Advances in Clinical Chemistry, Volume 47

ADVANCES IN CLINICAL CHEMISTRY VOLUME 47

Advances in CLINICAL CHEMISTRY Edited by GREGORY S. MAKOWSKI Clinical Laboratory Partners, LLC Newington, Connecticut USA

VOLUME 47

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

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Copyright ß 2009, Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-374796-9 ISSN: 0065-2423 For information on all Academic Press publications visit our website at www.elsevierdirect.com Printed and bound in USA 09 10 11 12 10 9 8 7 6

5 4 3 2

1

CONTENTS CONTRIBUTORS

................................................................................

ix

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

Amyloidosis KOSTANDINOS SIDERAS AND MORIE A. GERTZ 1. 2. 3. 4. 5. 6.

Abstract ... ................................................................................... Historical Perspective....................................................................... Pathogenesis ................................................................................. Diagnosis.. ................................................................................... Classification, Clinical Presentation, and Prognosis of Amyloidosis.................. Treatment . ................................................................................... References. ...................................................................................

2 2 3 14 20 28 36

Urinary Markers in Colorectal Cancer BO FENG, FEI YUE, AND MIN-HUA ZHENG 1. 2. 3. 4. 5.

Abstract ... ................................................................................... Introduction ................................................................................. Potential Urinary Markers for Colorectal Cancer ...................................... Analytical Techniques and Data Analysis ............................................... Conclusions .................................................................................. References. ...................................................................................

45 46 47 50 53 53

Effect of Hormone Replacement Therapy on Inflammatory Biomarkers PANAGIOTA GEORGIADOU AND EFTIHIA SBAROUNI 1. 2. 3. 4. 5. 6.

Abstract ... ................................................................................... Introduction ................................................................................. Inflammation and Vascular Disease ...................................................... Mechanisms of Action of HRT in Vascular Biology ................................... Effects of HRT on Inflammatory Markers .............................................. Conclusion ................................................................................... References. ...................................................................................

v

60 60 62 65 71 82 83

vi

CONTENTS

Personalized Clinical Laboratory Diagnostics KEWAL K. JAIN 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Abstract....................................................................................... Introduction.................................................................................. Basic Concepts of Personalized Medicine ................................................ Molecular Diagnostic Technologies for Personalized Medicine. ....................... Role of PCR in Development of Personalized Medicine.. .............................. Combined PCR–Enzyme-Linked Immunosorbent Assay (ELISA).................... Non-PCR Methods.......................................................................... Direct Molecular Analysis Without Amplification ...................................... SNP and Personalized Medicine ........................................................... Genetic Variations in the Human Genome Other Than SNPs ......................... Role of Biomarkers in Personalized Medicine ........................................... Application of Biochip Technology in Developing Personalized Medicine ...................................................................... Role of Nanobiotechnology-Based Diagnostics in Personalized Medicine ...................................................................... Role of Cytogenetics in Personalized Medicine .......................................... Integration of Molecular Diagnostics and Therapeutics ................................ Concluding Remarks and Future Prospects.. ............................................ References ....................................................................................

96 96 96 99 99 102 103 103 103 105 108 109 111 114 116 117 118

Verification of Method Performance for Clinical Laboratories JAMES H. NICHOLS 1. 2. 3. 4.

Abstract....................................................................................... Introduction.................................................................................. ISO Quality Management System: The Fundamentals of Quality..................... Laboratory Quality Standards in Regulations and Accreditation Guidelines ................................................................... 5. Comparison of Quality Requirements .................................................... 6. Performing Method Verification........................................................... 7. Summary ..................................................................................... References ....................................................................................

121 122 123 129 131 132 136 136

Interpreting the Proteome and Peptidome in Transplantation TARA K. SIGDEL, R. BRYAN KLASSEN, AND MINNIE M. SARWAL 1. 2. 3. 4. 5.

Abstract....................................................................................... Introduction.................................................................................. Application of Proteomics and Peptidomics in Transplantation....................... Important Issues ............................................................................. Conclusion ................................................................................... References ....................................................................................

140 140 155 160 162 163

CONTENTS

vii

Biomarkers in Long-Term Vegetarian Diets IRIS F.F. BENZIE AND SISSI WACHTEL-GALOR 1. Introduction ................................................................................. 2. Possible Nutritional Deficiencies in Association with Long-Term Vegetarian Diets. ............................................................................ 3. Biomarkers of Oxidant/Antioxidant Balance in Association with Vegetarian Diets....................................................................... 4. Biomarkers that Reflect Lower Risk of Disease in Long-Term Vegetarians ......... 5. Biomarkers to Differentiate the Vegetarian from the Nonvegetarian................. 6. Summary and Recommendations for Clinical Chemistry .............................. References. ...................................................................................

172 173 186 194 207 209 210

Effect of Caloric Restriction on Oxidative Markers JAN SˇKRHA 1. 2. 3. 4. 5. 6. 7. 8.

Abstract ... ................................................................................... Introduction ................................................................................. Foods and ROS Generation ............................................................... Mitochondria as a Source of Reactive Oxygen and Nitrogen Species ................ Caloric Restriction and Oxidative Stress ................................................. Oxidative Stress Markers by Caloric Restriction........................................ Data Interpretation ......................................................................... Conclusions .................................................................................. References. ...................................................................................

224 224 225 226 229 232 240 241 242

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COLOR PLATE SECTION AT THE END OF THE BOOK

249

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

IRIS F.F. BENZIE (171), Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong BO FENG (45), Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China PANAGIOTA GEORGIADOU (59), 2nd Department of Cardiology, Onassis Cardiac Surgery Center, Athens, Greece MORIE A. GERTZ (1), Division of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA KEWAL K. JAIN (95), Jain PharmaBiotech, Basel, Switzerland R. BRYAN KLASSEN (139), Department of Pediatrics—Nephrology, Stanford University Medical School, Stanford University, Stanford, California 94305, USA JAMES H. NICHOLS (121), Professor of Pathology, Tufts University School of Medicine and Medical Director, Clinical Chemistry, Baystate Health, Springfield, Massachusetts 01199, USA MINNIE M. SARWAL (139), Department of Pediatrics—Nephrology, Stanford University Medical School, Stanford University, Stanford, California 94305, USA EFTIHIA SBAROUNI (59), 2nd Department of Cardiology, Onassis Cardiac Surgery Center, Athens, Greece

ix

x

CONTRIBUTORS

KOSTANDINOS SIDERAS (1), Division of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA TARA K. SIGDEL (139), Department of Pediatrics—Nephrology, Stanford University Medical School, Stanford University, Stanford, California 94305, USA JAN SˇKRHA (223), Laboratory for Endocrinology and Metabolism and 3rd Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, U Nemocnice 1, 128 08 Prague 2, Czech Republic SISSI WACHTEL-GALOR (171), Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong FEI YUE (45), Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China MIN-HUA ZHENG (45), Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

