia PJ, Muccioli-Casadei G, Huls H, et al. Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation. Blood 2006; 108: 1797-1808. 17. Mutis T, Blokland E, Kester M, et al. Generation of minor histocompatibility antigen HA-1specific cytotoxic T cells restricted by nonself HLA molecules: A potential strategy to treat relapsed leukemia after HLA-mismatched stem cell transplantation. Blood 2002; 100: 547-552. 304

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18.Bondanza A, Valtolina V, Magnani Z, et al. Suicide gene therapy of graft-versus-host disease induced by central memory human T lymphocytes. Blood 2006; 107: 1828-1836. 19.Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: 2097-2100. 20.Edinger M, Hoffmann P, Ermann J, et al. CD4+ CD25+ regulatory T cells preserve graftversus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nature Medicine 2003; 9: 1144-1150. 21.Rieger K, Loddenkemper C, Maul J, et al. Mucosal FOXP3+ regulatory T cells are numerically deficient in acute and chronic GvHD. Blood 2006; 107: 1717-1723.

Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1.

After allo-HSCT the earliest lymphocyte population(s) to recover: a) NK lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) T CD4+ naïve T-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) T CD8+ memory T-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) All at the same time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.

Among the following lymphocyte phenotypic marker(s), which one is the most precise to define memory T-cells: a) CD45RA+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) CD45RO+ CCR7- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) CD45RA+ CCR7+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) CD16+ CD56+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.

Which is the main factor directly affecting thymic recovery after allo-HSCT? a) Sex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) CMV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) HLA mismatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) GvHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.

After allogeneic HSCT, the risk of EBV-induced proliferative disease (PTLD) is especially increased in case of which one of the following: a) T-cell depletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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b) Genoidentical sibling donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Sex-mismatch between donor and recipient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Aplastic anaemia as primary disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Among the following lymphocyte phenotypic marker(s), which is the most precise to define naïve T-cells: a) CD45RA+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) CD45RO+ CCR7- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) CD45RA+ CCR7+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) CD16+ CD56+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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*

CHAPTER 16

Psychosocial aspects of HSCT

A. Kiss, M. Kainz

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CHAPTER 16 • Psychosocial aspects of HSCT

1. Introduction HSCT has moved from an experimental treatment to become an accepted therapy. At the same time, the primary focus on increasing survival in previously lethal diseases has been enlarged to encompass psychosocial issues such as quality of life. In this Chapter, specific psychosocial aspects of the patient, the donor, the family, and the transplant team will be discussed in relation to the time trajectory of HSCT shown in Figure 1.

Figure 1: Trajectory of HSCT

Diagnosis Decision to transplant Donor search

HSCT In-hospital treatment Side effects, Toxicity Engraftment

Short-term follow-up Frequent controls Tx-related mortality GvH disease

Long-term follow-up Quality of life Return to "normal life" Relapse

2. Psychosocial aspects - the patient 2.1. Psychosocial morbidity The diagnosis of a deadly disease and the option of a treatment that may cure but which also has potentially lethal side effects puts the patient under heavy pressure and adjustment is difficult. Psychosocial morbidity is frequent, particularly adjustment disorders with symptoms of depression and anxiety. Approximately one third of patients report significant symptoms of intrusive and avoidance stress responses (1). The search for a suitable related or UD in allo-HSCT is a hard time for patients, for fear of not finding a donor. Coping mechanisms vary according to individual patients from fighting spirit to hopelessness and helplessness. Psychosocial evaluation systems such as the Transplant Evaluation Rating Scale (TERS) (2) have been proposed for assessing the psychosocial functioning of HSCT recipients, which could then allow early intervention in case of poor functioning. Psychiatric morbidity prolongs hospital stay independently of in-hospital somatic risk factors (1) and pre-transplant physical and mental functioning is strongly associated with self-reported recovery from stem cell transplantation (3). The greatest emotional distress occurs after admission to hospital and before the transplantation. Anxiety and depression decrease one week after the transplant. Psychosocial well-being after the transplant is heavily influenced by mucositis, toxicity, and other side effects, but conversely psychological factors such as anxiety, BMT-related distress, and social support also have a significant impact


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on how severely patients experience their mouth pain. Beside the usual medical treatments, psychosocial interventions such as hypnosis and muscle relaxation can substantially reduce nausea and pain. After discharge, many patients are disappointed by their low energy level (fatigue), high susceptibility to infections and the very slow return to normal life. In the first year after transplant psychological distress declines (4). However, elevated levels of anxiety and depression prior to transplantation predict more anxiety and depression in the follow-up period (4). Somatic risk factors for outcome after HSCT are well described, and include factors relating to the patient, the donor and the type of transplant. However coping strategies assessed pre-transplant such as emotional support, acceptance, taking control, and compensation also seem to have an influence. Compensation is reported to be associated with shorter, the other strategies with longer survival. Replication of the data is essential before clinical recommendations can be made (5). Patients suffering from cGvHD have lower QoL and may therefore be more vulnerable to depression and anxiety disorders. At long-term follow-up, some survivors still have to cope with a low energy level, some with fear of losing their job, and all have to deal with infertility and the fear of relapse and secondary malignancies. At 3 yrs post transplantation 80% of women and 29% of men report sexual problems (6). 2.2. Quality of life (QoL) Good QoL in HSCT has been reported repeatedly. However, it should be kept in mind that QoL is a poorly defined concept and instruments professing to measure QoL measure different things. The approaches that are most frequently used are shown in Table 1.

Table 1: Different approaches to the measurement of QoL in HSCT Approach

Examples

Generic

SF-36 (Medical Outcomes Survey - Short Form 36; EORTC QLQ-C30 (European Organisation for Research and Treatment of Cancer Core Quality of Life questionnaire), FACT (Functional Assessment of Cancer Therapy)

Disease-specific Leukaemia/BMT module of EORTC QLQ-C30, FACT-BMT (Bone Marrow Transplantation)

310

Utilitarian

TTO (Time Trade-Off)

Individualised

SEIQoL-DW (Schedule for the Evaluation of Individual QoL - Direct Weighting)

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A standardised multidimensional questionnaire (physical, psychological, functional and social dimensions) is often used in combination with a disease-specific module (7). Recently, a high-dose chemotherapy questionnaire module to supplement the European Organisation for Research and Treatment of Cancer Core Questionnaire (EORTC QLQ-C30) has been developed (8). The advantage of a generic approach is to obtain a numeric figure for a specific item, which can be used to assess the impact on QoL across different diseases or to assess changes in the same patient over time. However, the individual perception of a given symptom is neglected by these questionnaires: e.g. not being able to climb two flights of stairs may represent a substantial impairment for one patient but may have no impact on another patient. The utilitarian approach has only been used in solid organ transplantation but not for HSCT, as far we know. In this approach it is left to the patient to decide the areas in which the health-related QoL is compromised, as well as the severity of impairment. However, no information about the characteristics of the compromised dimension is given. An individualised approach not only lets the patient define the specific domains which are most important for his or her QoL, but also allows the patient to weight the importance of these domains individually (9). Such areas included positive aspects, e.g. a changed view of life and oneself (10). Cancer-related fatigue is one of the biggest constraints of QoL after HSCT. The majority of studies are cross-sectional and restricted to short follow-up. Compared to normal controls, HSCT survivors at a mean of 7 years after transplantation reported poorer physical, psychological, and social functioning but, conversely, more psychological and interpersonal growth, differences that appeared to persist many years after HSCT (11). In a study in survivors 10 years after HSCT, health problems were not focused on specific diseases or limited to survivors with readily identifiable risk factors. Musculoskeletal problems were frequent. Survivors require screening for sexual problems, urinary frequency, mood and need for antidepressants or benzodiazepines (12). However, it should be kept in mind even if QoL is measured more frequently nowadays, the data are often not understandable for the physician involved and therefore not used in the decision-making process with the patient (13). 2.3. Neglected issues 2.3.1. The patient with delirium Half of patients who undergo HSCT experience an episode of delirium during the 4 weeks post transplantation. Symptoms of delirium are poorly recognised by professionals because of the variability in the symptoms of confusion over the 24-


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hour period and also the patients’ tendency to dissimulate, as they do not want to be identified as “mad”. If symptoms are recognised there is a tendency to label them as difficulty in coping with an unbearable situation rather than a diagnosis of delirium. Transplant physicians are often inexperienced in the treatment of delirium, which can be successfully managed with a low dose of narcoleptics. Compared to patients without delirium, patients who experience delirium during myeloablative HSCT showed impaired neurocognitive abilities and persistent distress 80 days after transplantation (14). The long-term effect of delirium post HSCT remains to be determined. 2.3.2. The dying patient A substantial number of patients die despite HSCT and they represent the primary sources of stress in nurses and doctors working on a transplant unit (15). In a recent study about Advanced Care Planning (ACP) in HSCT patients, those patients least likely to have planned for poor outcomes were the ones most likely to face them (16). 2.3.3. The non-compliant patient Non-compliance in patients transplanted for solid organs is one the main factors of transplant failure. The prevalence of non-compliance in HSCT recipients is unknown (17). Given the complex medical regimen and the strict dietary and behavioural rules after transplantation, the medical team may incorrectly ascribe poor therapeutic outcomes to inadequacies in the regimen instead of non-compliance of the patients. They may prescribe more potent medicine with the potential for greater adverse reactions (17). Poverty is a potential factor in non-compliance that is often neglected: In the US underinsured/poor HSCT recipients have the same mortality during inpatient treatment as the non-poor, but a significantly higher mortality in the following 100 days. The authors hypothesise that the higher mortality is due to the poor patients’ inability to comply with or seek medical care because of deficient socioeconomic resources (18).

3. Psychosocial aspects - the donor 3.1. Related donor In contrast to living solid organ donors little is known about the decision-making process of HSC donors. Most donors do not even recognise a decision-making process, they have “no choice”, they just want to help the recipient (19). They believe the psychological aspects of the procedure outweighs the physical aspects of 312

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donation (19). Siblings of unsuccessful transplant recipients may feel guilty for their “bad marrow”, some donors have problems in coping, and difficulties within the affected families are not uncommon. 3.2. Unrelated donors Medical advances have made possible HSCT from UD. Many donor registries have been established in recent years. The motivations of UD donors may be different from related donors, as they do not know the recipients. Higher levels of ambivalence about BM donation are associated with joining during a recruitment drive for a specific patient, perceiving the recruitment staff as less informative, being discouraged from joining by others and not having an intrinsic commitment to donate (20). Unrelated female donors are mostly motivated by positive feelings, empathy and the desire to help someone.

4. Psychosocial aspects - the family The most significant social support for HSCT recipients comes from their family. Family members experience the similar distress as do the patients and report more impairments in family relationship than patients (21). Physical and emotional recovery after HSCT depends on the quality of family relationships as perceived by the recipient. They seem to have the most important role as a filter for stress. In a recent study in HCT survivor/partner pairs (n=177) spouses/partners experience similar emotional and greater social long-term costs of cancer and HCT than survivors without the potential compensatory benefits of post-traumatic growth (22).

