Advances in Cancer Research, Volume 3

ADVANCES IN CANCER RESEARCH Volume III

This Page Intentionally Left Blank

ADVA-NCES IN CANCER RESEARCH EDITED BY JESSE P. GREENSTEIN NationaE Cancer Institute, National Institutes of Health, U.S. Public Health Service, Bethesda, Maryland ALEXANDER HADDOW Chester Beatty Research Institute, Royal Cancer Hospital, London, England

Volume 1 I I

ACADEMIC PRESS INC., PUBLISHEHS NEW YORK, N.Y. 1955

COPYRIGHT 1955, BY ACADEMIC PRESS INC. 125 East 23rd Street, New York 10, N.Y.

All Rights Reserved No part of this book may be reproduced in any form, by photostat, microfilm, or any other means without written permission from the publishers. Library of Congress Catalog Card Number: 52-13360

PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME I11 RICHARD DOLL,Statistical Research ['?zit, The Medical Research Council, London School of Hygiene and Tropical Medicine, London, England

HAROLD P. MORRIS, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

A. PULLMAX, Institut du Radium, Paris, France R. PULLMAX, Institut d u Radium, Paris, France

P. ROSDONI,Cancer Institute, Milan, Italy MICHAEL B. SHIMKIN, National Cancer Institute, National Institutes of Health, Bethesda, Maryland SIDNEY WEINHOUSE,The Lankenau Hospital Research Institute and The Instal iite jor Cancer Research, Philadelphia, Pennsylcania

This Page Intentionally Left Blank

CONTENTS CONTRIBUTORS TO VOLUME 111. . . . . . . . . . . . . . . . . . . . . .

v

Etiology of Lung Cancer BY RICHARD DOLL.Statistical Research Crnit. The Medical Research Council. London S c h l of Hygiene and Tropical Medicine. London. England

1. Introdurtion . . . . . I1. Increase in Incidence . I11. Etiological Factors . . IV . Conclusion . . . . . . References . . . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

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

1 2 8 46 47

The Experimental Development and Metabolism of Thyroid Gland Tumors BYHAROLD P . MORRIS.IAhoratory of Biochemistry. National Cancer Institute. National Institutes of Health. Belhesda. Maryland

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Thyroid Gland Biochemistry and Metabolism . . . . . . . . . . . . . I11. Spontaneous Thyroid Tumors . . . . . . . . . . . . . . . . . . . . I V . Effect of Goitrogens and Iodine on Body Weight. and Thyroid Weight . . . V . Induction of Thyroid Gland Cancer by Chemical Carcinogens . . . . . . V I . Dietary Iodine Deficiency and Experimental Development of Thyroid Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII . Goitrogen-Induced Thyroid Gland Tumors in hlice . . . . . . . . . . VIII . Transplantability of Thyroid Gland Tumors in Mice . . . . . . . . . . IX. Thyroid-Pituitary Interrelationships . . . . . . . . . . . . . . . . . X . Effects of Ionizing Rndintions in Thyroid Gland Carcinogenesis . . . . . XI . Experiment.al Thyroid Tumors in Rats Produced by Goitrogens and Carcinogem . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI1. Transplantability and Xlrtnbolism of R a t Thyroid Gland Tumors . . . . XI11. Biochemistry of Thyroid Gland Tumors in Mice . . . . . . . . . . . XIV . Thyroid Gland Cancer in %Ian . . . . . . . . . . . . . . . . . . . XV . Summary nnd Conclusions . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

52 53 56 58 60

GO 62 62

65 68 75 81 80 102 109 112

Electronic Structure and Carcinogenic Activity and Aromatic Molecules New Developments BY A . PULLMAN A N D I3. PULLMAS. Institut du Radium. Paris. France 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 I1 . The Localization Theory of Chemical Reactions . . . . . . . . . . . . 118 111. Electronic Structure and Carcinogenic Artivity of Unsuhstituted Polynuclear Hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . .

122

IV . Extension of the Theory to Substituted Ilerivatives of Polycylic Hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 1‘. General Conclusions and Suggestions . . . . . . . . . . . . . . . . 156 Appendix . The Metabolic Renetivity of Carcinogenic Hydrocarbons . . . 161

References . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

167

viii

CONTENTS

Some Aspects of Carcinogenesis BY P. RONDONI. Cancer Institute. Milan. Italy

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 I1. Cancer as a Regressive Process in General Pathology . . . . . . . . . 172

I11. The Energy Changes in Carcinogenesis (The Concept of Entropy in Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . The Supposed Significance of Derangements of Oxidative Metabolism in Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . V . The Interaction between Some Carcinogenic Agents and Cell Constituents VI . Cancer as a Problem of Protein Chemistry . . . . . . . . . . . . . . VII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

174 180 185 194 213 214

Pulmonary Tumors in Experimental Animals

.

BY MICHAELB SHIMKIN.National Cancer Institute. National Institutes of Health. Bethesda. Maryland I . Historical Introduction . . . . . . . . . . . . . . . . . . . . . . . 223 I1. Frequency and Distribution of Pulmonary Tumors in Mice . . . . . . . 225 I11. Pulmonary Tumors in Other Animals . . . . . . . . . . . . . . . . 227 IV . Morphology and Biochemistry of Pulmonary Tumors in Mice . . . . . . 229 V . Histogenesis of Pulmonary Tumors in Mice . . . . . . . . . . . . . . 233 VI . Influence of Heredity in Pulmonary Tumors in Mice . . . . . . . . . . 235 VII . Polycyclic Hydrocarbons and Related Compounds . . . . . . . . . . . 237 VIII . Urethane and Related Compounds . . . . . . . . . . . . . . . . . 242 I X . Other Chemical and Physical Agents, Including Inhalants . . . . . . . 244 X . Factors Affecting Pulmonary Tumor Induction in Mice . . . . . . . . 248 XI . Mechanism of Induction of Pulmonary Tumors in Mice . . . . . . . . 252 XI1. Pulmonary Tumors in Man and General Discussion . . . . . . . . . . 256 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

Oxidative Metabolism of Neoplastic Tissues BY SIDNEYWEINHOUSE.The Lankenau Hospital Research Institute and The Institute for Cancer Research. Philadelphia. Pennsylvania I . The Concepts of Warburg . . . . . . . . . . . . . . . . . . . . . 270 11. The Pasteur Effect . . . . . . . . . . . . . . . . . . . . . . . . 274 111. Present Concept of Carbohydrate Oxidation . . . . . . . . . . . . . 278 IV . &Oxidation of Fatty Acids . . . . . . . . . . . . . . . . . . . . . 282 V . Mechanisms of Glycolysis in Tumors . . . . . . . . . . . . . . . . 283 VI . Electron Transport in Tumors . . . . . . . . . . . . . . . . . . . 288 VII . Oxidation in Tumor Homogenates . . . . . . . . . . . . . . . . . . 315 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 AUTHORINDEX . . . . . . . . . . . . . . . . . . . . . . . . . .