PREFACE I am pleased to present volume forty‐seven of Advances in Clinical Chemistry series. In this first volume for 2009, an array of relevant clinical laboratory topics is presented. The biochemistry of amyloidosis is explored with respect to the microenvironment, mechanisms of organ dysfunction, and the role of toxic intermediates. The importance of low molecular weight urinary biomarkers associated with colorectal cancer, one of the most commonly diagnosed cancers worldwide, is reviewed using a metabolomic approach. The role of hormone replacement therapy is investigated with respect to inflammatory biomarkers and vascular disease in women. It is noteworthy that cardiovascular disease risk is typically underestimated in the female population. A wonderful review on personalized clinical laboratory diagnostics is presented by a leader in the field of pharmacogenomics. Verification of method performance is reviewed with respect to a number of international quality standards, accreditation agencies, and regional laws. Another topic, application of the proteome to impact on organ transplantation outcomes, is also presented. This volume is concluded by two reviews on diet. In the first paper, biomarkers associated with vegetarian diets are explored. The second review investigates the role of caloric restriction on lifespan as evidenced by impact on oxidative markers. I extend my appreciation to each contributor of volume forty‐seven and thank colleagues who contributed to the peer review process. I extend my thanks to my Elsevier editorial liaison, Gayathri Venkatasamy. I sincerely hope the first volume of 2009 will be enjoyed by our diverse readership. As always, I warmly invite comments and suggestions for future review articles for the Advances in Clinical Chemistry series. In keeping with the tradition of the series, I would like to dedicate volume forty‐seven to my father‐in‐law Dr Gale R. Ramsby. GREGORY S. MAKOWSKI

xi

ADVANCES IN CLINICAL CHEMISTRY, VOL. 47

AMYLOIDOSIS Kostandinos Sideras and Morie A. Gertz1 Division of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Historical Perspective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Structure of the Amyloid Fibril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Amyloid Aggregation Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Interactions of the Amyloid Fibril with the Microenvironment . . . . . . . . . . . . . 3.4. Tropism of Amyloid Proteins for DiVerent Organs . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Mechanisms of Organ Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Suspecting the Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Screening for Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Establishing the Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Typing of Amyloid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Imaging of Amyloid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Classification, Clinical Presentation, and Prognosis of Amyloidosis . . . . . . . . . . . . . . 5.1. Primary Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Secondary Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Familial Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Senile Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Localized Amyloidosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Strategies Aimed at Eradicating the Production of the Amyloid Precursors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Native Protein Structure Stabilizing Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Amyloid Fibril Destabilizing Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Immunologic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Supportive Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Treatment of Localized Amyloidosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2 2 3 3 6 9 12 13 14 14 15 16 17 18 20 20 21 23 25 26 28 28 32 33 34 35 35 36

Corresponding author: Morie A. Gertz, e‐mail: [email protected] 1

0065-2423/09 $35.00 DOI: 10.1016/S0065-2423(09)47001-X

Copyright 2009, Elsevier Inc. All rights reserved.

2

SIDERAS AND GERTZ

1. Abstract Amyloidosis is a heterogeneous group of diseases in which an otherwise normal protein, with or without proteolytic cleavage, forms insoluble amyloid fibrils. These, in turn, deposit in various organs and cause dysfunction. A wide range of diseases are associated with amyloidosis such as Alzheimer’s disease, multiple myeloma, other plasma cell disorders, and chronic inflammation, either as a cause, or result, of amyloid production. This heterogeneity in cause and presentation leads to an incomplete understanding of the pathophysiology of amyloid disease. As such, study of this complicated disease process presents significant challenges. The purpose of this review article is to introduce the biochemistry of amyloidosis including ultrastructure analysis, models of monomer aggregation, the importance of the amyloid microenvironment, and the mechanisms of organ dysfunction, including the role of ‘‘toxic intermediates.’’ Pathophysiologic analysis of amyloidosis will focus on diagnostic tools as well as the classification of the various forms of amyloidosis. Finally, treatment of amyloidosis will be reviewed including traditional and established modalities. We will also introduce new and novel treatment options as they relate to the basic pathophysiology of this complex and heterogeneous disorder.

2. Historical Perspective Although the term ‘‘amyloid’’ was first used in botany as early as 1838 by Matthias Schleiden to describe plant starch, and subsequently in 1854 by Rudolf Virchow to describe abnormal macroscopic deposits, the disease of amyloidosis, though with diVerent names, was known at least since the mid‐ seventeenth century [1–3]. Anatomists and pathologists frequently described organs with a ‘‘lardaceous’’ or ‘‘waxy’’ appearance and a major debate in the mid‐nineteenth century consisted of whether the disease was caused by deposition of a lard‐like or a starch‐like substance. Professor Karl von Rokitansky from Vienna was the main proponent of the idea of ‘‘lardaceous change’’ (wax). On the other hand, the German (Berlin) professor Rudolf Virchow believed that a starch‐like substance was responsible for the abnormal spleens he examined. He had come to that conclusion after he stained the abnormal deposits with a combination of iodine and sulfuric acid, and found that, just like starch, the tissues stained pale blue, and thus must be carbohydrate, coining the term amyloid. Both Rokitansky and Virchow were wrong. George Budd found no lardaceous substance in the liver of a patient with amyloidosis and in 1859

AMYLOIDOSIS

3

Friedreich, Nicolau, and Kekule found no starch‐like substance in the spleens described by Virchow, suggesting, to their credit, that the ‘‘amyloid’’ substance was probably albuminoid in nature [4]. In 1920, Schmiedeberg described the similarity of amyloid to serum globulin, which strongly suggested its proteinaceous nature. Early reports of amyloidosis were invariably described in patients with chronic inflammatory conditions like tuberculosis, syphilis, leprosy, and rheumatoid arthritis. These were the early reports of secondary (AA) amyloidosis. However, occasional reports in patients without inflammatory conditions were also made. Sir Samuel Wilks is credited to be the first physician to describe such a patient, a 56‐year‐old with ‘‘lardaceous change.’’ This was probably the first report of a patient with primary (AL) amyloidosis. Soyka was the first to describe both cardiac amyloidosis in patients of advanced age and senile amyloidosis. The Congo red stain, which since 1884 was used in the textile industry to stain cotton, was used by Bennhold in 1922 to stain amyloid, for which it was found to have a strong aYnity [1]. However, it was Divry and Florkin who in 1927 found that Congo red stained amyloid exhibited green birefringence under polarized light. The first description of the amyloid fibril came from Cohen and Calkins in 1959 who noticed the fibrillar structure of amyloid when viewed under the electron microscope, thus being the first to definitely conclude that amyloid was not ‘‘amorphous’’ as suggested by its appearance under the light microscope [5]. Finally, in 1968, Eanes and Glenner discovered the b‐pleated sheet nature of the amyloid fibril which explained some of the resistance of the structure to the action of solvents [6].