5. Psychosocial aspects - the transplant team 5.1. Present and future of the transplant team There is great economic pressure for transplant teams to carry out more transplant procedures in less time with the same or even reduced staff. Earlier discharge and frequent unscheduled readmission may result. 5.2. Psychosocial well-being of team members The very nature of HSCT is characterised by the dominance of technology and the rapidity of decision-making and practice. Death is considered as a failure and is often due to the toxicity of the procedure itself or infections (23). Both doctors and nurses consider regular work with dying patients as the primary source of stress, besides other stressors such as interpersonal staff conflicts, excessive responsibilities and


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highly demanding patients and families (15). The death of a patient may be considered as a mismatch of high investment (high costs, time, emotional labour) and low returns (causing suffering without survival). Burnout in nursing and medical staff is high, half of them being emotionally exhausted, and 80% reporting feelings of low personal accomplishment (15). Doctors and nurses reported that the most frequent effects from prolonged stress were increased illness, reduced productivity and increased clinical errors. 5.3. Care for/of the team As the care of the patient is the primary focus for transplant teams, their own needs for care is are often denied and seldom addressed. Burnout must be considered as a permanent danger and not as an individual problem of the team member concerned. Time and resources have to be devoted to the care of the team as a prophylactic measure and not only after a team member has cracked under the pressure.

6. Personal conclusions and practical applications Psychosocial issues in patients, donors, families and transplant teams are not “soft data” but have a substantial impact on the morbidity of all persons involved and probably on the survival of patients. Communication and psychosocial skills are core competencies for doctors and nurses. They cannot be delegated to mental health professionals. Evidence-based training in these skills is available but seldom used by transplant teams. “The availability of specialised psychosocial care is necessary and, as with medical treatment, it should be carried out by specially trained staff” (24, 25). Every transplant unit must have a mental health professional who is a team member of the transplant team. A consulting psychiatrist coming solely on request as demanded by the present JACIE accreditation procedure is not sufficient. The tasks of a mental health professional regularly working in the transplant unit and its outpatient department is to care for individual patients and their families, and to support the transplant team.

References 1. Prieto JM, Blanch J, Atala J, et al. Psychiatric morbidity and impact on hospital length of stay among hematologic cancer patients receiving stem-cell transplantation. J Clin Oncol 2002; 20: 1907-1917. 2. Hoodin F, Kalbfleisch KR. Factor analysis and validity of the Transplant Evaluation Rating Scale in a large bone marrow transplant sample. J Psychosom Res 2003; 54: 465-473. 3. Andorsky DJ, Loberiza FR, Lee SJ. Pre-transplantation physical and mental functioning is strongly associated with self-reported recovery from stem cell transplantation. Bone 314

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Marrow Transplantation 2006; 37: 889-895. 4. Hjermstad MJ, Loge JH, Evensen SA, et al. The course of anxiety and depression during the first year after allogeneic or autologous stem cell transplantation. Bone Marrow Transplantation 1999; 24: 1219-1228. 5. Grulke N, Bailer H, Hertenstein B, et al. Coping and survival in patients with leukemia undergoing allogeneic bone marrow transplantation - long term follow-up of a prospective study. J Psychosom Res 2005; 59: 337-346. 6. Syrjala KL, Roth-Roemer SL, Abrams JR, et al. Prevalence and predictors of sexual dysfunction in long-term survivors of marrow transplantation. J Clin Oncol 1998; 16: 31483157. 7. Kopp M, Schweigkofler H, Holzner B, et al. EORTC QLQ-C30 and FACT-BMT for the measurement of quality of life in bone marrow transplant recipients: a comparison. Eur J Haematol 2000; 65: 97-103. 8. Velikova G, Weis J, Hjermstad MJ, et al. The EORTC QLQ-HDC29: a supplementary module assessing the quality of life during and after high-dose chemotherapy and stem cell transplantation. Eur J Cancer 2007; 43: 87-94. 9. Frick E, Borasio GD, Zehentner H, et al. Individual quality of life of patients undergoing autologous peripheral blood stem cell transplantation. Psychooncology 2004; 13: 116-124. 10.Wettergren L, Sprangers M, Bjorkholm M, Langius-Eklöf A. Quality of life before and one year following stem cell transplantation using an individualized and a standardized instrument. Psychooncology 2007 Jul 5 [Epub ahead of print]. 11.Andrykowski MA, Bishop MM, Hahn EA, et al. Long-term health-related quality of life, growth, and spiritual well-being after hematopoietic stem-cell transplantation. J Clin Oncol 2005; 23: 599-608. 12.Syrjala KL, Langer SL, Abrams JR, et al. Late effects of hematopoietic cell transplantation among 10-year adult survivors compared with case-matched controls. J Clin Oncol 2005; 23: 6596-6606. 13.Lee SJ, Joffe S, Kim HT, et al. Physicians’ attitudes about quality-of-life issues in hematopoietic stem cell transplantation. Blood 2004; 104: 2194-2200. 14.Fann JR, Alfano CM, Roth-Roemer S, et al. Impact of delirium on cognition, distress, and health-related quality of life after hematopoietic stem-cell transplantation. J Clin Oncol 2007; 25: 1223-1231. 15.Molassiotis A, van den Akker OB, Boughton BJ. Psychological stress in nursing and medical staff on bone marrow transplant units [published erratum appears in Bone Marrow Transplant 1995 Aug; 16: 328]. Bone Marrow Transplantation 1995; 15: 449-454. 16.Ganti AK, Lee SJ, Vose JM, et al. Outcomes after hematopoietic stem-cell transplantation for hematologic malignancies in patients with or without advance care planning. J Clin Oncol 2007; 25: 5643-5648. 17.Bishop MM, Rodrigue JR, Wingard JR. Mismanaging the gift of life: Noncompliance in the context of adult stem cell transplantation. Bone Marrow Transplantation 2002; 29: 875-880. 18.Selby GB, Ali LI, Carter TH, et al. The influence of health insurance on outcomes of relateddonor hematopoietic stem cell transplantation for AML and CML. Biol Blood Marrow


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Transplant 2001; 7: 576. 19.MacLeod KD, Whitsett SF, Mash EJ, Pelletier W. Pediatric sibling donors of successful and unsuccessful hematopoietic stem cell transplants (HSCT): A qualitative study of their psychosocial experience. J Pediatr Psychol 2003; 28: 223-230. 20.Switzer GE, Myaskovsky L, Goycoolea JM, et al. Factors associated with ambivalence about bone marrow donation among newly recruited unrelated potential donors. Transplantation 2003; 75: 1517-1523. 21.Siston AK, List MA, Daugherty CK, et al. Psychosocial adjustment of patients and caregivers prior to allogeneic bone marrow transplantation. Bone Marrow Transplantation 2001; 27: 1181-1188. 22.Bishop MM, Beaumont JL, Hahn EA, et al. Late effects of cancer and hematopoietic stemcell transplantation on spouses or partners compared with survivors and survivor-matched controls. J Clin Oncol 2007; 25: 1403-1411. 23.Futterman AD, Wellisch DK. Psychodynamic themes of bone marrow transplantation. When I becomes thou. Hematol Oncol Clin North Am 1990; 4: 699-709. 24.Fallowfield L, Jenkins V, Farewell V, et al. Efficacy of a Cancer Research UK communication skills training model for oncologists: A randomised controlled trial. Lancet 2002; 359: 650-656. 25.Gratwohl A, Apperley JF, Gluckman E, (eds). The EBMT Handbook. Blood and Marrow Transplantation. European School of Haematology; European Blood and Marrow Transplantation, 2000.

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CHAPTER 17

Indications and current practice for allogeneic and autologous HSCT for haematological diseases, solid tumours and immune disorders

P. Ljungman, A. Gratwohl for the European Group for Blood and Marrow Transplantation

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CHAPTER 17 • Indications and current practice

1. Introduction The EBMT has since 1996 regularly published special reports on the indications for and current practice of haematopoietic stem cell transplantation (HSCT) in Europe for congenital or acquired haematological diseases, solid tumours, immune disorders and inborn errors of metabolism (1–4). Major changes have occurred since the first report was published. Today, approximately 25,000 transplants (10,000 allogeneic and 15,000 autologous) are performed yearly by teams reporting to the annual EBMT survey (Table 1). Autologous and allogeneic HSCT are now established treatment options and have been incorporated into the treatment algorithms for many disorders. In addition, potential new indications have emerged such as autoimmune disorders and AL amyloidosis for autologous transplants and solid tumours for allogeneic transplants. Carefully conducted prospective studies hopefully will define the role of HSCT in these situations. On the other hand alternative non-transplant based treatment options are also making major progress, thus influencing the practice of HSCT. For example the number of transplants for previously important indications such as chronic myeloid leukaemia (CML) has been reduced with the introduction of the tyrosine kinase inhibitors. Furthermore, the technical developments made during the last decade have been impressive. Unrelated donor pools have expanded and alternative donors are now more extensively used. Stem cell sources include bone marrow, peripheral blood and cord blood. It is evident that recommendations are based on an ever-changing field. Long-term follow up is lacking for recently introduced methods, while available long term observations may relate to technologies no longer in use today. Still, a few principles remain valid.

2. Risk factors for transplant outcome The main risk factors for outcome can be defined today. They are based on stage of the disease, age of the patient, time interval from diagnosis to transplant and, for allogeneic HSCT, donor-recipient histocompatibility and donor-recipient sex combination. These risk factors are cumulative and can be modified by additional particularly good or poor prognostic features as exemplified in Table 2. The importance for outcome of this type of risk assessment has been shown for CML (5) and similar evaluations are in process for other diseases. In general, transplant related mortality increases and survival rates decrease with advanced disease stage, increasing age, increasing time from diagnosis to transplant, increasing histoincompatibility and in grafts involving male recipients with a female donor. All components have to be integrated into the risk assessment and the decision whether or not to perform a transplant. These factors are never absolute, for example, the age of an individual patient remains one of the most important determinants of


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Table 1: Number of patients treated with a first HSCT by indication, donor type and stem cell s

D Allogeneic TEAMS = 605

Leukaemias Acute myeloid leukaemia 1st complete remission not 1st complete remission Acute lymphatic leukaemia 1st complete remission not 1st complete remission Chronic myeloid leukaemia chronic phase not 1st chronic phase MDS incl. Sec AL MPS Chronic lymphatic leukaemia Lymphoproliferative disorders Plasma cell disorders - MM Plasma cell disorders - other Hodgkin's lymphoma Non Hodgkin lymphoma Solid tumors Neuroblastoma Soft tissue sarcoma Germinal tumours Breast cancer Ewing Renal cancer Melanoma Colon cancer Other solid tumours Non malignant disorders Bone marrow failure - SAA Bone marrow failure - other Haemoglobinopathies - thal Haemoglobinopathies - other Immune deficiencies Inherited disorders of metabolism Auto immune disease Others TOTAL 320

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BM 793 275 198 77 310 189 121 99 73 26 83 21 5 88 20 3 14 51 13 7 1

2

HLA-id PBPC 2449 1248 834 414 433 271 162 237 163 74 286 93 152 790 255 7 120 408 29 1 1 1 8 2 6 2

3 399 164 32 92 26 64 19 2 17

8 195 100 21 52 7 9 3 3 20

1310

3483

Cord 14 4 2 2 6 3 3 0

2 2 0

2 2

29 3 3 19 1 2 1

45

Family non-id BM PBPC 46 300 20 166 8 48 12 118 17 67 5 17 12 50 3 16 2 4 1 12 36 5 10 1 5 4 48 1 8 1 1 15 1 25 1 22 1 9 7 1 4