327

SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . .

339

Etiology of Lung Cancer RICHARD DOLL Statistical Research Unit of the Medical Research Council, London School of Hygiene and Tropical Medicine, London, England Page .......................... . . ................. 1 1 f Louvain Symposium... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Increase in Incidence.. . . . . . . . . . . . . . . , . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . 2 2 . - . . . . . ..................... 1. Extent of Increase.. . . . . . . . . . . . . . . 5 2. Changes in Sex Distribution.. . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . 5 3. Changes in Age Distribution.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4. Changes in Histological Distribution. . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.II. Etiological Factors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1. Tobacco _ . . . . . . ,. . . . . , , . , . . , . . . . . . , . . . . , . . . . . . . , . . . . . . . . . . . . . . . . A. Retrospective Inquiries. , . . . . . . . . , , , . , , , . . , . , . . . . , . . . . . . . . . . . . . . 8 B. Prospective Inquiries. . , . . . . . . . . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 C. Method of Smoking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 D. Extent of Risk.. , . . . , , . . . , , . . , , . . . . . . . . . . . . . 16 E. Difference between Histological Types. . . . . . . . . . . . . . 19 . F. Vital Statistics and Tobacco Consumption. . . . . . . . . . . 20 G. Identification of Carcinogenic Agent, . . . . . . , . . . . . . . . . . . . . . . . . . . . . 23 H. Various Criticisms. . . . . . . . . . . . . . . . . . . . . . . . . . 29 I. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 2. Industrial Hazards. . . . . ......................... 33 3. Atmospheric Pollution. . . . . . . - . . . . . . . . . . . . . . . . . 36 A. Mortality in Town 36 B. Pollution of Town Air .............................. 38 C. Differences in Urban an 40 D. Conclusion.. . . . . . . . ..................................... 41 4. Atmospheric Radioacti . . . . . . . . . . . . . . . . . . . . 41 5. Previous Respiratory I . . . . . . . . . . . . . . . . . . . . . . . . 44 IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... 46 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . . . . . . _ . 47

I. INTRODUCTION 1. Conclusions of Louvain Symposium Knowledge of the causes of lung cancer was reviewed a t an international symposium on the “ Endemiology of Lung Cancer” held at Louvain in 1952 (Council for International Organizations of Medical Sciences, 1953). The members of the symposium were unable to decide what factors were responsible for the majority of cases, but important conclusions 1

2

RICHARD DOLL

were reached on more limited problems. Firstly, it was agreed that ‘(a significant part” of the increase in mortality which had been reported from many countries “is absolute and represents a real increase in the number of people suffering from primary cancer of the lung”; secondly, “that there is now evidence of an association between cigarette smoking and cancer of the lung, and that this association is in general proportional t o the total consumption”; and thirdly, that “occupational hazards giving rise t o lung carcinoma have been demonstrated in a number of industries, in particular, in the handling of asbestos and chromates, in gas-works, in a factory refining nickel and in certain mines bearing radio-active ores.” Other possible etiological factors were considered-in particular, atmospheric pollution by effluvia and smoke from factories and domestic chimneys and by exhaust fumes from petrol and diesel engines. The possibility that carcinogenic agents might be absorbed through ingestion or skin contact was reviewed, as was the possibility that individuals might vary in their susceptibility to the environmental influences t o which they were exposed. No positive conclusions were reached with regard t o these latter problems. I n the last two years, however, much new evidence has been obtained, and it is now possible to give a more complete picture of the etiology of the disease.

11. INCREASE IN INCIDENCE I. Extent of Increase The highest death rate from lung cancer is recorded in Britain, where, in 1953, it was 342 per million persons. For both sexes taken together, lung cancer was the commonest type of fatal cancer, accounting for 17% of all cancer deaths; it accounted for 5% of male deaths from all causes a t all ages and, in the age group 45 to 64 years, for 10% of all male deaths. I n other countries for which detailed statistics are available the rate varies from a seventh to approximately two-thirds the British rate (Table I).* The disparity between the rates is mainly due to a disparity between the rates for men; with the exception of England and Wales, Scotland, and Finland the female rates are similar, varying only between 34 and 47 per million women. Each of the countries listed has experienced an increase in the mortality attributed t o lung cancer in the last half century, and the increase appears t o be still continuing (Fig. 1). * I n Table I, the figures for England and Wales and for Scotland are shown separately. The Scottish rate for all persons has usually been lower than the English and Welsh rate, but in 1953 the rate was slightly higher-346 per million against 342 per million.

3

ETIOLOGY O F LUNG CANCER

I n England and Wales the rate of increase has slackened in the last five years, and the death rate among men under the age of 50 years is now steady. On the assumption that the rates a t the younger ages remain steady and that the rates a t the older ages continue to increase until the age distribution of deaths from lung cancer resembles that of other extragenital epithelial cancers, Mackenzie (personal communication) estimates that the male death rate may increase to approximately 1350 per million men, ie., to more than twice its present level of 602 per million, before it stabilizes. By a similar method, Clemmesen, Nielsen, and Jensen (1953) TABLE I Crude Death Rate from Lung Cancer in Various Countries Crude Death Rate per 1,000,000 Country England and Wales Scotland Fioland Holland Switzerland U.S.A. Denmark Australia Canada France Sweden Norway Iceland

Year

Men

Women

Persons

1951 1951 1950 1951 1950 1951 1951 1951-52 1951 1950 1951 1951 1950

530 470 353 27 1 252 214 185 173 154 161 111 81

91 104 61 45 38 45 46 37 34 47 43 39

303 279 20 1 158 136 129 115 106 95 87 77 60 42

-

-

Rates have been shown for 1951, whenever possible, as data were available for t h e greatest number of countries around that year.

estimate that the death rate among men in Copenhagen may become even greater (ie., 2200 per million). Much of the recorded increase is due to the advancing average age of the population. This factor can, however, be allowed for. I n England and Wales, for example, the recorded death rate from lung cancer rose from 8 per million in 1900 t o 342 per million in 1953; ie., 43 times. But if the sex and age-specific death rates of 1953 had occurred in a population with the sex and age distribution characteristic of the population a t the beginning of the century, the total death rate would have been only 188 per million. The extent of the recorded increase after allowing for demographic changes is, therefore, 24 times, or little more than half the figure given by the comparison of the crude rates. Similar conclusions apply t o the increases recorded in other countries.