3. Pathogenesis 3.1. STRUCTURE OF THE AMYLOID FIBRIL Since Cohen and Calkins described the ‘‘fibrillar’’ nature of amyloid, multiple investigators used electron microscopy to further characterize the structure of the amyloid fibril [2]. Amyloid from diVerent human and animal sources was found to be composed of similar, rigid, nonbranching fibrils of indeterminate, long length (anywhere from 100 to 1600 nm), with an average width of 7.0–12 nm [2]. Each amyloid fibril in turn is composed of a number of b‐pleated sheets (protofilaments), each 2.5–3.5 nm in diameter, which run along the longitudinal axis of the amyloid fibril and slowly twist [7] creating a helical repeat of b‐pleated sheets of about every 11.5 nm. The b‐pleated sheets run perpendicular to the long axis of the fibril. This structure has

4

SIDERAS AND GERTZ

been called the continuous b‐sheet helix [8] or the cross‐b spine, and is responsible for the characteristic cross‐b X‐ray diVraction pattern of amyloid (Fig. 1). Further examination of the ultrastructure of the cross‐b spine has shown it to be a cross‐double b‐sheet, with side chains protruding from the two sheets forming a dry, tightly self‐complementing steric zipper, bonding the sheets [9]. Every segment is bound to its two neighboring segments through stacks of both backbone and side‐chain hydrogen bonds (Fig. 2). Despite this similarity at the protofilament level, the amyloid precursor proteins do not share a common sequence homology, size, function, or tertiary structure. At least 25 diVerent human proteins have been described as precursors to amyloid fibrils and cause a variety of diVerent amyloid‐ related diseases [10] (Table 1). Examples include the monoclonal l or k immunoglobulin light chain which causes AL amyloidosis (related to plasma cell disorders), the serum amyloid A protein (SAA) related to systemic inflammatory conditions (secondary or AA amyloidosis), and the Val30Met and Gly47Val transthyretin (TTR) variants related to hereditary amyloidosis. In addition to these 25 proteins that have the ability to form amyloid fibrils in vivo, there are a number of synthetic amyloid fibrils in existence which are used for research purposes. Moreover, there are many other proteins that are capable of forming fibrillar deposits when taken out of physiologic conditions but do not have the ability to cause amyloidosis in the human body. Another organizational diVerence between diVerent types of amyloid is at the number of protofilaments that form the amyloid fibril. The amyloid fibril, visible in vivo, is composed of anywhere from three to six protofilaments

115 Å 24 b-strands

FIG. 1. Molecular model of the common core protofilament structure of amyloid fibrils. A number of b‐sheets (four illustrated here) make up the protofilament structure. These sheets run parallel to the axis of the protofilament, with their component b‐strands perpendicular to the fibril axis. With normal twisting of the b‐strands, the b‐sheets twist around a common helical axis ˚ containing 24 that coincides with the axis of the protofilament, giving a helical repeat of 115.5 A b‐strands (this repeat is indicated by the boxed region). Reprinted from Ref. [7], Copyright 1997, with permission from Elsevier.

5

AMYLOIDOSIS

A Gln5

B

Gly1

Asn3

Asn2

Tyr7

Gln4

Asn6

Asn3 Gln5

4.87 Å

a

b

Gln5 Asn3

c C

Gln5

Asn3

Asn2

Tyr7

Gln4

b c D

a

a c

c

Wet interface Dry interface

E

Asn6

Asn2

Gln4

b

3.0 2.8

2.9

2.9

2.9

3.0 2.9 2.8

Gly1 Gln5

Asn3 2.9

2.9

2.8 2.9 2.8 2.9

3.1

3.1

Tyr7

3.2 2.8

2.8 3.0 2.9 2.7 3.2 2.9

2.9

2.8

2.8 3.0 2.9 2.7 3.2 2.9

2.9

3.2

2.8

a FIG. 2. Structure of GNNQQNY, a seven‐residue peptide segment from the yeast protein Sup35. Unless otherwise noted, carbon atoms are colored in purple or grey/white, oxygen in red, and nitrogen in blue. [9] (A) The pair‐of‐sheets structure of the fibril‐forming peptide GNNQQNY. The dry interface is between the two sheets, with the wet interfaces on the outside

6

SIDERAS AND GERTZ

which are laterally associated with each other. For example, the amyloid fibril of Val30Met TTR, the most common type of familial amyloidosis, is composed of four protofilaments arranged in a square array around an electron‐lucent center, whereas the amyloid fibril of amyloid Ab (which causes Alzheimer’s disease) is composed of five or six protofilaments [11]. 3.2. AMYLOID AGGREGATION MECHANISMS Despite this knowledge, advances in understanding the amyloid fibril at the atomic level have been more diYcult. It is not clear, for example, if the specific amino acid sequence plays a role in determining the ability of a protein to form a cross‐b spine, to what extent it aids in the stabilization of the amyloid fibril, and how this sequence aVects interaction of the amyloid fibril with the microenvironment (i.e., amyloid P component, heparan sulfate proteoglycans (HSPG), apolipoprotein E, extracellular matrix, lipid bilayer). A number of models have been proposed in this regard [12] (Fig. 3). Partially folded intermediates are thought to play an important role in the pathogenesis of amyloidosis. It is thought that thermodynamic instability of the protein native structure leads to partially folded intermediates several of which have been shown to form amyloid fibrils readily [13, 14]. The same mechanism is thought to lead to amorphous deposits of immunoglobulin light chains and it is unclear why some proteins favor deposition as amyloid fibrils versus amorphous deposits. The process appears to be dependent on the specific partially folded structure as diVerent intermediates of the same protein have been shown to deposit either as amyloid fibrils or amorphous deposits [15]. At a diVerent organizational level, oligomers can act as intermediates in the process of fibrilogenesis as well. For example, in the case of b‐2‐microglobulin, the formation of the final amyloid fibril is proceeded by the

surfaces. (B) The steric zipper viewed edge on (down the a‐axis). (C) The GNNQQNY crystal viewed down the sheets (i.e., from the top of panel a, along the b‐axis). Six rows of b‐sheets run horizontally. Peptide molecules are shown in black and water molecules are represented by redþ. ˚ ) alternating with a wider Notice that the sheets are in pairs, with a closely spaced pair (8.5 A ˚ ) pair. The wide spaces between sheets (wet interfaces) are filled with water spaced (15 A molecules, whereas the closely spaced interfaces (dry interfaces) lack waters, other than those hydrating the caroboxylate ions at the C‐termini of peptides. The atoms in the lower left unit cell are shown as spheres representing van der Waals radii. (D) The steric zipper. This is a close up view showing the remarkable shape complementarity of the Asn and Gln side chains protruding into the dry interface. (E) Views of the b‐sheets from the side (down the c axis), showing three b‐strands with the interstrand hydrogen bonds. Side chain carbon atoms are highlighted in yellow. Backbone hydrogen bonds are shown by purple or black dots and side chain hydrogen bonds by yellow ˚ units. Reprinted from Ref. [9], Copyright dots. The length of each hydrogen bond is noted in A 2005, with permission from Macmillan Publishers Ltd.