19 11

1 64 6 4 12 1 35 6

1

2

99

436

47 6 4 7

twin Cord 2

BM 5 0

2 2

2 1 1 0

0

PB 2 1 1 5 5 2 3 2 2

1 2

1

0

2 2

8 3

0

3 2 1

2

1 1

1 1

11

3

5 0

2

4

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and stem cell source in Europe in 2006 DONOR SOURCE No. of patients

neic

d

Autologous

Total

Unrelated twin BM 5 0

2 1 1 0

PBPC 23 15 10 5 5 2 3 2 2

1 2

1

2 2

8 3

3 2 1

5 0

BM 646 237 111 126 255 125 130 57 40 17 63 24 10 69 13 17 39 4 3 1

1 1

11

1 1

32

PBPC 2181 913 379 534 464 249 215 196 87 109 363 130 115 554 184 4 85 281 10

Cord 325 142 54 88 129 56 73 10 2 8 31 7 6 34 3 8 23 1

1 3 3 2

BM only 78 64 49 15 6 3 3 0

9 1

BM + PBPC 1101 747 632 115 145 93 52 13 4 9 37 9 150 12477 5918 251 1742 4566 1402 295 63 299 134 237 7

1 7 95 20 1 28 46 77 37 9 7

195 59 21 19 4 69 18 5 18

1 93 36 16 6

1 89 14 15 1

14 5

5 362 122

1

2

23 9 3 13

35 23 1 9

3 1 1

2 118 31

932

2851

458

256

15133

Cord 0 0

0

0

0

0

0

0

Allo

Auto

Total

6784 3020 1644 1376 1690 920 770 620 373 247 866 294 294 1597 489 15 260 833 85 25 10 4 15 7 8 2 0 14 1115 390 116 208 39 258 90 14 80

1179 811 681 130 151 96 55 13 4 9 38 9 157 12572 5938 252 1770 4612 1479 332 72 306 134 246 8 0 5 376 127 0 0 3 0 3 2 119 32

7963 3831 2325 1506 1841 1016 825 633 377 256 904 303 451 14169 6427 267 2030 5445 1564 357 82 310 149 253 16 2 5 390 1242 390 116 211 39 261 92 133 112

9661

15389

25050


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Table 2: Quantification of risk of transplant-related mortality Disease stage - Early (e.g. AML CR1) - Intermediate (e.g. AML CR2) - Advanced (e.g. refractory disease)

0 1 2

Age of patient - 40 yr

0 1 2

Time interval diagnosis to transplant - >12 months - >12 months (does not apply to patients in CR1)

0 1

Histocompatibility - HLA-identical sibling - Other donor

0 1

Gender combination - Other - Female donor for male recipient

0 1

Additional elements - Comorbidity / Karnofsky 50 years add - CMV not -/add - Syngeneic twin - Unrelated donor 10/10 high resolution matched

+1 +1 +1 -1 -1

outcome following both allogeneic and autologous HSCT-procedures. Generally, HSCT in children gives better results than in adults. Age cannot be seen as a single risk factor but must be taken together with other factors in the decision-making regarding HSCT. It should, however, be recognised that biological rather than chronological age is the more important determining factor for outcome and with reduced intensity conditioning regimens in allogeneic transplantation, the age limit has increased, permitting the inclusion of older patients.

3. The EBMT recommendations The EBMT recommendations are based on existing prospective clinical trials, EBMT registry data, and expert opinions. They are not formal evidence-based documents. Many potential indications are rare and will never be supported by evidence from an adequately powered randomised controlled trial. In addition, since thousands of patients survive long-term, issues of quality of life and late side effects are becoming increasingly important. This is especially 322

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important in children for whom late effects, such as growth retardation, sterility, impairment of intellectual ability, and secondary tumours have an even larger impact than in adults. It is, therefore, important when recommendations are made to integrate the possible survival gain from HSCT, the risk for late complications and the quality of life into the risk assessment strategy. Some recommendations were made based upon analogy, inference, and expertise. However, there is increasing knowledge from a number of well-designed prospective studies that have used standard randomisation procedures or the so called genetic randomisation technique. In the future, EBMT recommendations will incorporate evidence assessed in a more formal way. The EBMT recommendations are not meant to decide whether a transplant is the correct choice of procedure or not for an individual patient. They give guidance which must be considered together with the risk of the disease, the risk of the transplant procedure and the chances of strategies in the same situation. 3.1. Conditioning regimens Conditioning regimens vary in their intensity and are classified as standard intensity conditioning, reduced intensity conditioning, or intensified conditioning regimens. Reduced intensity conditioning (RIC) regimens can be used in the allogeneic setting with the intention of shifting the balance between risk of transplantrelated mortality and risk of relapse. During recent years approximately one quarter of all allogeneic HSCT were performed with RIC regimens. A wide variety of RIC regimens have been described in publications and RIC HSCT should preferably be performed with a previously published protocol to gain adequate experience with a few protocols. Extensive feasibility studies have been published and short-term results show that RIC HSCT can lower the risk for early transplant related mortality. This has been used as the main argument to use RIC HSCT for older patients and for patients with co-morbidities. Results have been published for related donor HSCT up to 75 years and for unrelated donor HSCT up to 70 years. The preferred stem cell source has been peripheral blood (90%). Experience with unrelated donors has been published with results comparable to those with related donors. No formal prospective or retrospective studies however, have so far shown superior long-term results with RIC HSCT compared to standard HSCT. A conventional transplant remains the therapy of choice for younger patients without co-morbidities in the absence of results from prospective, controlled trials. 3.2. Classification of indications An important aim of the indication documents has been to classify indications and to give advice about the settings in which these various types of transplants should be performed. They have been classified as “standard of care”, “clinical option”,


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“developmental” or “generally not recommended”. Respective examples are given in Table 3.

Table 3: Examples of transplant categories Standard of care

- AML CR2, allogeneic HSCT - Multiple myeloma, autologous HSCT

Clinical option

- Non-Hodgkin lymphoma, allogeneic HSCT

Developmental

- Autoimmune disease, autologous HSCT

Not recommended

- CML, blast crisis

3.2.1. “Standard of care” “Standard of care” transplants may be performed in any specialist centre with experience with HSCT procedures provided they have an appropriate infrastructure as defined by the EBMT and JACIE guidelines (6). The results of such transplants are reasonably well defined and compare favourably (or are superior to) results of non-transplant treatment approaches. Reporting of data to international transplant registries is considered as mandatory for EBMT members. Defining a transplant as the standard of care does not mean that it is necessarily the optimal therapy for a given patient in all clinical circumstances. 3.2.2. “Clinical option” The next category is transplants classified as a “Clinical option”. This is the most difficult category. It encompasses many rare diseases and the paucity of data relating to transplant outcome, the variability in transplant techniques and the contribution of patient factors such as age and co-morbidity makes the assessment of indications for transplantation much more complex. Our current interpretation of existing data for indications in this category supports HSCT as a valuable option for individual patients after careful discussions of risks and benefits with the patient. However the value of HSCT for patients included in this category needs further evaluation. Furthermore, it is necessary to carefully consider the potential impact of various prognostic factors such as the nature of the donor, the stem cell source, and the conditioning regimens used, since outcome is likely to vary depending on these choices. We believe that transplants for indications under the “Clinical option” heading should be performed in a specialist centre with major experience with HSCT procedures, with an appropriate infrastructure as defined by EBMT guidelines and, optimally, should meet JACIE standards (6, 7). It is also important 324

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that data from these procedures are reported to the international transplant registries in more detail (preferably on MED-B forms) that additional knowledge can be gained and used to further assess the value of HSCT in these indications. 3.3.3. “Developmental” The third category is “Developmental” and indications have been classified in this category if there is little experience with this particular type of transplant and when additional research is needed to define the role of HSCT. These transplants should be done within the framework of a clinical protocol in which patients are offered the opportunity to undergo allogeneic or autologous HSCT in the context of a study that has been designed for individuals who satisfy defined diagnostic criteria. The protocol may be performed in a single institutional study or may reflect national or international multi-centre collaboration. Protocols for “Developmental” transplants will have been approved by local research ethics committees and must be performed according to current international standards. It is implied that the results of the study are intended for presentation to and/or publication for the medical community at large. Centres performing transplants under the category of “Developmental” should meet JACIE standards (5). The reporting of MED-B data to the international transplant registries is a prerequisite to allow further assessment of the value of HSCT in these indications. 3.3.4. “Generally not recommended” Finally, we have also defined a “Generally not recommended” category. This category includes HSCT in early disease stages when results of conventional treatment do not normally justify the additional risk of transplant related mortality, or when the disease is so advanced that the chance of success is so small that the risk of the harvest procedure for the normal donor is difficult to justify. This grading may not apply to specific situations, e.g. where a syngeneic donor exists. This category also includes HSCT for a disease in a phase or status in which patients are conventionally not treated by HSCT. Therefore, there will be some overlap between “Generally not recommended” and “Developmental”. “Generally not recommended” does not exclude the possibility that centres with a focus on a certain disease can investigate HSCT in these situations. If a HSCT is performed for a “Generally not recommended” indication, the reporting of MED-B data is strongly recommended to allow further assessment of the value of HSCT in these indications.

4. Conclusion It is beyond the scope of this Chapter to discuss the specific indications and their grading. Instead, we recommend consulting the latest published version of the HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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indications document and the accompanying disease specific Chapters in this handbook. However, based on these recommendations and on individualised risk assessments, a risk adapted strategy should be developed at diagnosis. Depending on the assessment, the transplant can be planned as initial treatment, as rescue therapy or be rejected. With better knowledge of the disease risks and the transplant risks, those algorithms will become more and more refined.

References 1. Schmitz N, Gratwohl A, Goldman J. Allogeneic and autologous transplantation for haematological diseases, solid tumours and immune disorders: Current practice in Europe in 1996 and proposals for an operational classification. Bone Marrow Transplant 1996; 17: 471-477. 2. Goldman JM, Schmitz N, Niethammer D, Gratwohl A. Allogeneic and autologous transplantation for haematological diseases, solid tumours and immune disorders: Current practice in Europe in 1998. Accreditation Sub-Committee of the European Group for Blood and Marrow Transplantation. Bone Marrow Transplantation 1998; 21: 1-7. 3. Urbano-Ispizua A, Schmitz N, de Witte T, et al. Allogeneic and autologous transplantation for haematological diseases, solid tumours and immune disorders: Definitions and current practice in Europe. Bone Marrow Transplant 2002; 29: 639-646. 4. Ljungman P, Urbano-Ispizua A, Cavazzana-Calvo M, et al. Allogeneic and autologous transplantation for haematological diseases, solid tumours and immune disorders: Definitions and current practice in Europe. Bone Marrow Transplant 2006; 37: 439-449. 5. Gratwohl A, Hermans J, Goldman JM, et al. Risk assessment for patients with chronic myeloid leukaemia before allogeneic blood or marrow transplantation. Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Lancet 1998; 352: 1087-109. 6. Link H, Schmitz N, Gratwohl A, Goldman JM. Standards for specialist units undertaking blood and marrow stem cell transplants - recommendations from the EBMT. Bone Marrow Transplant 1995; 16: 733-763. 7. Joint Accreditation Committee. EBMT-EuroISHAGE (JACIE) Accreditation Manual (www.jacie.org).

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NOTES


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*

CHAPTER 18

Statistical evaluation of HSCT data

R.M. Szydlo

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CHAPTER 18 • Statistical evaluation of HSCT data

1. Introduction Stem cell transplantation (HSCT) is a widely accepted treatment modality, with both allogeneic and autologous HSCTs offering effective options for a number of diseases (eg. some leukaemias and severe aplastic anaemia) and curative potential for others (e.g. thalassaemia and CML). However, there is still much to be learnt, and the analysis of data generated from a stem cell transplant programme is not only fundamental to assessing the effectiveness of the treatment, but can provide invaluable information on the prognostic role of disease and patient factors. Thus, the appropriate analysis of such data is of paramount importance.