4

RICHARD DOLL

How much of the increase "is absolute and represents a real increase in the number of people suffering from primary cancer of the lung" and how much is merely due t o better diagnosis is uncertain. It is doubtful if the nature of the data concerned will ever permit a precise answer t o be given. Rigdon and Kirchoff (1953) still maintain that the whole increase may be spurious, but in this opinion they are almost alone.

ul

5

E N G L A N D 6 WALES (S C O T L A N D )

""4 300

.-0 2501 E L Q,

a c

1

200

/

L Q

5 150

'

( H o L L A N D)

SWITZERLAND

p'y'

U

2 100 C C

a

1920 1925

19'30

1935

1940 1945

1950 1953

Date

FIG.1. Increase in crude death rate from lung cancer in various countries, 19201953. The trend of the death rate in those countries shown in parentheses has been similar t o that in the countries against which they are placed.

Clemmesen, Nielsen, and Jensen (1953) in Denmark; Doll (1953a) and Stocks (1953a) in England; Kreyberg (1954b) in Norway; and Dorn (1954) in the United States have recently cited the reasons for believing that part of the increase is real. Three of the reasons are based on observations which are of special significance for the etiology of the disease. The observations are that the increase has fallen unevenly on: 1. The two sexes. 2. Different age groups. 3. Different histological types.

5

ETIOLOGY O F LUNG CANCER

2. Changes in Sex Distribution

National mortality statistics and autopsy series both agree th a t the change in incidence of the disease has been accompanied by a n increasing preponderance of male cases. Figure 2 shows how in different countries 350-

300. m

$a 2

250.

z 0

: J

200.

a W

0

w

+-4

150’

a x

2

W

100.

a J

d

a

z z

50.

d

L L0:l

2.0:I

3.011

4.011

RATIO OF MALE TO FEMALE

5.O:I DEATH

6.0:l

7.O:l

RATES

FIG. 2. Increase in ratio of male to female death rates with the increase in the crude lung cancer mortality in various countries.

the proportion of male to female deaths has become progressively greater as the total mortality has arisen. The reality and implication of this change are too well recognized to warrant further comment. 3 . Changes in Age Distribution

It has long been noted that the age distribution of lung cancer in men differs from that in women and from that of other extragenital epithelial tumors, in that, in countries with a high incidence, the male mortality rises t o a maximum comparatively early and falls off rapidly in the later age groups. The increase in mortality over the last 50 years did not affect all ages equally; a t first the younger age groups were principally affected

6

RICHARD DOLL

and the maximum mortality came to be between the ages of GO and 64 years; recently the increase has been most marked in the older age groups and the age of maximum mortality has risen. Korteweg (1951) has pointed out that these trends can be understood if comparisons are made between the age-specific death rates of groups of men all of whom were born a t a given period, rather than between groups of men living a t a given date, as is the normal custom. By this method of “cohort analysis” similar results have been obtained in Australia (Lancaster, personal communication), Denmark (Clemmesen, Kielsen, and Jensen, 1953), England and

0

10

20

30

50

40

60

70

80

90

Age

FIG.3. Male death rates from lung cancer in the U.S.A. by age in 1914, 1930-32, 1939-41, and 1949-50, showing (heavy lines) the increase in mortality with age for men born in 1850,1860, 1870, 1880, and 1890. (Reproduced from a paper by Dr. H. F. Dorn in Industrial Medicine and Surgery 23, 253-257, 1954.)

Wales (Korteweg, 1951, and-in a modified form-Stocks, 1953a), and the United States (Dorn, 1954). Dorn’s data are reproduced in Fig. 3. The dotted lines indicate the pattern of age-specific death rates when studied a t different dates (1914, 1930-32, etc.); the solid lines indicate the pattern when men who were born a t a given period (1850-59,1860-69, etc.) are followed throughout their lifetime. It is seen that for each (‘cohort” the mortality increases continuously with age, but that the later “cohorts” have a progressively higher mortality a t each age than the earlier ones. The changes in the shape of the customary age distribution curve for lung cancer can, therefore, be understood if it is postulated (1) that groups of men born a t each period suffer a mortality which

ETIOLOGY OF LUNG CANCER

7

increases in a way similar to that observed for other forms of extragenital epithelial cancer and (2) that men born a t successive periods were increasingly exposed to an environmental carcinogen. On the other hand, the observed changes cannot be explained, as Clemmesen (1954) has pointed out, if men of all age groups were equally exposed t o a new agent a t the same time.

4. Changes in Histological Distribution With the increase in lung cancer the proportions recorded as belonging t o the various histological types have altered ; adenocarcinoma has become relatively less common, and its incidence must, theref ore, be presumed to have increased less than that of other types. I n conformity with this and with the comparatively small increase of lung cancer in women, the sex ratio for adenocarcinoma has remained close to equality, whereas that for other types has shown a marked male predominance. Moreover, adenocarcinoma was not observed among the industrial tumors from which the Schneeberg and Joachimstal miners suffered (Schmorl, 1928; Hueper, 1942; Sikl, 1950). For these and other reasons, Womack and Graham (1938, 194l), Lickint (1953), and Kreyberg (1954a,b,c,d) have concluded that lung cancer may be divided into two essentially different types-endogenous and exogenous in origin. Kreyberg’s papers are particularly important because the data have been collected in a country where the total lung cancer mortality is low and during a period when changes similar to those which took place in Britain and the United States 20 to 30 years ago are only beginning to appear. Kreyberg classified his cases into two main groups: group I consisting of squamous and large- and small-cell carcinomas, and group I1 of adenocarcinomas, bronchiolar cell carcinomas, and benign and malignant adenomas and salivary gland type tumors. The group I tumors were predominantly male (273 M to 31 F) and, when related to the size of the Norwegian population in 1950, showed an age distribution similar t o that observed for all lung cancer in countries with a high incidencesave only that the characteristics of the distribution were more pronounced, ie., the “incidence” had an earlier peak (50 to 59 years) and fell off more sharply in the older age groups. The group I1 tumors were found almost equally often in each sex (81 M to 76 F) and showed an (‘incidence” which increased steadily with age in the case of adenocarcinoma and was approximately evenly distributed throughout the range of adult ages in the case of the adenomas and the salivary gland type tumors. When the cases were subdivided accordirlg to their date of occurrence, Kreyberg found that there had been no increase in the proportion of group I t o group I1 cases among women over the whole period 1925