7

AMYLOIDOSIS TABLE 1 SOME OF THE PROTEINS KNOWN TO CAUSE CLINICAL AMYLOID DISEASE IN HUMANS

Precursor protein

Human disease

Major causative association

Major clinical manifestation Renal, cardiac, GI, peripheral nervous system Much less frequent than AL amyloidosis Renal

Immunoglobulin light chain

Primary (AL) amyloidosis

Plasma cell disorders

Immunoglobulin heavy chain Serum amyloid A

Primary (AH) amyloidosis Secondary (AA) amyloidosis Familial

Plasma cell disorders

Transthyretin (TTR)

Inflammation Mutation of ATTR

Senile

Wild‐type ATTR, aging

Apolipoprotein AI

Familial

Mutation of Apo‐AI

Apolipoprotein AII

Familial

Lysozyme

Familial

Fibrinogen Aa‐chain

Familial

Gelsolin

Familial (Finish type)

Point mutation of stop codon leading to additional 20 amino acid residues Mutation of lysozyme Mutation of fibrinogen Aa‐chain Mutation of gelsolin

Cystatin C

Familial (Islandic type)

Mutation of cystatin

b‐2‐Microglobulin

Ab2M

Hemodialysis

Peripheral nervous system, heart Multiple organs, cardiac most clinically prominent Very slowly progressive disease Renal

Kidney, liver, lungs, and spleen Renal

Corneal dystrophy, cranial neuropathy, and cutis laxa Amyloid angiopathy and cerebral hemorrhage Joints

formation of dimeric, tetrameric, and hexameric intermediates in time scales much faster than the time scale of fibrilogenesis itself (minutes to hours versus weeks to months) [16]. In this model, the monomer (b‐2‐microglobulin) requires activation to an ‘‘activated state’’ (in the presence of copper) which then rapidly forms oligomers, by the sequential addition of dimmers, within minutes to hours. These oligomers form by the process known as domain swapping. Oligomers then form mature amyloid in timescales of weeks to months.

8

SIDERAS AND GERTZ

Model class

Native protein

Intermediate

Fibril

Refolding

Natively disordered

Gain-ofinteraction

FIG. 3. Cartoon depicting the three general types of models for the conversion of proteins from their native state to the amyloid‐like state. In refolding models, the protein unfolds and then refolds into a diVerent structure, which is stabilized largely by backbone hydrogen bonds. In natively disordered models, the cross‐b spine forms from protein segments that are poorly structured in the native state. In gain‐of‐interaction models, a change in the conformation of the protein frees a segment for interaction with segments from other molecules. An extensive portion of the native structure is maintained in the fibril. Reprinted from Ref. [12], Copyright 2006, with permission from Elsevier.

There are also kinetic intermediates of the amyloid fibril formation that are called protofibrils (not to be confused with the protofilaments we discussed earlier which are distinct substructural elements of the amyloid fibril) [17]. These protofibrils are in equilibrium with monomeric or dimeric forms of Ab molecules (in the case of Alzheimer’s disease), and tend to accumulate during fibrillogenesis on preformed fibrils and transition into mature fibrils themselves [16, 18]. In vitro, protofibrils have been shown to grow by both monomer elongation and by lateral association [19]. Recently, time‐resolved structure analysis of growing b‐amyloid fibrils, focusing on the first 2 h of fibrilogenesis, shows additional nonfibrillar intermediates present at these early stages in addition to the monomers and the protofibrils just described [20]. The propensity of diVerent proteins to form amyloid fibrils may be relatively common. However, what determines the ability of proteins to deposit in human tissues depends on the kinetics and thermodynamics of the specific ‘‘fibrillar state,’’ posttranslational modifications of the protein (i.e., proteolysis, as is the case with the immunoglobulin light chains and other proteins where only a fragment makes up the amyloid fibril), and interactions of the amyloidogenic protein with its microenvironment, namely the

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extracellular matrix, serum amyloid P (SAP) component, glycosaminoglycans, apolipoprotein E, and the lipid membrane bilayer itself [21]. In addition, the amyloidogenic protein is generally produced in suYcient amounts by an independent pathologic process (i.e., plasma cell proliferative disorders in the case of AL amyloidosis or inflammation in the case of AA amyloidosis) before a clinical amyloidosis syndrome can result from any given protein. This overproduction, however, does not always need to be the case since in certain situations, as in the case of some patients with senile amyloidosis, the plasma concentration of the amyloidogenic protein is in fact less than normal, but in suYcient quantity to result in the clinical syndrome [22]. Another mechanism for production of amyloidogenic proteins includes specific genetic mutations that make an otherwise normal protein carry the ability to interact with the microenvironment in a way that makes amyloid fibril formation and deposition possible as in the case of TTR. TTR also has the ability to form amyloid deposits in its wild form with advanced age and in this case it is unclear what triggers its deposition. Proteolytic cleavage is also an important part of amyloid formation for some of the amyloid‐forming proteins. For example, in AL amyloidosis it is usually only a variable region, a J‐segment and a variable part of the constant region that forms the amyloid fibril rather than the full length immunoglobulin light chain. SAA, which is responsible for amyloid formation in response to inflammation (secondary or AA amyloidosis), does so only after cleavage of the 76‐residue N‐terminal part of the protein. The requirement for proteolytic cleavage need not be exact and fragments of variable length can be found in amyloid deposits. In the case of Alzheimer’s disease, however, the proteolytic cleavage of the amyloidogenic protein APP needs to be at exact locations before the pathologic fragments Ab 1–40 and Ab 1–42 are formed. Figure 4 schematically depicts some of these mechanisms of amyloid production, from abnormalities at the genetic level, to requirements for posttranslational modification, to overproduction of the amyloidogenic protein.

3.3. INTERACTIONS OF THE AMYLOID FIBRIL WITH THE MICROENVIRONMENT Despite the knowledge of the proteinaceous nature of the amyloid fibril, as early as 1894 glycosaminoglycans were suspected to be part of amyloid deposits by the Italian surgeon and anatomist Ruggero Oddi. In fact, later immunohistochemical analysis of amyloid deposits, found other nonfibril‐

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X

Abnormal gene Amyloid fibril Post-translational events (s)

Expression

Degradation

Amyloid fibril

FIG. 4. Mechanisms for amyloid fibril formation are shown schematically. Open circles represent normal protein molecules, solid circles are amyloidogenic protein molecules, and overlapping circles are the fibrils made from these proteins. Reprinted from Ref. [154], reproduced with permission from Elsevier.

forming proteins in close association with the amyloid fibril. The glycoprotein ‘‘SAP component,’’ glycosaminoglycans (mainly heparan sulfate), and apoprotein E have been found in amyloid deposits of diVerent types of amyloidosis [2]. Amyloid P component is a pentameric (thus the P) globular protein that binds reversibly in a Ca2þ dependent fashion to amyloid fibrils [23] and it is a universal constituent of amyloid deposits. It constitutes anywhere from 12% to 20% of the dry weight of amyloid deposits [24, 25]. It is a normal plasma protein (SAP component) which, like C‐reactive protein, is a small pentraxin produced in the liver. It appears to function physiologically as a key component of innate immunity and inflammation [26]. Occasionally, it can form fibrillar deposits itself and causes a form of amyloidosis secondary to inflammation. However, its main interest lies with its nonfibrillar native form and the nature of its association with the amyloid fibril. When 123I‐labeled SAP component is injected, patients with amyloidosis clear the radiolabeled SAP from the plasma more rapidly, but retain it in tissues for significantly longer periods, indicating its rapid localization to amyloid deposits [27]. In fact, this property of amyloid P component has been used clinically

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for purposes of diagnosis by using scintigraphy to detect the accumulated amyloid P component [28] (Fig. 5). The diagnostic specificity in one study was 90% for AL and AA amyloidosis and only 48% for ATTR‐type amyloidosis with a sensitivity of 93%.