2. Outcomes Patients who undergo a HSCT procedure require considerable support and supervision, which in turn, allows the treatment modality to be reviewed in a variety of ways. Key events are assessed at varying times post-HSCT and these can be used to calculate a number of outcomes defined below: • Survival - the probability of survival irrespective of disease state • Disease-free survival (DFS) - the probability of being alive and free of disease (in leukaemia this outcome could also be termed leukaemia-free survival (LFS) • Graft vs. host disease (GvHD) - the probability of developing GvHD (the severity of disease being estimated would need to be clearly stated) • Graft failure (GF) - the probability of primary graft failure • Transplant related mortality (TRM) - the probability of dying without recurrence of disease • Relapse - the probability of disease recurrence • Progression-free survival (PFS) - the probability of being alive and with a disease stage not advanced of that at the time of transplantation • Neutrophil engraftment - defined as the first of 3 consecutive days post HSCT where values above a specified level are achieved (e.g. ≥0.5 x 109/L) • Platelet engraftment - defined as the first of 3 consecutive days post HSCT where values above a specified level are achieved (e.g. ≥50 x 109/L) Probability curves describing these outcomes fall into two categories: Survival, DFS/LFS and PFS involve events with decreasing cumulative probabilities over time, whilst GvHD, TRM, GF and relapse involve events that result in increased cumulative probabilities over time.

3. Survival analysis The outcomes outlined above require careful consideration before a statistical analysis can be considered. Each event of interest may occur at variable times post transplant,


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so in statistical terms it has two components - whether it occurs at all and, if it does, the length of time from transplant to the event. However, inherent in many studies is the problem that the event of interest is seldom observed in all of the patients. Thus, a patient who has not yet had the event of interest at the time of analysis, or who is lost to follow-up, would be “censored” at the time of last contact. The inclusion of data that is censored precludes the use of simple statistical methods such as chi-squared analysis or rank methods and requires a statistical treatment known as survival analysis, which can be applied to a variety of end points. 3.1. Kaplan-Meier method There are a number of methods for analysing survival data, and though these depend on the precision of the recorded time interval, are usually summarised as survival or Kaplan-Meier (1) curves which are derived from calculated tables commonly known as life tables (constructed on the basis of a series of conditional probabilities). The term life table is also frequently used to describe data where the results are grouped into time intervals, often of equal length, and this method of calculation is described as actuarial. In fact the terms “actual” and “actuarial” are often used mistakenly to describe survival probabilities generated by Kaplan-Meier methods. Figure 1 shows typical survival data from fifteen consecutive patients transplanted at a single centre. Four have died and eleven were still alive at various time points post-transplant. If the data are rearranged in order of time, then a life-table can be calculated by the method of Kaplan-Meier as shown in Table 1.

Patient number

Figure 1: Survival data from fifteen patients who received a haematopoietic stem cell transplant

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15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0,0

Alive Dead

0


40

80

120 160 200 240 280 320 Days post BMT

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Table 1: Life table for fifteen patients who received an allogeneic haematopoietic stem cell transplant Time (days)

Status (0=alive, 1=dead)

Number at risk

Probability of survival

Standard error

0 1 1 0 1 0 1 0 0 0 0 0 0 0 0

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

1.00 0.93 0.86

0.069 0.094

0.78

0.113

0.69

0.129

16* 26 66 69* 74 82* 88 89* 117* 133* 144* 172* 252* 291* 305* * censored observation

The data presented in Table 1 can be used to produce a survival curve, also known as a cumulative survival rate, or a survival function (Figure 2). Vertical tick marks on the curve represent censored individuals who make no contribution to the curve after that particular time point. The curve is an estimated probability of survival, and using appropriate methods to compute the standard error, 95% confidence intervals (95%CIs) can be calculated. In common with many analyses of small data sets, the standard error calculated from day 88 post BMT has yielded a large 95% CI, and so the survival curve must therefore be interpreted with some caution. 3.2. Cumulative incidence procedure The following outcomes–relapse, TRM, GvHD and GF are subject to the problem of “competing risks” (for example, in the case of calculating a relapse probability, a patient who dies in remission cannot relapse), and so the most appropriate method of analysing such data is to produce a cumulative incidence curve (2). Although this methodology is not included in most commercial statistical packages, it is present in the statistical package NCSS (Statistical analysis & data analysis software), and


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Figure 2: Probability of survival following a haematopoietic stem cell transplant (n=15)

Probability of survival %

100 80

69%

60 40 95% confidence interval 20 0

0

28

56

84

112 140 168 196 224 252 280 308

Days post BMT

macros are available to allow such curves to be calculated using the statistical packages SAS and R (3). Although it is possible to use the Kaplan-Meier method for each of these outcomes, this is likely to produce an overestimate of the true probability as calculated by the cumulative incidence approach (4). This discrepancy will be largest where the event of interest occurs later after HSCT (TRM, relapse and chronic GvHD - see Figure 3) and may be negligible with early outcomes (acute GvHD and graft failure).

4. Other methods for describing outcomes Recent advances in statistical methodology have enabled LFS and DFS curves to be modified to take into account durable remissions achieved after relapse (5). The generation of current leukaemia-free survival (CLFS) curves does however require detailed follow-up data and the use of macros designed for the statistical software package SAS. If the exact times to the onset of GvHD are not known, then simple proportions of grades of disease can be presented for those patients who survived long enough potentially to develop the disease (thus patients who died within 100 days postHSCT are not eligible for chronic graft versus disease). Engraftment times can either be described with a median and range, or with cumulative probability curves. Comparisons between GvHD groups should be made using the chi-squared test or chi-squared trend test, whilst the Mann-Whitney or Kruskal-Wallis test are applicable for engraftment data. An example of data presentation from 45 consecutive patients is illustrated in Figure 3.

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Figure 3: Probability curves of 6 different possible outcomes

5. Composite outcome diagrams An interesting new way of graphically representing how outcome probabilities change with time post-HSCT has been developed by Ronald Brand and is included in a recent paper (Figure 4) (6). Cumulative incidences of relapse, and of non-relapse


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Figure 4: Composite outcome diagram 1.00 0.90 0.80 Disease Free Survival

0.70

Overall Survival

0.60 Alive after relapse

0.50

Relapse Incidence

0.40 Dead after relapse

0.30 0.20

NRM

0.10 0

350

720

Days following transplantation

death are estimated and simultaneously plotted with the Kaplan-Meier survival probability. The resulting diagram thus provides the 4 possible patient states after a HSCT - alive without relapse of disease (white), alive after relapse (light blue, an outcome not normally calculated), dead after relapse (dark blue) and non-relapse death (grey). In addition, the proportion of patients relapsing (relapse incidence) is provided by the sum of the horizontal and diagonal hashed groups, the interface between the alive and dead components represents overall survival, and the interface between alive with and without relapse – relapse-free survival. One is thus able to view in one diagram the relative importance of all these possible outcomes. This is especially useful for illustrating differences between groups identified from univariate or multivariate analyses as being of prognostic significance. Specialist software is not required to create such diagrams, as macros are available for the statistical package SPSS-14.

6. Comparison of survival curves Survival curves provide a visual assessment for disease course and/or outcome of a particular treatment or disease course. In order to establish whether there is a survival advantage between, for example, two treatments, it is necessary to perform a statistical test to compare the two life tables. This is achieved using the logrank or Mantel-Cox test (7). In this test each observation is given an equal “weight”. However, in the transplantation setting, where there may be considerable early mortality, it may be more useful to “weight” early observations and in this context the Breslow test (8) may be more appropriate (this test is also less sensitive to late 334

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events when few subjects may be present in the study). The Tarrone-Ware test (9) is a compromise between the Mantel-Cox and Breslow tests. An example of the relative merits of these tests is provided in Figure 5. Here survival data from two hypothetical groups are presented. The proportion of survivors could be compared using a standard chi-squared test (treatment A 20/30 vs. treatment B 12/30), yielding a significant result (p=0.04), and suggesting treatment A to be the superior. However, this analysis ignores the time to an event, and gives a misleading result as exemplified by Figure 5. There is a clear survival advantage in the first four years post BMT for treatment A, but for long-term survival, treatment B may be better. The curves presented in Figure 5 do however highlight another analytical problem. In order to perform a log-rank test, the groups being tested should run in parallel and not cross over. A more sophisticated approach for analysing such data should therefore be undertaken (4). In addition to comparing treatments, the log-rank test can be used to compare selected sub-groups within one treatment or disease category e.g. males vs. females, patient age 35 years) as a prognostic factor. SR patients were by definition younger than 35 years without adverse prognostic factors. Phnegative patients with a donor had a superior OS (53%) compared to those without a donor (45%), mainly due to a lower RR. The difference was particularly evident in SR (OS 62 vs. 52%) but not in HR (OS 41 vs. 35%) patients (4). The TRM reached 20% even in the young SR patients and 39% in the older HR patients. Two conclusions can be drawn: - The outcome of HSCT is better in younger patients - In older HR patients the outcome is similarly poor with chemo and HSCT.


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It is likely that outcome with optimal chemotherapy is similarly good or even better than HSCT in young SR patients, as demonstrated in paediatric studies. For older HR patients the results underline the need to improve conditioning regimens and reduce pre-transplant morbidity and TRM.

7. Role and outcome of haploidentical transplant For haploidentical HSCT experience is restricted mainly to paediatric patients, where it may be considered in patients without donor and with urgent need of HSCT. In adults haploidentical HSCT is an experimental approach and should be restricted to specialised centres and later stage of disease, and should preferably be performed within clinical trials.

8. Role and outcome of cord blood transplant (UCB) The experience with UCB transplantation in ALL mainly comes from paediatric patients. In adults the limited cell dose is one of the major obstacles. Initial registry results for younger adults with acute leukaemia indicate however that UCB (single or double) can be considered as an alternative source, if available (9).

9. Conditioning There is also no standard for the conditioning regimen before HSCT in ALL. Most regimens are based on total body irradiation (TBI). The usual dose is 12 Gy. TBI is most frequently combined with cyclophosphamide (Cy) or VP16. A recent analysis of EBMT registry patients showed no difference in outcome for SIB-HSCT with TBI/VP16 or TBI/Cy in CR1. In second remission the TBI/VP16 combination was associated with a lower relapse risk (10). Clearly inferior results were reported for busulfan-based preparative regimens compared to TBI based regimens. With RICHSCT one third of patients may survive if transplant is performed in 1st CR (11).

10. Nature and role of MRD monitoring after transplant After transplantation regular evaluation of chimerism and MRD shows the individual course of disease. The outcome after HSCT is influenced by the status of MRD before and after HSCT. In Ph-positive ALL it has been demonstrated that in patients with MRD after HSCT the survival was significantly superior in those who rapidly responded to imatinib compared to those who did not respond (12). The outcome of patients with a high level of MRD before HSCT is poor. Therefore MRD status must be considered both before and after HSCT, to decide on additional treatment either before HSCT in order to reduce tumour load or after HSCT to prevent relapse.

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CHAPTER 21 • ALL in adults

10.1. Nature and role of any additional cellular or chemotherapy post-transplant In early relapse, preferably molecular relapse, immunologic treatments such as reduction of GvHD prohylaxis and/or application of donor lymphocytes are promising approaches for preventing overt relapse. In Ph-positive ALL post-transplantation treatment with imatinib – either up-front or after detection of MRD – appears to be a very successful approach.

11. Stem cell transplantation in Ph+ ALL Due to the poor outcome with intensive chemotherapy, HSCT has always been the treatment of choice for Ph+ ALL. The survival after allo-HSCT in first CR ranges between 27–65% (13). The RR is higher than in Ph-negative ALL and the outcome is compromised by TRM due to the higher median age of Ph+ ALL patients. Nowadays the majority of patients with Ph+ ALL receive imatinib as front-line therapy. Apparently there is no increase in TRM if HSCT is performed thereafter. The outcome of HSCT in Ph-positive ALL is strongly correlated with the level of MRD before and after HSCT and with the use of imatinib as part of the post-transplantation strategy (see above).