8

RICHARD DOLL

t o 1953, despite the fact that the standardized mortality rate for women increased four and a half times. On the other hand, group I tumors became relatively much more frequent among men compared with group I1 tumors, while the standardized male mortality rate increased sevenfold. Despite the considerable difference in the total mortality experience of the two sexes, the sex ratio for the group I1 tumors remained close t o equality. It is easy to criticize Kreyberg’s material on the grounds that it was heterogeneous in origin (part collected from clinical and part from autopsy series) and that the relative amounts collected in the different ways varied over the period studied. Moreover, it is likely that his cases provided a larger sample of those occurring in the younger age groups than in the older groups. Nevertheless, the characteristics of the histological types varied so markedly and the observations agree so well with the trend of the data obtained in other countries, that it would be unreasonable to dismiss the material because it falls short of perfection. Kreyberg interpreted his findings to mean that the group I tumors were largely the result of the introduction of some new carcinogenic agent into the environment, t o which men were more exposed than women, whereas the adenocarcinomas “are probably caused by comparatively weak carcinogenic influenccs, evenly distributed over large areas, well established in the society and striking both sexes with equal force.” The recorded increase in mortality in women in Norway may, he suggests, indicate the extent of the increase due t o better diagnosis, and the total mortality in women (including a small proportion due to group I tumors) may, with present knowledge, be regarded as “unavoidable” cancer. I n contrast, the increased mortality in men additional to that recorded in women and attributed solely to group I tumors can be regarded as “avoidable” cancer. It may well prove that these conclusions are of general significance and also apply to many countries other than the one in which the data were collected. 111. ETIOLOGICAL FACTORS

I. Tobacco A. Retrospective Inquiries. When the Louvain symposium concluded (‘that there is now evidence of an association between cigarette smoking and cancer of the lung,” it did so on the basis of evidence which was derived entirely from retrospective studies of patient’s histories. I n these studies the histories given by patients with lung cancer had been compared with the histories given by patients without lung cancer who, in one or other way, had been selected as ‘(controls.” Many studies of this general type have been reported, and the principal results obtained from

9

ETIOLOGY OF LUNG CANCER

them are summarized in Table 11. All agree in showing that there are more heavy smokers and fewer nonsmokers among patients with lung cancer than among patients with other diseases. With one exception (the difference between the proportions of nonsmokers found by McConnell, TABLE I1 Principal Characteristics of Smoking Histories of Men with and without Lung Cancer, Reported by Various Authors

Numbcr of Men

Author hliiller Schairer and Schoniger Wassink Schrek et al. Mills and Porter Levin et al. Wynder and Graham McConnell et al. Doll and Hill Sadowsky et al. Wynder and Cornfield Koulumies Lickint Breslow et al. Watson and Conte

Gsell Randig

Percentage of Percentage of “Nonsmokers” “Heavy Smokers” among Men among Men

With Without With Without With Without Lung Lung Lung Lung Lung Lung Date Cancer Cancer Cancer Cancer Cancer Cancer 1939 1943 1948 1950 1950 1950 1950 1952 1952 1953 1953 1953 1953 1954 1954 1954 1954

86 93 134 82 444 236 GO5 93 1357 477 63 812 224 518 265 135 415

86 270 100 522 430 481 780 186 1357 615 133 300 1000 518 277 135 381

3.5 3.2 4.5 14.6 7 15.3 1.3 5.4 0.5 3.8 4.1 0.6 1.8 3.7 1.9 0.7 1.2

16.3 15.9 19.0 23.9 31 21.7 14.6 6.5 4.5 13.2 20.6 18.0 16.0 10.8 9.7 1F.7 5.8

65 52 55 18

36 27 19 9

-

-

51 35 25

19 22 13 29 31 29 42 57 33 18

68 66 74 74 73 86 34

Note. It has not been possible to make all the figures in this Table completely comparable. Some series include, for example, a few women; in others the proportions of heavy smokers are based on totals which are different from those used to calculate the proportion of nonsmokers. One series excludes adenocarcinoma. The individual papers should be referred to before any detailed use is made of the figures.

Gordon, and Jones), the differences are large enough to be important. More detailed results of two of the investigations are shown in Tables 111 and IV. From these it is seen (1) that there is a steady increase in the relative proportions of lung cancer to control patients as the amount smoked daily increases, and (2) that the difference in smoking habits between persons with and without the disease is more marked for men than for women.

10

RICHARD DOLL

TABLE I11 Average Amount of Tobacco Smoked Daily: Lung Carcinoma Patients and Control Patients with Other Diseases*

% Smoking a Daily Average for 20 Years of: Sex

Disease Group

No. of Patients

% Nonsmokerst

10 g.-

16 g.- 21 g.-

1.8

2.3

10.1

35.2

30.9

20.3

14.6

11.5

19.0

35.6

11.5

7.6

40.0

4.0

16.0

24.0

8.0

8.0

79.6

9.2

6.9

3.2

0.6

0.6

Lung Carcinoma 605 (squamous or (100.1%) undifferentiated) 780 Other Diseasest (99.8%)

M

Lung Carcinoma 25 (squamousor (100.0%) undifferentiated) Other Diseases$ 522 (IOO.IoJ,)

F

* After Wynder and

___

1 g.-

35 g.+

Graham, 1950.

t Nonsmokers defined as persons smoking an average of less than I cigarette a day (or its equivalent in pipe tobacco or cigars) over the previous 20 years. $ The age distributions of the control patients were different from those of the lung carcinonla patients; the percentages quoted were therefore obtained by weighting the age groups so as to make them have the same relative importance as they had in the group of 605 men with squamou.8 cancer.

TABLE IV Average Amount of Tobacco Smoked Daily: Lung Carcinoma Patients and Control Patients with Other Diseases* ~

% Smoking a Daily Average for 10 Years of: Sex

Disease Group

No. of Patients

% Nonsmokerst

< 5 g.

5 g.-

15 g.-

25 g.- 50 g.

0.5

4.0

36.0

35.0

21.6

2.8

4.5

9.5

42.0

31.8

11.3

0.9

108 (100.0%) Other Diseases1 108

37.0

14.8

22.2

13.0

13.0

0.0

(100.0%)

54.6

23.1

16.7

5.6

0.0

0.0

1357 (99.9%) Other Diseases1 1357 (100.0%)

+

Lung Carcinoma

Lung Carcinoma

* After Doll and Hill, 1952.

t Nonsmokers defined as persons who had never consistently smoked as much as 1 g. of tobacco a day for as long as one year. $ Patients with other diseases matched to be within the same five-year age group and to be in haspitals of the same type and in the same region at approximately the same tiineasthelungcarcinomapatients.