FIG. 5. Total body 123I‐SAP scan, anterior (A) and posterior (P) view, 24 h after injection. (A) Male control. Blood pool activity as well as minor nonspecific uptake can be seen in the (blocked) thyroid, nasopharynx, stomach, urine in bladder, and testicles. (B–F) Five patterns of organ uptake in AA amyloidosis: (B) kidney uptake only (and prostatism with urine retention in bladder); (C) intense splenic uptake only; (D) spleen and kidney uptake; (E) spleen, kidney, and adrenal gland; (F) spleen, kidney, and liver uptake (and nonspecific uptake in the stomach adjacent to the left liver lobe, providing the illusion of liver enlargement). (G–I) Three examples of organ uptake in AL amyloidosis: (G) joints only (8%); (H) spleen and liver (20%); (I) spleen, liver, and bone marrow (10%). Reprinted from Ref. [28], Copyright 2006, with permission from Elsevier.

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The role of SAP component is not understood but it appears to prevent the degradation of amyloid fibrils by proteolytic enzymes [25]. It is unclear how it is structurally related to the amyloid fibril and whether it plays a role in the formation of the amyloid fibril or simply stabilizes the fibril once it is formed. HSPG have also been found to be closely associated with amyloid fibrils. Glycosaminoglycans have been isolated from water extracted amyloid fibrils as well as directly from amyloidogenic tissues [29]. Glycosaminoglycans have been shown to promote the aggregation of amyloidogenic proteins [30]. Again the precise mechanism of interaction is unclear. One theory suggests that the chemical properties of glycosaminoglycans as polyanions have an important role in catalyzing protein aggregation and stabilization of amyloid fibrils. This is supported by the evidence that nucleic acid, another polyanion, has been isolated from the brains of patients with Alzheimer’s disease, a form of amyloid deposition disease [31]. In fact other polyanions, like ATP, DNA, and heparin are also able to promote aggregation of amyloidogenic proteins [30]. Although the precise role and interaction of these nonfibrillar components is not well understood, it is noteworthy that in vitro amyloid can be formed without the aid of glycoproteins and glycosaminoglycans adding confusion to their role in the pathogenesis of amyloid. 3.4. TROPISM OF AMYLOID PROTEINS FOR DIFFERENT ORGANS Very little is understood on why diVerent amyloidogenic proteins ‘‘prefer’’ to deposit in diVerent organs. Although multiple associations have been made, we have been unable to explain the reasons behind amyloid tropism. For example, familial TTR amyloidosis aVects primarily the peripheral nervous system while native TTR, which causes senile amyloidosis, aVects primarily the heart when present in suYcient quantities to cause disease. Mutations of Apolipoprotein AI lead to a rare form of familial amyloidosis. Patients with mutations at the amino‐terminal present with renal, hepatic, and occasional cardiac amyloid, while patients with mutations at the carboxy‐terminal present with cardiac, cutaneous, and laryngeal involvement [32]. No success in explaining this striking observation has yet been made. It is also well known that certain subtypes of l light chain amyloidosis carry a propensity to aVect diVerent organs [33]. In a retrospective study of 60 patients with AL amyloidosis patients with clones derived from the 6a V (lambda VI) germ line gene were more likely to present with dominant renal involvement, whereas those with clones derived from the 1c, 2a2, and 3r V (lambda) genes were more likely to present with dominant cardiac and multisystem disease [33]. Patients with V (kappa) clones were more likely to have dominant hepatic involvement and patients with multiple myeloma

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were more likely to present with dominant cardiac involvement independent of which germ line gene was responsible for the plasma cell clone producing the amyloid [33]. Again, despite this knowledge, no specific theory has explained these associations. 3.5. MECHANISMS OF ORGAN DYSFUNCTION One of the ways amyloid can cause organ dysfunction is through anatomic interference or destruction of the involved organ. In this case, the organ dysfunction is directly proportional to the amount of amyloid deposited. For example, in the heart, amyloid deposition can occur within the cardiac muscle interfering with the hearts ability to contract properly leading to heart failure. Deposition within the conducting system leads to fatal arrhythmias, a main cause of death for this population. Deposition of amyloid in the kidney interferes with the structure and function of the glomerulus causing heavy proteinuria, or interferes with the tubular system causing azotemia and nephritis. Deposition in the arterial vasculature causes friable vessels which can result in life threatening bleeding (another cause of bleeding diathesis involves the deposition of the procoagulant factor X into the amyloid deposits leading to systemic deficiency of factor X). However, the organ dysfunction is not always proportional to the amount of amyloid present. For example, even small amounts of light chain can cause organ dysfunction. In this case it is thought that the amyloid fibril is ‘‘toxic’’ to the organ involved. This is especially true in the case of AL amyloidosis and is part of the reason that a partial response after treatment of the disease may not lead to clinical improvement of the involved organs. Organ improvement is usually the case only after a relatively complete response and elimination of the production of amyloid. After disease relapse, the clinical manifestations of organ dysfunction return. In fact there is ample evidence to suggest that, at least in certain situations, it is the protofibril and oligomeric intermediates that are toxic to organs rather than the mature amyloid fibril itself. For example, transgenic mice that overexpressing the Ab‐peptide of Alzheimer’s disease have evidence of neural degeneration prior to the formation of amyloid plaque, suggesting the presence of toxic intermediates [34]. In fact, nonfibrillar oligomers and protofibrillar intermediates of the Ab‐peptide have been shown to directly induce neurotoxicity [35, 36]. These findings may explain why the plaque load in Alzheimer’s disease does not correlate well with the severity of disease. In another example when neuroblastoma cell lines have been treated with TTR, the protein that causes familiar amyloidosis, it is the immature amyloid which has not yet properly aggregated that causes cell death rather than the mature amyloid fibril [37].