12. Prognostic factors Prognostic factors are not only used to identify HR patients who could benefit from HSCT. To an increasing extent prognostic factors have been described which help to estimate the risks of the HSCT itself, such as patient age, donor characteristics, degree of matching etc. Bringing both risk estimates together will in future allow a more accurate definition of indications for HSCT. Besides age, scoring of comorbidities may assist decision-making regarding indication for HSCT and the intensity of conditioning.

13. Future options in HSCT Large national and even international study groups are committed to the development of chemotherapy schedules and optimal integration of HSCT in front-line therapy. One important point is the balance between efficacy and toxicity of pre-transplant treatment and preparative regimens, in order to reduce TRM. Also the optimal timing of HSCT has to be defined. For an improvement of outcome after allogeneic HSCT a reduction in both RR and TRM is required. High resolution HLA typing and improved infection prophylaxis are important in this respect. For patients without a donor or with a contraindication to conventional HSCT alternative approaches need to be explored. MRD evaluation before and after HSCT is of interest, particularly in order to decide


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on maintenance therapy and immunotherapy such as donor-lymphocyte infusions. The prophylactic application of donor lymphocytes may be considered in patients with no or low GvHD.

References 1. Gökbuget N, Hoelzer D. Treatment of adult acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program 2006: 133-141. 2. Bachanova V, Weisdorf D. Unrelated donor allogeneic transplantation for adult acute lymphoblastic leukemia: A review. Bone Marrow Transplant 2007 Oct 29; [Epub ahead of print]. 3. European Leukemia Study Registry. www.leukemia-net.org 2008. 4. Goldstone AH, Richards SM, Lazarus HM, et al. In adults with standard-risk acute lymphoblastic leukemia (ALL) the greatest benefit is achieved from a matched sibling allogeneic transplant in first complete remission (CR) and an autologous transplant is less effective than conventional consolidation/maintenance chemotherapy in All patients: Final results of the international ALL trial (MRC UKALL XII/ ECOG E2993). Blood 2007 Nov 29; [Epub ahead of print]. 5. Hahn T, Wall D, Camitta B, et al. The role of cytotoxic therapy with hematopoietic stem cell transplantation in the therapy of acute lymphoblastic leukemia in adults: An evidence-based review. Biol Blood Marrow Transplant. 2006; 12: 1-30. 6. Yanada M, Matsuo K, Suzuki T, Naoe T. Allogeneic hematopoietic stem cell transplantation as part of postremission therapy improves survival for adult patients with high-risk acute lymphoblastic leukemia: A metaanalysis. Cancer 2006; 106: 1657-1663. 7. Chaidos A, Kanfer E, Apperley JF. Risk assessment in haemotopoietic stem cell transplantation: Disease and disease stage. Best Pract Res Clin Haematol. 2007; 20: 125154. 8. Loberiza F. Summary Slides 2003 - part III. IMBTR/ABMTR Newsletter 2006; 10: 6-9. 9. Rocha V, Labopin M, Sanz G, et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004; 351: 2276-2285. 10.Marks DI, Forman SJ, Blume KG, et al. A comparison of cyclophosphamide and total body irradiation with etoposide and total body irradiation as conditioning regimens for patients undergoing sibling allografting for acute lymphoblastic leukemia in first or second complete remission. Biol Blood Marrow Transplant 2006; 12: 438-453. 11.Arnold R, Massenkeil G, Bornhauser M, et al. Nonmyeloablative stem cell transplantation in adults with high-risk ALL may be effective in early but not in advanced disease. Leukemia 2002; 16: 2423-2428. 12.Wassmann B, Pfeifer H, Stadler M, et al. Early molecular response to posttransplantation imatinib determines outcome in MRD+ Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Blood 2005; 106: 458-463. 13.Fielding AK, Goldstone AH. Allogeneic haematopoietic stem cell transplant in Philadelphiapositive acute lymphoblastic leukaemia. Bone Marrow Transplant. 2007 Oct 29; [Epub ahead of print].

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CHAPTER 21 • ALL in adults

Multiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Which post-transplantation strategy is most successful in Ph/BCR-ABL positive ALL a) Imatinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Mercaptopurine/methotrexate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) No treatment (to avoid toxicities) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) DLI only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Is non-myeloablative HSCT an option in adult ALL? a) Not at all . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Generally preferable due to lower toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Within studies focussed on older and/or patient with comorbidities . . . . . d) Based on individual decision and patients wish . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. What are the results of allo sibling HSCT compared to matched unrelated SCT (MUD) in adult ALL? a) Sibling SCT clearly superior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) MUD SCT clearly superior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Similar overall results with higher relapse rate and lower TRM for sibling SCT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Similar for matched or mismatched donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. What are the results of autologous HSCT compared to chemotherapy? a) Similar in most studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Inferior compared to chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Superior compared to chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Independent of minimal residual disease and post-transplant maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Which is the standard for conditioning in adult ALL? a) TBI based regimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Busulfan based regimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Non-myeloablative regimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Use of ATG and prophylactic DLI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HAEMATOPOIETIC STEM CELL TRANSPLANTATION

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*

CHAPTER 22

HSCT for myelodysplasia in adults

T. de Witte, G. Sanz

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CHAPTER 22 • Myelodysplasia in adults

1. Introduction Myelodysplasia (MDS) consists of a heterogeneous group of clonal stem cell disorders. The spectrum of MDS varies from a disease with an indolent course over several years to a form with rapid progression to acute myeloid leukaemia (AML). Since 1982, myelodysplasia has been classified according to the French–American–British (FAB) criteria (1). The new World Health Organization (WHO) classification system for MDS corrected several limitations of the original FAB classification in 1997 (2). An international workshop generated an International Prognostic Scoring System (IPSS) in 1997 (3). MDS usually occurs without a preceding provoking event (primary MDS), but treatment with radiotherapy and certain chemotherapeutic agents promotes the development of therapy-related (t-)MDS. Both alkylating substances and drugs targeted at topoisomerase II are capable of inducing t-MDS and t-AML. The majority of MDS patients are older than 60 years. For these patients supportive care, including new biologic response modifiers, is the mainstay of therapy. Allogeneic haematopoietic stem cell transplantation (HSCT) is usually considered the treatment of choice for most young MDS patients who have a histocompatible donor. Long-term disease-free survival (DFS) can be attained by these patients. For patients who lack a compatible donor, the outcome with autologous HSCT appears comparable with allogeneic SCT. Many clinicians consider allogeneic HSCT as the only curative treatment option. However, a retrospective study evaluating intensive chemotherapy alone versus chemotherapy followed by HSCT did not show a clear benefit for either chemotherapy alone or chemotherapy followed by HSCT.

2. Role of reduced-intensity conditioning (RIC) regimens in myelodysplasia The general age of patients with MDS is higher than 60 years and co-morbidity is rather common. For these reasons, RIC regimens have recently been increasingly used in MDS. The initial reports in MDS showed an encouraging low TRM compared with conventional conditioning. Kröger et al. reported on 37 MDS patients who were ineligible for conventionally conditioned HSCT. The actuarial DFS rate at 3 years was 38%, with a median follow-up of 20 months (4). More favourable results were reported following conditioning with fludarabine, busulfan, and alemtuzumab in 62 MDS patients. The 1-year DFS rates were 61 and 59% in patients with matched sibling donors (n=24) and unrelated donors (n=38), respectively (5). The EBMT analysed the outcomes of 215 RIC patients, and standard myeloablative conditioning (SMC) in 621 patients. In a multivariate analysis, the 3-year relapse rate was significantly increased after RIC (hazard ratio [HR] 1.61, P=0.001), but the 3-year non-relapse mortality (NRM) was decreased after RIC (0.61, P=0.015) (6).


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It is difficult to evaluate the contribution of RIC regimens to the improved outcome of allogeneic HSCT for MDS patients in view of the recently improved outcomes of HSCT with marrow ablative regimens and the heterogeneity of the patient populations (age, comorbidity, stage of disease). Therefore, the EBMT has launched a prospective randomised study comparing RIC regimens with standard conditioning regimens (for details, see: http://www.ebmt.org/5WorkingParties/CLWP/clwp8.html).

3. Role and outcome of autologous stem cell transplantation In view of the high relapse rate after chemotherapy alone, transplantation with autologous stem cells has been applied in an attempt to intensify post-remission therapy. One of the first reports by the EBMT on autologous HSCT in MDS showed a 2-year DFS of 34%. In a prospective study, 24 of the 39 candidates received an autologous HSCT, resulting in a median DFS of 29 months from transplantation (7). A European study compared the results of 100 patients who had entered CR after remission–induction chemotherapy and who were candidates for allogeneic and autologous HSCT, depending on the availability of an HLA-identical sibling. The 4year DFS rates in the group of patients with or without a donor were 31 and 27%, respectively (HR 0.93, 95% CI 0.57–1.52). This outcome suggested that patients with high-risk MDS might benefit from either allogeneic or autologous HSCT (8). A successful autograft is theoretically restricted to patients who achieve CR following induction chemotherapy, and in whom a suitable autologous harvest can be collected. Stem cell mobilisation was feasible in only 44/102 patients (43%) in the recovery phase after chemotherapy with G-CSF (9). This relatively low yield of a sufficient number of stem cells might reflect the low number of residual normal stem cells or the damage to the bone marrow stroma caused by pro-apoptotic cytokines produced by the MDS clone. It is clear that better mobilisation schedules and approaches should be developed before autologous peripheral HSCT can be recommended as part of the post-remission treatment of MDS patients treated with intensive anti-leukaemic therapy.

4. Role and outcome of HLA-identical sibling transplants Results of allogeneic HSCT have improved over time. The International Bone Marrow Transplant Registry (IBMTR) reported a 3-year DFS rate of 40% for 452 patients who underwent HLA-identical sibling HSCT for MDS performed between 1989 and 1997 (10). Deeg et al. reported favourable results in MDS patients treated with a busulfan-based regimen in which the busulfan dosage was adjusted to maintain blood levels at 800–900 ng/mL. The 3-year non-relapse mortality (NRM) rate was 28% with related donors (11). 382

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CHAPTER 22 • Myelodysplasia in adults

Cytogenetic abnormalities have a major influence on the outcome after HSCT. Using cytogenetic risk categories defined by the IPSS, the event-free survival rates for the poor-risk, intermediate-risk, and good-risk groups were 6, 40, and 51%, respectively (12). More advanced age (continuous variable) and long disease duration (>12 months) before HSCT were associated with an increased risk for treatment-related death after HSCT. This mandates consideration of HSCT early in the disease course. However, Cutler et al. showed that delayed HSCT results in maximised overall survival (OS) for low and intermediate-1 IPSS groups. They hypothesised that the optimal timing of HSCT in this cohort is at the time of development of a new cytogenetic abnormality, the appearance of a clinically important cytopenia, or an increase in the percentage of marrow blasts (13). Whether patients with advanced stage MDS should receive remission-induction chemotherapy prior to HSCT conditioning remains a point for debate. Retrospective analyses have reported conflicting data. Interpretation of the data is hampered by different selection biases in the two treatment approaches. Details regarding the type of chemotherapy administered are lacking in most studies. Preliminary analysis of the Criant study presented in 2005 showed that the majority of patients with an identified donor who were treated with remission-induction and consolidation chemotherapy received the planned HSCT (47/50). The 4-year DFS rate of the donor group was 46% – encouraging when compared with large-registry data (9). However, only prospective randomised studies can prove the benefit of remissioninduction therapy prior to transplant conditioning. The EBMT launched such a study in December 2006 (see http://www.ebmt.org/5WorkingParties/CLWP/clwp8.html for full details).