ETIOLOQY OF LUNG CANCER

11

The conclusions t o be drawn from these investigations depend on whether the comparisons between the smoking histories of the various groups of lung cancer and control patients are valid and on the extent to which the control patients were representative of the populations from which the lung cancer patients were drawn. I n some of the earlier investigations there were reasons for doubting whether the comparisons were valid-for example, when the histories of the two groups of patients were recorded by different methods. Other investigations, in which the patients were interviewed by the same persons and by the same methods throughout and in which the control patients were chosen to “match” the lung cancer patients with regard to sex and age, the date of interview, and the hospital in which they were treated, were not open to objection on this score. Nevertheless, there was the possibility that bias of one or another sort could have entered into the selection of the patients or the recording of the results. The various types of bias which might have occurred were considered in detail by Doll and Hill (1950, 1952). They concluded that bias could not be responsible for their results and that the only logical explanation was that the observed association between the smoking of tobacco and the development of lung cancer was real. Further important evidence has been obtained from the preliminary results of two “prospective” inquiries. These inquiries have been conducted on a different principle and are not subjected to the types of bias which might theoretically have occurred in the retrospective studies. Since they lead to the same conclusion, it is not now necessary to give further detailed consideration to the evidence from which it was deduced that the association shown by the retrospective studies was real. B. Prospective Inquiries. I n the prospective inquiries, the smoking habits of large numbers of “normal” persons have been recorded, and the subjects have subsequently been watched to see what diseases they developed. Preliminary results of studies of this type have been reported by Doll and Hill (1954a) and by Hammond and Horn (1954). I n Hammond and Horn’s inquiry, a large number of people who volunteered to help the American Cancer Society were each asked t o interview approximately 10 white men, aged between 50 and 69 years, to be chosen from among acquaintances with whom they expected t o remain in contact for several years. The smoking histories obtained a t the interview were recorded on a standard questionnaire. Subsequently, on the 1st of November each year, the interviewers filled in a follow-up form stating whether the men were alive or dead or had been lost sight of. The State Health Department was then asked to supply an abstract of the death certificate of each man reported to have died. When cancer was certified as the cause of death, an attempt was made to obtain further

12

RICHARD DOLL

details from the certifying physician. A total of 204,547 questionnaires was collected, of which 14,413 were eliminated because they referred to inappropriate subjects or to subjects who were interviewed outside the specified period 1.1.52 to 31.5.52, or because they were inadequately completed. Of the subjects corresponding to the remaining questionnaires, 187,766 (98.8%) were successfully traced a t 1.11.53. Altogether, 4854 TABLE V Lung Cancer Death Rates among Men by Type of Smoking and by Amount Smoked* All Cases Reported as Primary Lung Cancer

Type of Smoking

Population

Never smoked or occasional only Cigar and/or pipe smoking but never smoked cigarettes regularly History of regular cigarette smoking

Total Regular cigarette smoking; less than 1 pack a day at time of questioning Regular cigarette smoking; 1 pack or more a day a t time of questioning

* Reproduced from the Journal

No. of Deaths

Microscopically Proved Lung Cancer (Excluding Adenocarcinoma)

Death Rates

No. of Deaths

Death Rates

44,091

12

27.2

4

9.1

35,853

12

35.5

3

8.4

107,822

143

132.6

45

41.7

187,766

167

88.9

52

27.7

54,799

62

113.1

17

31.0

25,497

61

239.2

24

94.1

-

of the American Medical Association (IIammond and Horn, 19.54).

men were reported to have died, and the certified cause of death was obtained for 4710 (ie., in 97%). Cancer of the lung was certified as the cause in 167 instances. According to the authors, “The evidence at present at hand does not warrant presenting the findings in any greater detail than is shown in Table 13 (reproduced above as Table V). The lung cancer death rate was higher among men with a history of regular cigarette smoking than among men who had never smoked regularly and even higher among men who currently smoked one pack or more of cigarettes a day a t the time of questioning. The differences are statistically

13

ETIOLOGY O F LUNG CANCER

significant (P = 0.002 or less). I n fact, even the men smoking less than one pack of cigarettes daily have significantly higher death rates from lung cancer than those who have never smoked regularly ( P = 0.03 or less). The best estimate that can be made a t the present time (at the 5 % level of confidence) is that lung cancer deaths are from 3 to 9 times as common among men with a history of cigarette smoking as among men who have never smoked regularly and that lung cancer deaths are from 5 to 16 times as common among men who smoke one pack or more per day.” Differences in the age distributions of the men in the different categories have not been allowed for in Table V. Such differences cannot, however, be responsible for the results, because Hammond and Horn have also shown that the proportion of nonsmokers is greater and the proportion of cigarette smokers is smaller in the older age groups in which lung cancer is more common. If, therefore, an allowance for age differences is made it will be found that the real difference in mortality between cigarette smokers and nonsmokers is, in fact, even greater than would appear from the above data. The investigation reported by Doll and Hill (1954a) was on a smaller scale and was organized differently, but the trend of the results is similar. A postal questionnaire was sent to nearly 60,000 men and women on the British Medical Register. Just over 40,000 replied, giving details of their smoking habits. Subsequently the national offices for the registration of deaths notified the causes of death of all doctors, and clinical details of the deaths attributed t o lung cancer were obtained through the physicians who had signed the death certificates. I n the first 29 months following the date when the questionnaires were sent out, 789 deaths occurred among the 24,389 male doctors, aged 35 years and above, whose smoking habits had previously been recorded and classified. The numbers of deaths from lung cancer which occurred among men in the different smoking categories are shown in Table VI, in comparison with the numbers which would TABLE VI Number of Deaths from Lung Cancer, Observed and Expected, among Doctors Smoking Different Amounts of Tobacco * Most Recent Amount Smoked Daily t

No. of Observed Deaths No. of Expected Deaths Observed as Percentage of Expected

* After Doll and Hill, 1954a.

0

1-14g.

15-24g.

25 g.f

0 3.77

12 14.20

14 10.73

13 7.33

0

85

130

177

t Defined as the amount smoked a t the time of completing the questionnaire or, if smoking had been stopped, immediately before stopping.