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4. Diagnosis 4.1. SUSPECTING THE DIAGNOSIS One of the greatest challenges in amyloidosis is suspecting the diagnosis. The majority of the signs and symptoms are not pathognomonic. On the contrary, signs and symptoms are nonspecific and they are shared by many other more common diseases. The symptoms are generally directly related to the organs involved. As a result renal involvement, which is the most commonly involved organ in amyloidosis, is expected to present with nephrotic range proteinuria resulting in edema, weight loss, and fatigue. Peripheral nerve involvement causes paresthesias, and cardiac involvement commonly causes dyspnea, edema, and weight gain. Gastrointestinal involvement can cause a variety of symptoms including malabsorption with resulting weight loss, diarrhea, or less commonly pseudo‐obstruction of the upper gastrointestinal tract. Senile amyloidosis commonly aVects the heart, and in the case of Alzheimer’s disease amyloid deposits in the brain lead to cognitive decline. In less common situations amyloidosis has been known to involve almost any organ, and in these cases the clinical manifestations are more atypical. Pathognomonic findings exist in amyloidosis but they are uncommon, and when they occur they are easily overlooked. For example, tongue enlargement, periorbital purpura, and periarticular amyloid infiltration (shoulder pad sign), although specific to amyloidosis, are only present in 15% of patients with AL amyloidosis. Thus, a high index of suspicion is needed by any clinician faced with these symptoms. In certain clinical scenarios, on the other hand, the diagnosis of amyloidosis should always be suspected. Nondiabetic patients who present with nephrotic range proteinuria should always be screened for amyloidosis since 10% of these patients are found to have the disease [38]. Patients with evidence of cardiomyopathy without symptoms of ischemia or evidence of atherosclerotic heart disease should also be screened for amyloidosis. Findings in the electrocardiogram such as low voltage and the pseudo‐infarction sign as well as specific findings in the echocardiogram such as wall thickening and poor filling in the absence of systemic hypertension, can lead clinicians to the diagnosis. Other clinical scenarios include peripheral neuropathy in a nondiabetic patient, unexplained hepatomegaly, patients with tongue enlargement, and patients with unexplained malabsorption, weight loss, or pseudo‐obstruction. Finally, patients with established multiple myeloma and symptoms that are not consistent with myeloma should also be screened always.

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4.2. SCREENING FOR AMYLOIDOSIS Tissue biopsy is the only method available for establishing the diagnosis of amyloidosis. However, tissue biopsy sometimes is diYcult to perform and can carry significant risks especially if organs like the heart, kidney, or liver are involved. In these cases screening for amyloidosis first is desirable. However, it needs to be kept in mind that noninvasive screening is available only for AL amyloidosis. This is due to the fact that in AL amyloidosis detection of the underlying plasma proliferative disorder and immunoglobulin light chains is possible. No such neoplastic underlying conditions exist for AA amyloidosis, hereditary amyloidosis, or localized amyloidosis, and as a result biopsy is the only means of detecting the disease. If screening is negative in a patient where there is a high index of suspicion for amyloidosis, as is the case of a patient with characteristic echocardiographic findings of cardiac amyloid but negative screening, a biopsy needs to be performed. The current standard for screening for AL amyloidosis is testing for the presence of immunoglobulin light chains by immunofixation of both the serum and urine and by a free immunoglobulin light chain assay. A commonly occurring error in clinical practice is screening for amyloidosis by serum or a urine protein electrophoresis without immunofixation. The reason is that although serum protein electrophoresis is a good test for screening for the circulating heavy chain produced by multiple myeloma cells, it generally does not detect light chains, because light chains are in small quantities in the serum and do not produce a spike on the electrophoresis. On the contrary, immunofixation allows for the identification of monoclonal proteins in the serum and/or the urine in 90% of cases of AL amyloidosis. Also, the reason for testing both the serum and the urine is that 25% of patients with AL amyloidosis that have a negative serum immunofixation will be found to have a positive urine immunofixation alone. However, the most sensitive test for screening for amyloidosis is an immunoglobulin free light chain assay [39]. This technique, which uses a nephelometric assay with antibodies that recognize only free light chains not bound to heavy chain, has the ability of detecting a quantitatively abnormal k or l free light chain population or an abnormal k to l ratio in 99% of patients with AL amyloidosis. In patients with a negative serum but positive urine immunofixation, the free light chain assay detects abnormalities in over 80% of patients. Even in patients with known amyloidosis, and both negative serum and urine immunofixation, the quantitative immunoglobulin light chain assay can detect abnormalities in 86% of patients with k and in 30% of patients with l light chain amyloidosis.

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4.3. ESTABLISHING THE DIAGNOSIS The diagnosis of amyloidosis can only be confirmed with tissue biopsy. The pathognomonic finding on biopsy is the presence of green birefringence on Congo red staining. The diagnosis requires pathologists experienced with the technique since it is not uncommon for patients to be given a diagnosis of amyloidosis and in further review of the biopsy to be found that the patient does not in fact have amyloidosis. Over fixation and trapping of the Congo red stain can result in false positives. Also staining of collagen and elastin in the skin and fat can be confused with amyloid tissue; however, in these cases there is no green birefringence. Congo red staining is a highly specific technique for diagnosing amyloidosis with a specificity of 100% in experienced hands. However, it is not as sensitive nor does it allow typing of the amyloid protein. The technique can be combined with immunohistochemistry and Congo red fluorescence to improve sensitivity. Congo red fluorescence can detect small amounts of amyloid deposits, does not interfere with immunohistochemical staining [40], and can be successfully applied in frozen sections. Typing of the amyloid (identifying the specific protein involved) cannot be done with the Congo red stain since all forms of amyloid deposits are identical at this level. Immunohistochemical staining of tissues with commercially available antisera is needed for such identification. Amyloid P component, which is present in all amyloid deposits irrespective of the specific protein involved, acts as a positive control. In contrast, apolipoprotein E does not appear to be uniformly present [41]. Immunohistochemical analysis has sometimes identified patients with amyloidosis of diVerent kinds that just happen to have an underlying plasma cell proliferative disorder. In one particular study as many as 10% of patients who were thought to have AL amyloidosis were found to have diVerent types of amyloidosis [42]. This included 5% of patients who had fibrinogen amyloid and 4% who had hereditary amyloidosis with mutations of TTR. Although biopsy of the organ involved is highly specific for the diagnosis of amyloidosis, this is not always desirable. Commonly involved organs include the kidney, heart, and liver and biopsy of these organs can lead to significant morbidity. Although fine needle aspirate can occasionally replace core needle biopsy in the diagnosis of amyloidosis thus reducing the risk of the procedure, biopsy of easily accessible sites is more desirable [43, 44]. For example, amyloidosis in patients with renal disease can be successfully diagnosed with duodenal biopsies, which are easier to perform [45]. Similarly, labial salivary gland biopsy has been used to diagnose amyloidosis in patients presenting with polyneuropathy [46]. Other sites that have been routinely used for the diagnosis of amyloidosis include the rectum and the skin.

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However, the most commonly used technique today for the diagnosis of amyloidosis is a subcutaneous fat aspirate [47]. Fat aspiration is an easy technique to perform, causes minimal discomfort to the patient, allows for diagnosis within 24 h and has an acceptable sensitivity ranging from 58% to 84% with a specificity of 99–100% [47–49]. Although weak nonspecific staining and collagen birefringence can lead to false positives [50], fat aspiration is the established diagnostic technique for suspected amyloidosis.