5. Role and outcome of matched unrelated allogeneic transplants Several reports have demonstrated that transplantation from matched unrelated donors (MUD) is a feasible and curative strategy for MDS patients. The National Marrow Donor Program (NMDP) reported on 510 MDS patients, transplanted between 1988 and 1998. At 2 years, the probability of DFS was 29%, and the cumulative incidence of TRM was 54% (14). A more recent report described the results obtained with a conditioning regimen of oral busulfan targeted to plasma concentrations of 800–900 ng/mL plus cyclophosphamide. The 3-year relapse-free survival of 64 MDS patients who underwent HSCT from MUDs was 59% and the NRM was 30% (11). The status of the disease at transplantation and the time from diagnosis to transplantation have shown close relationships with DFS after MUD transplantation for MDS. Patients with MDS secondary to chemo/radiotherapy have poorer DFS rates than patients with de novo MDS. There also appears to be an improvement in DFS


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rates in patients who have been transplanted in recent years (>1992). Other characteristics that have been associated with better DFS rates in some series are younger recipient age (continuous variable), greater cell dose, CMV seronegativity, and grades 0–I acute GvHD.

6. Role and outcome of cord blood transplants Experience with CB transplantations from unrelated donors for MDS patients is still very scarce. Ooi et al. from the University of Tokyo have published the outcomes of 13 patients with advanced MDS with a median age of 40 years (15). Despite the Figure 1: Algorithm for the management of MDS patients candidates for intensive therapy A

Marrow blasts 10 year survivorship at least in a subset of patients. Nevertheless, the US study (SWOG 9321), the French MAG91 study and the Spanish PETHEMA group, although they confirmed the benefit of auto-HSCT in terms of response rate and EFS, did not find superiority in terms of survival as compared to SDT (4, 5, 6). These discrepancies


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can be, at least in part, explained by: 1) differences in the studies’ design (the Spanish study randomised patients responding to initial therapy while, in the others, randomisation was performed up-front), 2) differences in the conditioning regimens and, particularly, 3) differences in the intensity and duration of the chemotherapy arm (the dose of alkylating agents and steroids were higher in the SWOG and Spanish trials, which may explain why overall survival for conventionally treated patients was longer in these two studies as compared to IFM and MRC trials). Finally, two recent meta-analyses did not provide evidence of an overall survival advantage for HDT as compared to SDT (7, 8). In spite of these discrepancies, HDT is currently considered as standard of care for younger patients with multiple myeloma, mainly based on the benefit on response rate and EFS. Nevertheless, the availability of highly efficient new drugs may challenge this statement. Thus, novel drugs combinations based on thalidomide, bortezomib or lenalidomide, have shown to be superior to VAD-like regimens as debulking agents prior to auto-HSCT, with response rates >80%, including up to 10–30% CR rates. The next question is whether or not auto-HSCT is able to up-grade the response obtained with novel agents. In six pilot studies based on bortezomib-induction regimens, it was observed that the CR rate was improved following auto-HSCT, which suggests that the two approaches (induction with novel agents and auto- HSCT) are complementary rather than alternative. Regarding tandem auto-HSCT, its use will decrease for two reasons: 1) according to IFM (9) and Italian (10) experience only patients achieving a very good partial response with the first transplant benefit from the second and 2) a similar benefit is obtained upon using thalidomide as consolidation/maintenance therapy (11). In contrast, second transplant at relapse may be increasingly used, providing that the duration of the response to first transplant has lasted for more than 2–3 years.

4. Role and outcome of HLA-identical sibling transplant Although HDT followed by autologous transplantation allows long-term survival, at least in a subset of patients, there is no clear plateau on the survival curve. Moreover, patients displaying poor prognostic features such as IgH translocations plus Rb or p53 deletions and advanced stage according to the International Staging System display poor prognosis after HDT. On the contrary, allogeneic transplantation remains the only curative therapeutic approach which may offer long-term disease free survival. Unfortunately, although TRM has been

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CHAPTER 26 • MM in adults

reduced in the last years, conventional allogeneic transplantation is still associated with a high TRM. In the SWOG 9321 randomised trial, patients with a suitable donor received allogeneic transplantation after conditioning with melphalan 140 mg/m2 plus TBI and the arm was closed due to a 1 year TRM of 53%. Interestingly, 7 year estimated progression free survival in this subset of patients was 39%, similar to that reported for patients receiving autologous transplant or SDT, and this was due to a low relapse rate among patients receiving allogeneic transplant. In order to decrease TRM different RIC regimens have been developed (allo-RIC). In a prospective randomised trial, the French group compared double auto-HSCT to auto-HSCT followed by allo-RIC among patients displaying poor prognostic features (high B2microglobulin and monosomy of chromosome 13). Unfortunately, there were no event free survivors at 5 years either after double auto-HSCT or after auto-HSCT followed by allo-RIC (12). By contrast, the Italian group, using a similar approach, has recently described an improvement in terms of overall survival among patients receiving auto-HSCT followed by allo-RIC as compared to double auto-HSCT (13). In addition to differences in patient characteristics, the different GvHD prophylaxis and conditioning regimens used could explain these differences. Currently available data suggest that the use of RIC rather than myeloablative conditioning regimens may decrease TRM, but no prospective comparison is so far available. In a retrospective study from the EBMT comparing MC vs. allo-RIC, TRM was 37 vs. 24% and cumulative incidence of disease progression were 27 vs. 54% for MC vs. allo-RIC, respectively, which resulted in similar overall survival (51 vs. 38%). In high risk MM patients, including those displaying poor cytogenetic features (t(4;14), t(14;16) and t(14;20), Chr 13 deletion by conventional cytogenetics, p53 deletion, complex karyotype or hypodiploidy) or those with progressive disease during induction therapy, the use of novel agents as induction therapy followed by a tandem transplant, auto-HSCT and allo-RIC, represents an attractive approach, although these type of studies should be conducted within well controlled clinical trials. Regarding rescue therapy, in a series of 54 patients, 14 patients obtained complete remission or partial response out of 19 patients with refractory disease undergoing auto-HSCT followed by RIC allogeneic transplant (14). Unfortunately, a significant proportion of these patients finally relapse, leading to a poor event free survival, especially among patients who had relapsed after a prior autograft or with active disease at the time of allogeneic transplantation (15) (Table 1).


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Table 1: Results of randomised trials Trials (Ref.)

EFS / PFS

OS

p

Standard dose chemotherapy (SDT) vs. autologous HSCT IFM(2) SDT Auto-HSCT

18 months (10% 5 years) 27 months (28% 5 years)

37 months (12% 5 years) not reached (52% 5 years)

S

MRC(3) SDT* Auto-HSCT *

19 months 31 months

42 months 54 months

SWOG(4) SDT* Auto-HSCT *

14% 7 years 17% 7 years


PETHEMA(5) SDT* Auto-HSCT *

33 months 42 months

66 months 61 months

MAGG(6) SDT* Auto-HSCT *

19 months 25 months

47 months 47 months

S

NS

NS

NS

Single vs. double autologous HSCT IFM(9) Single* Double*



S

Cavo(10) Single Double

23 months 35 months

46 months 43 months

NS

Autologous vs. allogeneic HSCT IFM(12) Auto-HSCT (double) 30 months (0% 5 years) Auto-HSCT plus RIC-allo 25 months (0% 5 years)


NS

Bruno(13) Auto-HSCT (double)* 29 months Auto-HSCT plus RIC-allo * 35 months

54 months 80 months

SWOG(4) Auto-HSCT Allo-HSCT (MC)


S

NS 17% 7 years 22% 7 years

(p) differences for OS: (NS) non significant; (S) significant differences. (*) results expressed as median OS: overall survival; EFS: event-free survival; PFS: progression-free survival; MC: myeloablative conditioning

5. Role and outcome of matched unrelated donor (MUD) allogeneic transplant In the unrelated donor setting, the use of fludarabine and melphalan as conditioning regimen plus ATG as graft-versus-host disease prophylaxis was associated with a 90% 418

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response rate (40% complete and 50% partial) in a series of 21 MM patients. TRM was 26% at 1 year and 2 year overall and progression free survival were 74% and 53% (16). In another series of 17 patients, receiving MUD after fludarabine plus 2 Gy TBI, including 71% with chemotherapy resistant disease, 42% achieved complete remission and 17% partial response after transplant. Outcome was significantly better among those receiving tandem auto-HSCT followed by allo-RIC as compared to those who directly proceeded to allogeneic transplantation (51 vs. 11% progression free survival, respectively) (17).

6. Other sources: role and outcome of haploidentical transplant and cord blood transplant Data using alternative sources of progenitor cells in MM patients are too limited to draw any firm conclusion.

7. Nature and role of any additional cellular or chemotherapy posttransplant Donor lymphocyte infusions (DLI) given for relapsed myeloma following allogeneic transplantation induce responses in 30–50% of patients, the most common approach being the use of escalating dose at a usual starting dose of 1 x 107 CD3/kg (106 in the unrelated setting). In a recent multicentre analysis, the most important prognostic factors for response to DLI after RIC were the development of acute and chronic GvHD. Interestingly, the combination of DLI with thalidomide or bortezomib may improve the response rate and contribute to modulate the immune response, although further studies are required to confirm these data (18).

8. Nature and role of minimal residual disease monitoring after transplant A high relapse rate has been reported among MM patients receiving transplantation even in the allogeneic setting. For this reason, MRD monitoring might allow individualised treatment strategies. In this regard, PCR may contribute to identify patients at high risk of relapse; thus, in a series of MM patients undergoing transplantation, among 16 PCR negative patients no relapses were observed as compared to 100% among 13 patients with positive PCR (19). Unfortunately, a high proportion of patients develop extramedullary relapses without bone marrow involvement (20) indicating that, although the disease may be under control in the bone marrow milieu, extramedullary spread may occur. For this reason, MRD monitoring in bone marrow may not allow an early identification of all patients at risk of relapse and other tools, such as PET/MRI should be considered for a better follow-up of these patients.