14

RICHARD DOLL

have been expected to occur if smoking had been unrelated t o the disease. (As in the American investigation, the proportions of nonsmokers, of pipe smokers, and of light cigarette smokers were greatest in the oldest age groups, so that the expected numbers had to be calculated separately for each age group and added for all ages.) The number of cases so far studied is small, but there is a steady and striking increase in the ratio between the numbers of cases observed and expected in each smoking category as the amount smoked increases. When this biologically important trend is taken into account, the differences are statistically highly significant (P < 0.01). The similarity of the quantitative relationships between smoking and mortality which have been estimated from the retrospective and the prospective inquiries is also striking (Doll and Hill, 1952, 1954a). The mortality rates estimated by the two methods for each of the smoking categories have been expressed as percentages of the unweighted averages of the four rates, and a comparison of the relationships between them is shown in Fig. 4. The slopes of the two graphs are almost identical. Four explanations are theoretically possible. 1. Doll and Hill’s results might have been produced if heavy smokers who suspected that they had lung cancer had replied to the questionnaire more readily than nonsmokers or lighter smokers in a similar situation. If this had been so, the effect would necessarily wear off as the duration of time increased between the completion of the questionnaire and death. I n fact, no such diminution in the strength of the relationship occurred over the first 29 months of the inquiry. It is, in any case, unlikely that a similar form of selection could have entered into the choice of subjects for interview in Hammond and Horn’s inquiry. 2. Certification of the cause of death may have been biased by knowledge of the subject’s smoking history. If, however, there was a tendency t o diagnose lung cancer more readily in heavy smokers, the death rate attributed to other causes among men in this category would be expected to be proportionately less than average, and this was not so in either investigation. I n fact, by no means all doctors are convinced of the reality of the association-as was shown, for example, in response t o a questionnaire sent t o Massachusetts physicians by Snegireff and Lombard (1954) ; bias in diagnosis, if it existed a t all, may well have operated in the opposite direction. 3. Smoking may be associated with lung cancer only indirectly, being linked with another factor which is associated with it directly. Such an indirect link may, perhaps, account for some of the association found by Hammond and Horn, since both smoking and the disease may be commoner in certain social and occupational groups within the population,

15

ETIOLOGY O F LUNG CANCER

Such factors are unlikely to have contributed to Doll and Hill’s results, since the population studied was entirely composed of doctors and was, therefore, comparatively homogeneous. In both inquiries an indirect link may have arisen because cigarette smoking and lung cancer are both commoner in towns than in the countryside. But this cannot account for 225Retro-spedive inquiry X-x Pro-specllve lnqulry @---a

200-

175-

150Y

125-

c U C L

I

Nonsmokers

Light Smokers ( I 149 a day)

-

Moderate Smokers (15-249. a day)

Heavy Smokers ( 259. or more a Qy)

FIG.4. Standardized death rate from lung cancer among men smoking four different amounts of tobacco, expressed as a percentage of the unweighted average of the four rates: ( I ) estimated from a retrospective inquiry into patients smoking histories (Doll and Hill, 1952), and (2) observed during a prospective inquiry into the mortality of doctors (Doll and Hill, 1954a).

much of the observed differences, since the association between smoking habits and place of residence (Doll and Hill, 1952; Hammond and Horn, 1954) is much weaker than the association between smoking and the disease. 4. There remains, therefore, the possibility that the association is real and direct. C. Method of Smoking. The evidence suggests that all forms of smoking are not equally associated with the disease. Pipe smoking and cigar smok-

16

RICHARD DOLL

ing are less closely associated with it than cigarette smoking (Wynder and Graham, 1950; Levin et al., 1950; Schrek et al., 1950; Doll and Hill, 1952,1954a; Sadowsky, Gilliam, and Cornfield, 1953; Breslow et al., 1954; Watson and Conte, 1954; and Hammond and Horn, 1954). Only McConnell, Gordon, and Jones (1952) and Randig (1954) failed to find any distinction between the various methods of consumption of tobacco. From the mortality rates estimated by Doll and Hill (1952) it would appear that the risk among “pure pipe smokers” may be as much as two-thirds the risk among ‘ I pure cigarette smokers,” but they hesitated to draw any precise conclusion because of the variation in the average amounts of tobacco smoked by the different types of smoker and because of the difficulty of separating with certainty a group of smokers who had never smoked cigarettes a t all. Sadowsky, Gilliam, and Cornfield (1953) found a greater difference between pipe and cigarette smokers, and Hammond and Horn (1954) found that the mortality among smokers who had never smoked cigarettes was practically identical with that among nonsmokers (see Table V). Hammond and Horn’s evidence is particularly important because it was obtained from a prospective inquiry in which great care had been taken to eliminate from the pipe and cigar group all men who had ever smoked as much as ten packs of cigarettes in their entire lives. The investigation was on such a scale that even with this definition it was still possible to secure a large group for study, and these results are likely to be more reliable than those of other workers who defined the categories of smokers less strictly. Few people (outside South Africa) have smoked filter-tipped cigarettes or used cigarette holders regularly for any length of time, and it has, therefore, been difficult t o obtain evidence regarding the possible protective effect of these methods of smoking. The data obtained by Doll and Hill (1952) are shown in Table VII. A smaller proportion of the lung cancer patients than of the control patients had used holders and a smaller proportion had smoked filter-tipped cigarettes, but the numbers are small and it would be unwise to draw any positive conclusions from this very limited evidence. It might be that the use of cigarette holders and of filter-tipped cigarettes are both associated with light smoking, but this did not appear to be the explanation among the patients referred to above. D. Extent of Risk. The results of the two prospective inquiries which have been reported do not as yet permit direct measurements to be made of the full extent of the lung cancer mortality among smokers of different quantities of tobacco. Persons who were seriously ill when the inquiries were started are relatively unlikely to have been included in the initial population, and consequently the mortality rates recorded in the first year

17

ETIOLOGY OF LUNG CANCER

or two of follow-up are almost certainly too low. Until a few more years have elapsed, estimates of the risks to which the different categories of smokers are exposed must, therefore, still be based on the data derived from the retrospective studies. Estimates have been made by Doll and Hill (1952), Heady and Barley (1953), Sadowsky, Gilliam, and Cornfield (1953), and Wynder and Cornfield (1953). The results obtained in Britain and the United States have TABLE V I I Use of Cigarette Holders and of Filter-Tipped Cigarettes: Male Lung Cancer Patients and Matched Control Patients* Male Lung Cancer Patients

hfale Control Patients

No.

%

No.