4.4. TYPING OF AMYLOID Because the treatment of the various forms of amyloidosis varies considerably, and multiple patients are known to have received the wrong therapy (i.e., chemotherapy for patients with secondary, hereditary, or localized amyloidosis who happen to also have an unrelated paraproteinemia) typing of the amyloid fibril is of the utmost importance in situations when a paraproteinemia is not found or the patient presents in an atypical location of disease. For example, involvement of the skin, larynx, tracheobronchial tree, vocal cords, bladder, urethra, ureter, macula, orbits, conjunctiva, atria of the heart, lung, pleura, and articular cartilage should raise the suspicion of localized amyloidosis. Renal amyloidosis could be due to immunoglobulin light chain deposition, AA amyloid deposition, as well as various hereditary mutant proteins making the need for proper typing important in situations when the diagnosis is not clear [51]. Because correct typing requires considerable amount of tissue, several microtechniques have been developed aiming to use smaller amounts of protein. Microextraction and micropurification techniques have successfully been used to extract and purify amyloid fibrils both from fresh tissue as well as from formalin fixed tissue [52, 53]. Several techniques have been used to identify the purified amyloid deposits. Although immunohistochemistry is the most commonly used of these techniques, there are several pitfalls that make its use controversial. The antibodies used for immunohistochemistry have poor sensitivity in detecting light chain amyloid fibrils although generally the sensitivity is much better for other types of amyloidosis. Although various synthetic peptides have been developed aiming at increasing the ability of immunohistochemical techniques to recognize light chain amyloid fibrils, in up to a third of the patient’s, immunohistochemistry can be negative or equivocal [54, 55]. This is partly due to the fact that the amyloid deposits are composed of the N‐terminal fragment of the light chain where most commercial antisera recognize the constant portion of the light chain. Inconsistent immunolabeling reactions and nonspecific background staining leads to some of these problems as well.

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Attempts at developing suitable antibodies against immunoglobulin light chains for use in immunohistochemistry are underway [56]. AA amyloidosis deposits are easily recognized by immunohistochemistry and thus the diagnosis is easily confirmed or excluded by this method. However, staining quality and observer experience always remain an issue and attempts to develop techniques with improved diagnostic accuracy are ongoing. One such technique involves quantification by ELISA of SAA using monoclonal antihuman serum amyloid A antibodies which was recently reported to have a sensitivity and specificity of 84% and 99%, respectively [57]. In another study ELISA has been used successfully in typing amyloid from subcutaneous fat biopsies correctly in 14 out of 15 patients studied [49]. Various other laboratory techniques exist for typing amyloid deposits [58]. This includes deposits in formalin fixed specimens [59]. Recent improvements in micromethods have enabled tandem mass spectrometry to precisely identify the protein nature of the pathologic deposits [60]. Mass spectroscopy, looking for TTR molecules with abnormal molecular weight, has been particularly helpful in classifying deposits in cardiac amyloidosis. Gene expression analysis can identify unique molecular profiles that can be used to discriminate AL amyloidosis from other subtypes [61]. Restriction fragment length polymorphism, various polymerase chain reaction techniques, single strand confirmation polymorpism, and nucleotide sequencing can recognize mutations in proteins that cause familial amyloidosis [58]. Ultrastructural studies of abdominal fat samples using immuno‐electron microscopy has shown significant specificity by correctly identifying amyloid deposits in 15 out of 15 patients in one study [62]. Western blot analysis combined with specific amyloid fibril protein antibodies has characterized successfully 32 out of 35 abdominal fat biopsies with amyloid deposits [63]. Proper typing, using one or a combination of the above techniques, is of the outmost importance in familial amyloidosis. This is true because family history is an inaccurate screening tool since half the patients have no family history and misdiagnosing patients with true familial amyloidosis as having AL amyloidosis is not uncommon when an unrelated monoclonal gammopathy is present [64, 65]. In the case of a previous unknown mutation a combination of the above techniques is necessary to accurately provide the diagnosis. 4.5. IMAGING OF AMYLOID Echocardiography, magnetic resonance imaging, radionuclide imaging, and radioiodinated amyloid P component scan are various imaging techniques used to detect amyloidosis in tissues. Echocardiography can diVerentiate amyloid heart disease from other types of cardiomyopathy. In general,

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thickening of the left ventricular wall with reduced left ventricular compliance (diastolic dysfunction) is the most common finding. This is clinically followed by restrictive cardiomyopathy and systolic dysfunction. Amyloid fibrils depositing into the myocardium can cause local ischemia and disruption of the conducting system which eventually leads to arrhythmias, a common cause of death in these patients. Specific echocardiographic findings associated with cardiac amyloidosis include thickened interventricular septal wall and ventricular wall, reduced left ventricular systolic and diastolic diameters, restrictive physiology, and a characteristic granular appearance of the myocardium. When increased interventricular septum wall thickness is combined with the electrocardiographic findings of low voltage the pattern is highly suggestive of amyloid infiltrative cardiomyopathy [66]. When conventional echocardiography is combined with newer techniques, such as tissue Doppler and myocardial strain rate imaging, earlier stages of cardiac pathology can be detected [67, 68]. Similarly, with cardiac MRI, characteristic patterns of gadolinium kinetics and subendocardial late enhancement are specific to amyloid cardiomyopathy and thus cardiac MRI has found a clinical role in the diagnosis of this disease [69–71]. MRI can also aid in the identification of amyloid in bone thus helping to diagnose amyloid arthropathy [72]. Various radionuclide imaging techniques have been successful in detecting amyloid deposition with impressive results. Mean washout rates of Thallium‐ 201 from the heart can correlate with the severity of the disease [73]. Single proton emission computed tomography (SPECT) using Gallium‐67 and Thalium‐201 can diVerentiate active from inactive amyloid deposits in patients with dialysis‐associated amyloidosis [74]. Technetium labeled N2S2 conjugates of chrysamine G appear to have specificity for renal amyloidosis [75]. Technetium aprotinin has been shown to diVerentiate TTR‐related amyloidosis from AL amyloidosis of the heart [76]. Radioiodinated SAP component (I‐123 SAP) localizes in amyloid infiltrated tissues in proportion to the amount of disease. Patients with amyloidosis clear I‐123 SAP from the plasma quicker and show increased extravascular retention and interstitial exchange rate of I‐123 SAP. This characteristic has been used for evaluating the distribution of amyloidosis for prognostication and identifying possible biopsy sites as well as for monitoring the disease in a safe and noninvasive way [27, 77, 78]. Recent advantages in molecular imaging have been made through the use of small molecules that bind amyloid fibrils in a specific manner. Four of these small molecules (18F‐FDDNP, 11C‐PIB, 11C‐SB13, and 11C‐BF‐227), called positron emission tomography (PET) ligands, have found their way into imaging of amyloid tissue in patients with Alzheimer’s disease [79]. Specifically, 11C‐PIB, otherwise called Pittsburg compound B, has been

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shown in several studies to be highly retained in frontal, temporal, parietal, and occipital cortices and the striatum of patients with early Alzheimer’s disease compared to healthy controls [80]. It is noteworthy that as years go by the retention of the Pittsburg compound B in the brains of Alzheimer’s patients does not increase despite clinical worsening of the disease [81]. Thus, since the amyloid load in any given patient does not seem to directly correlate with clinical disease status, neural damage seems to be caused very early in the disease process. Also, as we discussed previously in Section 3.5, these findings could argue for the presence of toxic intermediates that do the damage instead of the mature amyloid fibril.