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References 1. Moureau P, Facon T, Attal M, et al. IFM. Comparison of 200 mg/m2 melphalan and 8 Gy total body irradiation plus 140 mg/m2 melphalan as conditioning regimens for peripheral blood stem cell transplantation in patients with newly diagnosed multiple myeloma: Final analysis of the IFM 9502 randomized trial. Blood 2002; 99: 731-735. 2. Attal M, Harousseau JL, Stoppa AM, et al. A prospective randomised trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Eng J Med 1966; 335: 91-97. 3. Child JA, Morgan GJ, Davies FE, et al. Medical Research Council adult Leukemia Working Party. High dose chemotherapy with hematopoietic stem cell rescue for multiple myeloma. N Eng J Med 2003; 348: 1875-1883. 4. Barlogie B, Kyle RA, Anderson KC, et al. Standard chemotherapy compared with high dose chemoradiotherapy for multiple myeloma: Final results of a phase III US intergroup trail S9321. J Clin Oncol 2006; 24: 929-936. 5. Blade J, Rosignol L, Sureda A, et al. PETHEMA. High dose therapy intensification compared with continued standard chemotherapy in multiple myeloma patients responding to the initial chemotherapy: Long term results from a prospective randomised trial from the Spanish cooperative group PETHEMA. Blood 2005; 106: 3755-3759. 6. Fermand JP, Katsahian S, Divine M, et al. Group Myeloma Autogreffe. High dfose therapy and autologous blood stem cell transplantation compared with conventional treatment in myeloma patients aged 55 to 65: Long term results of a randomised control trial from the MAG. J Clin Oncol 2005; 23: 9227-9233. 7. Levy V, Katsehian S, Fermand JP, et al. A meta-analysis on data from 575 patients with multiple myeloma randomly assigned to either high-dose therapy or conventional therapy. Medicine (Baltimore) 2005; 84: 250-260. 8. Koreth J, Cutler CS, Djulbegovic B, et al. High dose therapy with single autologous transplantation versus chemotherapy for newly diagnosed multiple myeloma. A systematic review and meta-analysis of randomised controlled trials. Biol Blood Marrow Transplant 2007; 13: 183-196. 9. Attal M, Harousseau JL, Facon T, et al. IFM. Single versus double autologous stem cell transplantation for multiple myeloma. N Eng J Med 2003; 349: 2495-2502. 10.Cavo M, Tosi P, Zamagni E, et al. Prospective, randomized study of single compared with double autologous stem-cell transplantation for multiple myeloma: Bologna 96 clinical study. J. Clin Oncol 2007; 25: 2434-2441. 11.Attal M, Harouseau JL, Leyvraz S, et al. Maintenance therapy with thalidomide improves survival in patients with multiple myeloma. Blood 2006; 108: 3289-3294. 12.Garban F, Attal M, Michallet M, et al. Prospective comparison of autologous stem cell transplantation followed by dose-reduced allograft (IFM99-03 trial) with tandem autologous stem cell transplantation (IFM99-04 trial) in high-risk de novo multiple myeloma. Blood 2006; 107: 3474-3480. 13.Bruno B, Rotta, M, Patriarca F, et al. A Comparison of Allografting with Autografting for Newly Diagnosed Myeloma. N Engl J Med 2007; 356: 1110-1120. 14.Maloney D, Molina A, Sahebi F, et al. Allografting with nonmyeloablative conditioning 420

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following cytoreductive autografts for the treatment of patients with multiple myeloma Blood 2003; 102: 3447-3454. 15.Kröger N, Perez-Simon J, Myint H, et al. Influence of timing allogeneic stem cell transplantation after dose-reduced melphalan/fludarabine conditioning in multiple myeloma. Biol Blood and Marrow Transplant 2004; 10: 698-708. 16.Kröger N, Sayer H, Schwerdtfeger R, et al. Unrelated stem cell transplantation in multiple myeloma after a reduced-intensity conditioning with pretransplantation antithymocyte globulin is highly effective with low transplantation-related mortality. Blood 2002; 100: 3919-3924. 17.Georges G, Maris M, Maloney D, et al. Nonmyeloablative unrelated donor hematopoietic cell transplantation to treat patients with poor-risk, relapsed, or refractory multiple myeloma. Biology of Blood and Marrow Transplantation 2007; 13: 423-432. 18.Van de Donk N, Kröger N, Hegenbart U, et al. Prognostic factors for donor lymphocyte infusions following non-myeloablative allogeneic stem cell transplantation in multiple myeloma. Bone Marrow Transplant 2006; 37: 1135-1141. 19.Corradini P, Cavo M, Lokhorst H, et al. Molecular remission after myeloablative allogeneic stem cell transplantation predicts a better relapse-free survival in patients with multiple myeloma. Blood. 2003; 102: 1927-1929. 20.Pérez-Simón J, Sureda A, Fernández-Avilés F, et al. Reduced intensity conditioning allogeneic transplantation is associated with a high incidence of extramedullary relapses in multiple myeloma patients. Leukemia 2006; 20: 542-545.

Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Regarding auto-HSCT, which of the following sentences is wrong? a) Patients receiving melphalan 200 mg/m2 display a better median overall survival as compared to patients treated with melphalan 140 mg/m2 in combination with TBI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Two recent meta-analyses have not provided evidence of an overall survival advantage auto-HSCT as compared to standard dose therapy . . . . c) Novel drugs combinations based on thalidomide, bortezomib or lenalidomide, have shown to be superior to VAD-like regimens as debulking treatment prior to auto-HSCT, with response rates >80%, including up to 10–30% CR rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Auto-HSCT does not improve the response rate obtained with bortezomib-based induction regimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


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2. Regarding auto-HSCT, one of the following answers is incorrect: a) MM is currently the most common indication for auto-HSCT in North America and Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) In the Spanish (PETHEMA) and American (SWOG) trials, the dose of alkylating agents and steroids used in the chemotherapy arm were higher than in the French (IFM) and UK (MRC) trials, which may explain why the survival was similar to that obtained with auto-HSCT . . . c) According to two randomised trials, patients achieving complete remission with the first auto-HSCT do benefit from the second transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Second transplant in relapsing patients offers no benefit to those patients in whom the duration of the response to first transplant has lasted less than 1 year . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Choose the correct answer: a) In the SWOG 9321 randomised trial, patients with a suitable donor received allogeneic transplantation after conditioning with melphalan 140 mg/m2 plus TBI and the arm was closed due to a 1 year TRM of 53% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Seven year estimated overall survival in this subset of patients was 39%, similar to that reported for patients receiving auto-HSCT or standard dose chemotherapy, and this was due to a low relapse rate among patients receiving allogeneic transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The IFM compared double auto-HSCT to auto-HSCT followed by RIC-allo among patients displaying poor prognostic features (high B2microglobulin and monosomy of chromosome 13). There were no event free survivors at 5 years in either arm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) In the IFM trial, the conditioning regimen among patients receiving allo-RIC consisted of fludarabine and melphalan . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Regarding allogeneic transplantation one of the following answers is incorrect: a) Median overall survival among patients receiving double auto-HSCT was 54 months as compared to 80 months among those receiving auto followed by allo-RIC in a multicentre prospective randomised Italian trial conducted by Bruno et al. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422

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b) This study included only patients displaying poor cytogenetics . . . . . . . . . . . c) In a retrospective study from the EBMT comparing myeloablative conditioning vs. allo-RIC or RIC, the TRM was lower with RIC-allo but the cumulative incidence of disease progression was higher, which resulted in similar overall survival in both subgroups . . . . . . . . . . . . . . d) In that study, the use of Campath in a subset of patients receiving RIC-allo was associated with a significant increase in the risk of relapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Regarding allogeneic transplantation which of the following answers is correct: a) Response rates ranging from 70 to 90% has been reported even among patients with refractory MM undergoing auto followed by RIC-allo transplant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) DLI given for relapsed myeloma following allogeneic transplantation induce responses in >80% of patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) The most common approach is the use of escalating dose at a usual starting dose of 1 x 108 CD3/kg (107 in the unrelated setting) . . . . . . . . . . d) The combination of DLI plus thalidomide or bortezomib may improve the response rate as compared to DLI alone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


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*

CHAPTER 27

HSCT for primary amyloidosis in adults

J. Esteve

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CHAPTER 27 • Amyloidosis in adults

1. Introduction Primary systemic amyloidosis (AL) is a misfolding protein disease which leads to the extracellular deposition of abnormal protein fibrils in various tissues, including kidney, heart, liver, gastrointestinal tract, and peripheral nervous system, causing organ dysfunction. Amyloid fibrils in AL are constituted by a insoluble monoclonal light chain (LC) which aggregates forming b-pleated sheets. Diagnosis of AL is based on the recognition of amyloid substance in an appropriate tissue (subcutaneous fat tissue, rectum, bone marrow, involved organ), revealed by the characteristic staining pattern with Congo red dye, and further identification of amyloid fibril composition. Adequate identification of fibril precursor protein, a monoclonal LC, allows differentiation from other types of amyloidoses (familial type, secondary amyloidosis, dialysis-associated, senile). In most patients a monoclonal gammopathy is detected in serum and/or urine. Since AL is a clonal plasma cell disorder responsible for the synthesis of amyloidogenic LC, therapeutic agents used for AL therapy are similar to those used against multiple myeloma. Despite the usual low tumour burden characteristic of this disorder, AL is a poor-prognosis disease, with only a modest response pattern to standard chemotherapy, which cannot prevent progression of tissue damage. In contrast, intensive therapy with high-dose melphalan with auto-HSCT produces a high proportion of responses, followed by significant amelioration of organ damage in most responding patients. Unfortunately, this procedure is associated with an exceedingly high toxicity, reflecting the underlying organ damage secondary to amyloid deposits (1).

2. Indications Treatment with high-dose melphalan followed by peripheral blood stem cell rescue (auto-HSCT) should be considered in younger patients, up to 65–70 years, diagnosed with systemic AL, with no limiting organ damage, i.e., in the absence of uncompensated cardiac failure, severe renal failure or marked increase in bilirubin, and suitable for such an intensive procedure.

3. Stem cell mobilisation and collection, and conditioning regimen Stem cell mobilisation and leukapheresis in patients with AL is associated with unusual morbidity and with some reports of fatal events, due to the impaired organ and cardiovascular reserve of these patients. Thus, a syndrome of hypoxia and hypotension has been described both during mobilisation with G-CSF and during the leukapheresis procedure itself, probably as a result of diverse causes such as a capillary leak syndrome triggered by G-CSF, platelet activation during SC collection, and the release of inflammatory cytokines. Therefore, use of reduced doses of G-


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CSF (5–6 mg/kg ever 12 hours) and careful monitoring during the leukapheresis procedure is highly encouraged, with admission if necessary to an Intensive Care Unit, in order to avoid or correct immediately any sudden volume imbalance (hypovolemia or fluid overload) that may arise during the SC collection process. Conditioning regimens in AL are based on high-dose melphalan. The usual melphalan dose is 200 mg/m2, although a “risk-adapted” approach, with dose reduction to 140 mg/m2, has been proposed in higher risk patients in order to decrease transplantrelated toxicity (2). Proposed criteria for defining high risk patients are age >60, increased creatinine level, performance status (PS) 2 or compensated cardiac failure. Reduced melphalan dose has been associated with a decreased response rate in some studies (2, 3), although this observation has not been confirmed in other studies (4).

4. Results: toxicity, response, and long-term outcome Auto-HSCT is associated with a remarkably high risk of morbidity and mortality in patients with AL, with a TRM ranging from 11 to 43% (Table 1). Cardiogenic shock, fatal arrhythmias, gastrointestinal bleeding, and infections are the most frequent complications involved in procedure-related deaths during this phase. Infrequent causes such spontaneous splenic rupture or DMSO-triggered cardiac arrest have also been reported in this setting. Furthermore, renal insufficiency develops frequently after auto-HSCT, occurring in approximately 20% of patients. As regards activity against AL, high-dose melphalan results in significant responses in 50–60% of cases, with complete responses in about one third of patients. CR is defined, in this setting, by a negative immunofixation and normal free Table 1: Summary of the outcome of patients with primary amyloidosis undergoing autotransplant according to largest series Source

No. of pts

Overall response TRM (%) rate (CR) %

Overall survival (%)

Boston (US) (3) CIBMTR (multicenter) (11)

205 107

NR (40) 32 (16)

13 (100-day) 18 (30-day) 27 (1-yr)

60 (3-yr) 66 (1-yr) 56 (3-yr)

UK (multicenter) (12)

92

64 (35)

23 (100-day)

50 (5-yr)

Mayo Clinic (7)

282

71 (33)

11 (100-day)

60 (5-yr)

French Intergroup (MAG-IFM) (9)

50

49 (30)

24 (100-day)

45 (3-yr)

CR: complete response; TRM: transplant-related mortality; NR: not reported; CIBMTR: Center for International Blood and Marrow Transplant Research; UK: United Kingdom; MAG-IFM: Myélome Autogreffe-Intergroupe Francophone du Myélome 426

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immunoglobulin light-chain (FLC). Interestingly, haematologic responses are followed by organ responses, i.e. improvement of involved organ function, in most cases. Although median time to achieve a response is between 3–4 months, responses can take several months, up to 2 years, in some patients. On the other hand, some relapses are observed during follow-up, although the overall incidence of relapses is relatively low, especially among patients who achieve CR after autoHSCT. Thus, the relapse incidence at 10-years among CR patients was 21% in the Boston series (3). These combined results translate into a long-term survival between 45–60%, according to larger series (Table 1). Guidelines for an adequate evaluation of potential candidates to an autotransplant, recommended clinical care following transplant and basic criteria for assessment of response after auto-HSCT are summarised in Table 2.