%

Use of Cigarette Holders Regularly Occasionally Never

10 15 479

2.0 3.0 95.0

27 27 413

5.8 5.8 88.4

Total Cigarette Smokers

504

100.0

467

100.0

Use of Filter-Tipped Cigarettes Ever regularly Never regularly

3 501

0.6 99.4

15 452

3.2 96.8

504

100.0

467

100.0

Type of Smoker

Total Cigarette Smokers

_~

-

Sniokers FVho Had Never Smoked Cigarettes Nonsmokers __ Total men

15 4

30 26

523 t

523 t

--

--

* After Doll and

Hill, 1052 total numbers of men are diffeient froin the numbers shown in Table I V , because questions about the use of cigarette holders and filter-tipped cigarettes were only Introduced in the last part of the inquiry.

t The

been compared by the two latter groups of authors and, in a more detailed fashion, by Cutler and Loveland (1954). Cutler and Loveland’s estimates are summarized in Table VIII. From the table it appears that for a man aged 40 years who smokes 20 or more cigarettes a day (1) the risk of dying of lung cancer before the age of 80 years is of the order of 8% and that (2) this risk is some 6+ to 30 times as high as that among nonsmokers. The reliability of these estimates depends on the validity of certain

18

RICHARD DOLL

assumptions which had to be made before the rates could be calculated. These are: 1. That the deaths recorded nationally as being due to lung cancer provide a fair estimate of the actual number of deaths due to the disease. 2. That the smoking habits recorded by patients with lung cancer were, at each age and in each sex, typical of all those persons who died of the disease during the period of the survey. 3. That the smoking habits recorded by the ((control” patients without lung cancer were similarly representative of those of all members of the population from which the lung cancer patients were drawn. TABLE VIII Risk of Developing Lung Cancer among Men Smoking Different Amounts of Tobacco, Estimated from the Results of Three Groups of Investigators* Estimated Risk of Developing Lung Cancer by the Age of 80 Years, per 1000 Men Aged 40 Years

Amount Smoked

Sadowsky, Gilliam, and Cornfield (1953)

Wynder and Graham (1950)

Doll and Hill (1952)

Combined Results

10

3

5

6

22

19

34

25

46

52

48

49

65

90

86

80

Nonsmokers Smokers, of under 10 g. a day Smokers of l(f20 g. a day Smokers of more than 20 g. a day

* After Cutler and Loveland,

1954.

Further assumptions are also required about the trend of future changes in mortality in order t o present the risks in the form chosen by Heady and Barley (1953) and by Cutler and Loveland (1954), but these are of minor importance in that they have little effect on the relative sizes of the risks for the different smoking categories. Whether the assumptions are justified is impossible to say with certainty, and the rates must be regarded as provisional. I n view, however, of the conformity of the estimates calculated from data from independent investigations in different countries and the further confirmation of the relative sizes of the risks by the preliminary results of the prospective inquiries, it is unlikely that the estimated rates are seriously in error. The correspondence between the results of Doll and Hill’s two investigations has been shown previously in Fig. 4, and Hammond and Horn’s

ETIOLOGY OF LUNG CANCER

19

“best estimate . . . that lung cancer deaths are from 5 to 16 times as common among men who smoke one pack or more per day” as among nonsmokers largely overlaps the estimates made by Cutler and Loveland (Table VIII). On the other hand, the fact that smoking habits are not invariable and that the amounts which have been related to mortality have been recorded only a t one point in time must have blurred the differences between the smoking categories, so that the estimated rates for light smokers are probably somewhat overestimated, whereas the rates for heavy smokers are likely to have been underestimated. The estimated rate for nonsmokers is low but it is not intrinsically unreasonable. At ages 45 to 74 the rates calculated from the English data are slightly lower than the rates which actually occurred among women in rural areas in England (Doll, 1953b), and they are similar t o the rates now recorded among women in Denmark and several other countries (see Table I). If these rates represent the mortality risk in the absence of smoking, then the number of deaths from lung cancer attributable to causes other than smoking among persons aged 25 to 74 years in England and Wales in 1950 would have been about one-fifth of the total number actually recorded. E. Diference between Histological T y p e s . In the foregoing discussion no consideration has been given to the possibility that the different histological types of carcinoma of the lung may have different etiological relationships to smoking. It has, however, been suggested that the relationship with smoking holds only for squamous, oat-cell, and anaplastic carcinomas. This qualification makes little difference to the conclusions which have already been drawn, because, wherever lung cancer is common, the great majority of cases are of the squamous, oat-cell, or anaplastic types. The distinction is, however, of considerable theoretical interest. Three reports have paid special attention to histological differences. Wynder and Graham (1950) found that the smoking habits of 39 men and 15 women with adenocarcinoma were closely similar t o those of control patients with diseases other than lung cancer. Doll and Hill (1952) reported that there was “ n o statistically significant difference between the amounts smoked by patients in the different histological groups in either sex. The number of cases of adenocarcinoma is, however, too small (33 male and 10 female) to conclude that no difference exists. There were, in fact, relatively more non-smokers and very light smokers . . . among the patients with adenocarcinoma in both sexes.’’ Breslow et al. (1954) noted that “Six out of 46 (13 per cent) of the cases of adenocarcinoma did not smoke cigarettes; whereas only 28 out of 472 (6 per cent) of the patients with other types of carcinoma . . . did not smoke ciga-

20

RICHARD DOLL

rettes.” The interpretation of these data is complicated by the inclusion of men and women in a single series. When, however, the sexes are separated, the distinction still persists (Breslow, personal communication). The most striking evidence has, however, been obtained by Kreyberg (personal communication), and the author is indebted to him for permission to cite his results obtained up to the end of 1954. Smoking histories were taken from patients in the wards of the Rikshospitalet, Oslo, before operation, and the histological typing of the tumors was made independently without knowledge of the patient’s history, sex, or age. The most recent amount of tobacco smoked daily by men whose tumors were classified as belonging to the two main histological groups (see page 7) was as follows: Type of Tumor Group 1 Group 2 Ratio of group 1 t o group 2 tumors

No. of Nonsmokers 3 2 1.5/1

No. of Men Smoking Daily: 1-9 g. 10-19 g. 20 g. 40 98 52 11 14 5

+

3.6/1

7 . 0/1

10.4/1

Total No. of Men 193 32 6 0/1

Finally, Wynder (1954) has studied the problem by collecting details of the histology of the cases of lung cancer which are reported to have occurred among male nonsmokers. Twenty nine per cent (i.e., 14 out of 48) were adenocarcinomatous, whereas only 5% (i.e., 54 out of 1019) were adenocarcinomatous among male smokers with lung cancer in his personal series. It must, therefore, be concluded that adenocarcinoma of the lung is less closely related t o smoking habits than are the squamous, oat-cell, and undifferentiated types of cancer, and it may well prove that smoking plays no part a t all in its production. F. Vital Statistics and Tobacco Consumption. The sharp increase in the number of deaths attributed to lung cancer during a period when tobacco consumption was also increasing has been cited as one reason for believing that tobacco is a cause of the disease. I n fact, correlations in time may be-and often are-entirely irrelevant, so that they are of no value in proving the existence of a causal relationship. On the other hand, if a relationship can be demonstrated by other means, i t is reasonable to test its significance by seeing if it is consistent with such temporal changes as are observed t o occur. The changes which have taken place in tobacco and cigarette consumption and in lung cancer mortality in England and Wales in the last 70 years are illustrated in Fig. 5 and Table IX. Whether the correlation between cigarette consumption and mortality is as close as would be expected if cigarettes were one of the principal causes of the disease is

21

ETIOLOGY OF LUNG CANCER

P

3

7.0

.---. x-* 0......0

CANCER DEATH RATE TOBACCO CONSUMPTION CIGARETTE CONSUMPTJON

1920

)

1930

1940

1950

P

I

FIG.5. Crude death rate from lung cancer in England and Wales and per capita consumption of cigarettes and of all tobacco products in Great Britain, 1900-1953.