5. Classification, Clinical Presentation, and Prognosis of Amyloidosis 5.1. PRIMARY AMYLOIDOSIS Patients with AL amyloidosis have a detectable (by immunofixation) monoclonal light chain in the serum or urine and/or a detectable circulating free light chain level in addition to a clonal plasma cell disorder in the bone marrow. The bone marrow clonal plasma cell proliferative disorder, responsible for the generation of the amyloid paraprotein need not be of large quantity, as in the case of multiple myeloma, and in fact it is typically small. Patients with amyloidosis do not go on to develop multiple myeloma if it is not present at the time of diagnosis indicating the separate pathogenesis of these diseases. Three quarters of patients with amyloidosis present with l light chain restricted disease in contrast to MGUS where two‐thirds of patients are k light chain restricted, indicating an intrinsic amyloidogenic potential of l light chain immunoglobulin fragments. This appears to be due to the increased amyloidogenic potential of certain l light chain genes. Specifically, the genes 3r and 6a, belonging to the lIII and lVI families, encode 42% of amyloid variable l regions and can potentially account for the overrepresentation of the l restricted disease seen in AL amyloidosis [82]. Two‐thirds of patients with AL amyloidosis are male, and the median age of presentation at the Mayo Clinic is 67 years [83]. The most common organs involved clinically include the heart in 37% (with about half presenting with symptoms of heart failure), the kidney in 27% (with nephrotic range proteinuria being the most common manifestation), and the peripheral nervous system in 15% of the patients (presenting as paresthesias, carpal tunnel syndrome, and pain). The peripheral neuropathy is frequently axonal and demyelinating [84]. Hepatomegaly is seen in 17% of the patients but it is

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clinically dominant only in about 5%. Finally, gastrointestinal involvement in the form of pseudo‐obstruction, bleeding, and diarrhea is seen in 7% of patients. Other rarer presentations include involvement of the tongue, joints, and soft tissues. Of all the known prognostic factors cardiac involvement is the most important. Median survival of patients with cardiac amyloidosis is measured in months, where if there is no significant cardiac involvement the median survival is measured in years [85]. A prognostic model using biomarkers of cardiac injury (Troponin T or I and N‐terminal probrain natriuretic peptide) has been developed in patients with AL amyloidosis [86]. Specifically, patients are separated into three stages depending whether none (Stage I), one (Stage II), or both (Stage III) of these cardiac biomarkers are elevated with a median survival of 26.4, 10.5, and 3.5 months for each stage, respectively [87]. This model holds true for patients with amyloidosis undergoing bone marrow transplantation as well [86]. In bone marrow transplantation specifically the absolute value of the immunoglobulin free light chain is another important prognostic indicator [88]. Another important prognostic marker includes b‐2‐microglobulin despite the fact that in amyloidosis, unlike multiple myeloma, the overall disease burden is low indicating that b‐2‐microglobulin is not simply a reflection of neoplastic burden. 5.2. SECONDARY AMYLOIDOSIS Secondary amyloidosis is caused by the deposition of SAA fragments during a sustained inflammatory state. Serum amyloid A is an acute phase reactant whose levels can increase 1000‐fold during an acute inflammatory reaction [89]. Serum amyloid A is a 104 amino acid long apolipoprotein that binds to high‐density lipoprotein (HDL) particles during inflammation and replaces apolipoprotein A‐I which normally is the major apolipoprotein bound to HDL [90]. The reason for this binding is unclear but the SAA‐rich HDL may be involved in manipulating cholesterol metabolism and macrophage chemoattraction during inflammation [91, 92]. The amyloidogenic N‐terminal fragment of serum amyloid A can be produced by macrophage‐ induced proteolytic cleavage of SAA at position 76 in vitro [93]. Thus macrophages appear to have a central role in the pathogenesis of AA amyloidosis. SAA is actually a family of proteins with SAA1 and SAA2 being the two loci at the genetic level thought to be involved with amyloidogenesis due to their function as acute phase reactants. AA amyloidosis is seen during longstanding chronic inflammatory conditions such as rheumatoid arthritis, juvenile idiopathic arthritis, and ankylosing spondylitis, during chronic infections such as tuberculosis, osteomyelitis,

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syphilis, bronchiectasis, leprosy, and chronic infections associated with the paraplegic state (pressure sores, urinary tract infections), during periodic fever syndromes such as familial Mediterranean fever, TNF‐receptor‐ associated periodic fever syndrome, hyperimmunoglobulinemia D and periodic fever syndrome, cryopyrin‐associated periodic syndrome, and during other inflammatory conditions such as Crohn’s disease, cancer, Castleman’s disease, vasculitis, etc. In the western world AA is more strongly associated with rheumatoid arthritis in contrast to the rest of the world where infections are the most common cause. Amongst the periodic fever syndromes familial Mediterranean fever is the most frequent entity [94]. The most frequent organ involved is the kidney, in over 80% of patients and renal dysfunction has been described in up to 97% of patients [95]. The classical presentation of AA amyloidosis is a patient with rheumatoid arthritis who presents with nephrotic range proteinuria, although proteinuria is not the only renal manifestation of AA amyloidosis seen. Cardiac involvement can occur, although less commonly than in patients with AL amyloidosis. Hepatic involvement is clinically apparent in 9% of patients although SAP scintigraphy shows evidence of disease in up to 23% of patients [95]. Neuropathy is considered rare. Although the prognosis of AA amyloidosis is generally considered much better than AL amyloidosis, selected patients can have a rapidly deteriorating clinical course and poor prognosis. The predictors of poor outcomes are likely related to the ability to adequately control the underlining inflammatory condition. Cardiac involvement, as in the case of AL amyloidosis, again appears to predict for worse outcome with a 5‐year survival of 31% versus 63% for patients without cardiac involvement in a retrospective series of 42 patients from Japan [96]. In addition, the degree of renal involvement is important, with patients who have elevated creatinine levels doing worse compared to patients with a normal creatinine. The pattern of renal involvement is also important. Specifically, glomerular involvement with amyloid and fibrosis appear to have a clinical course characterized by deteriorating renal function compared to patients with other types of renal involvement [97, 98]. Generally, however, the median survival is over 5 years. In the largest prospective series of 334 patients from the United Kingdom (which excluded patients with cardiac involvement) elevated absolute annual median SAA concentration was strongly associated with poor outcome [95] (Table 2). End stage renal disease (RR 2.97) and older age (RR 1.53) were also associated with poor outcome where evidence of radiographic improvement by SAP scintigraphy (RR 0.13) and underlying periodic fever syndrome (RR 0.36) were associated with better outcome.

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AMYLOIDOSIS TABLE 2 UNADJUSTED RELATIVE RISK OF DEATH ASSOCIATED WITH THE MOST RECENT MEDIAN ANNUAL SAA CONCENTRATION [95] SAA (mg/l)

Relative risk (95% CI)

P value