5. Prognostic factors Several factors have been related to transplant outcome in AL. Thus, cardiac involvement, as assessed by several methods (congestive heart failure, thickened intraventricular wall on ultrasonography), is invariably identified as an adverse prognostic factor. In this regard, measurement of cardiac troponins (cTnT, cTnI) and pro-brain natriuretic peptide (NT-proBNP) provides a refined surrogate marker of myocyte damage in AL and these cardiac biomarkers are strong predictors of survival after auto-HSCT, with a significant shortened life expectancy among patients with increased levels (5). Concurrent renal failure is also associated with shorter survival after transplant and, in general terms, auto-HSCT is contraindicated in patients with severe renal failure. Variables related to disease extent also predict outcome after transplant. Thus, involvement of more than two organs correlates with an unfavourable prognosis (6). More recently, baseline level of FLC has been identified as a prognostic factor in this setting, with an increased risk of TRM in patients with higher pre-transplant levels (7). Of note, the degree of response achieved after transplant showed a striking correlation with long-term outcome, with the most favourable outcome observed in patients who achieved CR (8). On the contrary, patients who fail to achieve a significant response show a poor outcome, with rapid disease progression.

6. Role of auto-HSCT in the management of the disease: comparison with other treatment approaches Long-term survival after auto-HSCT appears to be prolonged, especially in patients who achieve CR after transplant, and compares favourably with historic controls. This observation, however, should be interpreted with caution as it might reflect


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Table 2: Specific considerations regarding evaluation of auto-HSCT in patient with primary amyloidosis Diagnostic accuracy

1. Demonstration of amyloid deposition in an appropriate tissue (Congo Red staining) 2. Confirmation of primary origin AL: - Presence of monoclonal light chain in amyloid fibril - (or) Detection serum/urine monoclonal light-chain

Is the patient a candidate to an autotransplant?

1. Age up to 65–70 years 2. Adequate performance status (£2) 3. Absence of limiting organ damage: - No compensated cardiac failure - Severe renal failure - Markedly increased bilirubin

Assessment of risk factors

1. Assessment of cardiac involvement: - Determination of cardiac biomarkers (cTnT, cTnI, NT-proBNP) - Echocardiogram (interventricular wall thickness) 2. Assessment of amyloid deposition: - Number of involved organs (renal, cardiac, hepatic, gastrointestinal, peripheral & autonomic neuropathy) - Serum FLC measurement

Recommended care during procedure

1. Monitor stem cell mobilisation and collection. Consider admission to an Intensive Care Unit 2. Adequate risk-adapted conditioning: - Standard dose: melphalan 200 mg/m2 - Reduced dose (if concurring risk factors): melphalan 140 mg/m2 3. Careful post-transplant management: - Close monitoring of cardio-vascular function - Prevention of mucosal & gastrointestinal bleeding: specific platelet transfusion policy (maintain > 50 x 109/L)

Adequate assessment of post-transplant response

1. Haematologic response: - CR: negative serum & urine immunofixation; normal k/l ratio & absolute value of FLC - PR: 50% reduction of serum M component, urine light chain & FLC 2. Organ response. Re-assessment of specific parameters of pre-transplant involved organ: - Renal (24-hour urinary protein, creatinine level) - Heart (functional class, septal thickness, ejection fraction) - Liver (alkaline phosphatase, liver size) - Nerve (nerve conduction)

merely a selection bias, since candidates for auto-HSCT constitute a selected population with better prognosis. Therefore, the precise impact of auto-HSCT in the management of the disease remains controversial; there are only a few studies 428

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comparing auto-HSCT with standard dose chemotherapy and the results of these studies are conflicting. Thus, on the one hand, a case control-study conducted by the Mayo Clinic, which compared the outcome of 63 patients who had received an auto-HSCT with that of 63 patients treated with standard therapy, showed a better outcome in the intensively treated subgroup, with a 4-year survival of 71 vs. 41% (9). This study, despite matching for main variables, has some limitations derived from the retrospective nature of the study and therapy administered in the control group, which mostly consisted of melphalan and prednisone. In contrast, recently published results of the only randomised trial comparing high-dose melphalan with standard dose melphalan and dexamethasone showed a better outcome in patients not randomised to high-dose melphalan, when analysed on an intentionto-treat basis (10). This study, however, also has some limitations, such as the relatively small number of patients included, 50 per arm, and the low compliance with the assigned therapy, since only two-thirds of patients randomised to intensive therapy finally underwent auto-HSCT. When the analysis was restricted to patients who actually received the assigned therapeutic option, no major differences were observed between the arms. Therefore, the potential benefit of high-dose melphalan and the exact target population of AL patients remain to be clarified in further studies. Moreover, the potential improvement in the “control arm” (i.e., based on nonintensive therapy) with the introduction of newer agents such as immunomodulators (thalidomide, lenalidomide) or the proteasome inhibitor bortezomib, should be considered while assessing the therapeutic role of auto-HSCT in this disease.

7. Future perspective and conclusions Given the relevance of achieving a response after transplant, several approaches for increasing the proportion of responses after auto-HSCT have been proposed. Thus, the Boston group is conducting a trial of tandem transplants, with performance of a second transplant with melphalan at a dose of 140 mg/m2, in patients not achieving CR after first transplant. The administration of post-transplant maintenance therapy with newer agents is another potential strategy for improving control of the disease. Finally, an allogeneic procedure using reduced-intensity conditioning has been performed in a minority of patients, with the aim of exploiting a possible “graft-versus-amyloidosis” effect. In conclusion, auto-HSCT results in haematological responses and organ improvement in a significant proportion of patients with AL. Moreover, responding patients show a relatively prolonged survival. Nonetheless, this procedure is associated with a exceedingly high mortality, with a TRM of at least 10%, reflecting the fragile condition of patients due to multiorgan damage caused by amyloid deposition. Therefore, careful selection of patients for transplant is critical. In this regard, a


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more refined assessment of cardiac involvement or the extent of the disease, by means of measurement of cardiac biomarkers and quantification of serum FLC, might contribute to a more accurate evaluation of patients prior to auto-HSCT. Nonetheless, the long-term effectiveness of high-dose melphalan for the management of the disease is currently unclear, and comparison with standard-dose agents shows conflicting results. Finally, development of new strategies to intensify and/or prolong response after auto-HSCT might improve the outcome. In this regard, further prospective studies are required to elucidate the role of auto-HSCT in AL.

References 1. Comenzo RL. AL amyloidosis. In: Multiple Myeloma and Related Disorders. Gahrton G, Durie BGM, Samson DM (eds), London, UK: Arnold; 2004: 400-419. 2. Gertz MA, Lacy MQ, Dispenzieri A, et al. Risk-adjusted manipulation of melphalan dose before stem cell transplantation in patients with amyloidosis is associated with a lower response rate. Bone Marrow Transplant 2004; 34: 1025-1031. 3. Skinner M, Sanchorawala V, Seldin DC, et al. High-dose melphalan and autologous stem cell transplantation in patients with AL amyloidosis: An 8-year study. Ann Int Med 2004; 140: 85-93. 4. Perfetti V, Siena S, Palladini G, et al. Long-term results of a risk-adapted approach to melphalan conditioning in autologous peripheral blood stem cell transplantation for primary (AL) amyloidosis. Haematologica 2006; 91: 1635-1643. 5. Dispenzieri A, Gertz M, Kyle RA, et al. Prognostication of survival using cardiac troponins and N-terminal pro-brain natriuretic peptide in patients with primary systemic amyloidosis undergoing peripheral blood stem cell transplantation. Blood 2004; 104: 1881-1887. 6. Gertz MA, Lacy MQ, Dispenzieri A, et al. Stem cell transplantation for the management of primary systemic amyloidosis. Am J Med 2002; 113: 549-555. 7. Dispenzieri A, Lacy MQ, Kartzmann JA, et al. Absolute values of immunoglobulin free light chains are prognostic in patients with primary systemic amyloidosis undergoing peripheral blood stem cell transplantation. Blood 2006; 107: 3378-3383. 8. Gertz MA, Lacy MQ, Dispenzieri A, et al. Effect of hematologic response on outcome of patients undergoing transplantation for primary amyloidosis: Importance of achieving a complete response. Haematologica 2007; 92: 1415-1418. 9. Dispenzieri A, Kyle RA, Lacy MQ, et al. Superior survival in primary systemic amyloidosis patients undergoing peripheral blood stem cell transplantation: A case-control study. Blood 2004; 103: 3960-3963. 10.Jaccard A, Moreau P, Leblond V, et al. High-dose melphalan versus melphalan plus dexamethasone for AL amyloidosis. N Engl J Med 2007; 357: 1083-1093. 11.Vesole DH, Pérez WS, Akasheh M, et al. High-dose therapy and autologous hematopoietic stem cell transplantation for patients with primary systemic amyloidosis: A Center for International Blood and Marrow Transplant Research Study. Mayo Clin Proc 2006; 81: 880-888. 12.Goodman HJ, Gillmore JD, Lachmann HJ, et al. Outcome of autologous stem cell transplantation for AL amyloidosis in the UK. Br J Haematol 2006; 134: 417-425. 430

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Mutiple Choice Questionnaire To find the correct answer, go to http://www.esh.org/ebmt-handbook2008answers.htm 1. Regarding diagnosis of primary AL, indicate the correct answer: a) Diagnosis of AL relies exclusively on Congo Red positivity . . . . . . . . . . . . . . . . b) A monoclonal light chain is rarely detected in serum . . . . . . . . . . . . . . . . . . . . . . c) Congo Red staining must be performed in the involved organ . . . . . . . . . . . . d) AL is generally a low burden monoclonal gammopathy, with a low level of plasma cell bone marrow involvement and M spike. . . . . . . . . . . . . . . .

2. All of the following statements about TRM in patients with AL undergoing autotransplantation are true, except one. Indicate the incorrect answer: a) Is exceedingly high, between 10–20%. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Is related to increased level of pro-brain natriuretic peptide (NT-proBNP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Is correlated with the heavy chain immunoglobulin idiotype of associated monoclonal gammopathy (IgG, IgA, IgM) . . . . . . . . . . . . . . . . . . . . . . d) Correlates with the number of involved organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Concerning response after autotransplant, which of the following is the correct answer? a) Haematologic response assessment is based on serum and urine immunofixation and serum free light chain measurement . . . . . . . . . . . . . . . . . b) Organ responses can be observed in many patients who do not achieve a significant haematologic response. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) Long-term survival is similar in patients achieving a complete response and those who obtain a partial response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d) Organ response are always observed in the early period post-transplant, i.e., no longer than 3 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. Which of the following complications can be observed in AL patients undergoing autotransplant? a) Frequent gastrointestinal bleeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


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b) DMSO-triggered cardiac arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c) G-CSF induced respiratory failure during mobilisation . . . . . . . . . . . . . . . . . . . . . . d) All the previous have been reported in this setting . . . . . . . . . . . . . . . . . . . . . . . . 5. Which of the following answers is not appropriate for describing outcome after autotransplant in AL patients? a) Long-term follow-up shows a significant proportion of long-term survivors, between 50–60%. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b) Relapse rate in patients achieving CR is relatively low,

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