TABLE I X Crude Lung Cancer Death Rate and Consumption of Cigarettes and Other Tobacco Products for Men and Women Separately in England and Wales, 1881-1950 Annual Consumption, Lbs. per Adult (aged 15 years +) Men Period 1881-90 1891-1900 1901-10 1911-20 1921-30 1931-40 1941-50

Lung Cancer Death Rate per 1,000,000 Persons (aged 15 years $)

Women

Cigarettes Other Tobacco Cigarettes Date 0.006 0.4 1.8 3.8 5.1 6.9 8.3

6.1 6.2 4.9 4.3 3.7 2.7 2.4

Men

Women

25 74 256 624

13 27 68 111

0.0

0.0 0.0

0.0 0.2 0.8 2.4

1920 1930 1940 1950

Note. The figures shown in this table are derived from a different source from those shown in Fig. 5 and differ in that the estimates of tobacco consumption exclude the amounts consumed duty-free in the Merchant Navy and in the Armed Forces abroad. Except in wartime these amounts are negligible. The figures also differ in that the table shows rates per adult (or per 1,000,000 adults) and the figure shows rates per person (or per million persons).

22

RICHARD DOLL

uncertain, because many of the relevant facts are unknown. It is, for example, not known: 1. What proportion of the increase in recorded mortality is real. 2. What are the relative risks attached to the smoking of tobacco in cigarettes and in other forms. 3. What is the biological relationship between the dose of cigarette smoke and the development of the disease.

On the basis of present knowledge one may, perhaps, suggest more or less reasonable solutions t o the first two problems; although it must be admitted that no estimate of the extent of the real increase in mortality can be more than an intelligent guess. Save, however, that the evidence indicates that mortality varies in direct arithmetical proportion with the amount smoked at a given time, we are completely ignorant of the third. We neither know the induction time of the tumor nor the relative effects of the same dose a t different periods of life; and different hypotheses about either of these must lead to gross differences in the temporal relationship between consumption and mortality. If, for example, it is postulated that the mechanism of carcinogenesis is of the type suggested by Nordling (1953), Stocks (1953b), and Ambrose (1954), i t might well be that the effect of a dose of cigarette smoke a t a given time is proportional to the fourth or fifth power of the time elapsing after its administration (Armitage and Doll, 1954). On such a hypothesis a reasonable agreement between the figures in Table IX can be demonstrated. I n the present state of ignorance, however, it is probably better not to attempt any exact correlation; but to note only that if smoking is a major cause of lung cancer it will be difficult to account for the figures unless there is also a considerable difference in the relative effects of smoking cigarettes and pipes. It is almost equally difficult to decide whether differences in smoking habits are adequate to account for the difference in mortality observed in men and women. At first sight it would seem unlikely that they were, since women have been responsible for an increasing proportion of the total amount smoked and, in all countries, the preponderance of men among subjects of the disease has become more marked. The difficulty, however, is the same as was encountered previously; that is, we do not know the induction time of the disease nor the relative importance of smoking a t different periods of life. From the figures which are available for Britain (Table IX) it would seem that so long as the effect of smoking does not reach its maximum till after 20 years, differences in smoking habits could readily account for a large and still increasing difference in the mortality of men and women. I n fact, it may well be that the cases of

ETIOLOGY OF LUNG CANCER

23

lung cancer among women still include only a small proportion specifically related to tobacco and that the major increase in female mortality is to come. An alternative approach is to estimate the mortality among male and female nonsmokers. According to Doll (1953b) the proportions of men and women found among the nonsmokers with lung cancer in Doll and Hill’s (1952) series are consistent with the hypothesis that in the absence of smoking (and exposure to certain industrial carcinogens) the death rates are equal in the two sexes. The attempt t o compare mortality and tobacco consumption in different countries is even more hazardous, for not only do standards of death certification vary but so do methods of smoking. It is said, for example, that few Europeans throw away as large an unsmoked butt as is commonly discarded in the United States. It is, however, of interest to make the comparisons, provided that the deficiencies of the data are recognized. Statistics for 8 countries have been collected by Nielsen and Clemmesen (1955), and these have largely been drawn on, in the preparation of Figs. 6 and 7. I n Fig. 6, the male death rate from lung cancer in 11 countries in 1950 (or in the nearest year for which the information is available) has been plotted against the annual consumption of all tobacco products per head of the population 20 years earlier; in Fig. 7 it has been plotted against the annual consumption of cigarettes per head 20 years earlier. In fact, nearly all the tobacco consumed in 1930 was consumed by men (even in the Scandinavian countries the amount of tobacco smoked by women a t that period was small) so that it is not unreasonable to compare the male death rate with the per capita consumption. In nearly all the countries the consumption per man in 1930 is likely to have been approximately double the consumption per person. Figure 6 fails to show any relationship between lung cancer mortality and total tobacco consumption in the various countries, but from Fig. 7 it would appear that the data (with the exception of those from the United States) are not inconsistent with the existence of a relationship with cigarette consumption. To a small extent the anomalous position of the United States can be explained by the high proportion of young people in its population; whether the sort of consideration which has been referred to above can account for the rest is a matter for conjecture. What is certain is that the observed facts fit the hypothesis of a relationship between the disease and cigarettes much better than the hypothesis of a relationship with tobacco generally. G. Identification of Carcinogenic Agent. Numerous attempts have been made t o induce cancer with tobacco products in animals. A significantly increased incidence of the common pulmonary adenoma of mice was ob-

24

RICHARD DOLL

served on one occasion by exposing animals with a high spontaneous incidence of the tumor t o strong concentrations of cigarette smoke (Essenberg, 1952), but no change, or very little change, in incidence has been observed by others (Passey, 1929; Campbell, 1936; Loren2 et al., 1943).

0’

I

I

1.0

I

I

2.0

I

I

1

3.0

Annual tobacco consumption, kg. per person (1930) FIG.6. Crude male death rate from lung cancer in 1950 and per capita consumption of all tobacco products in 1930 in various countries: (1) Great Britain, (2) Finland, (3) Switzerland (tobacco consumption estimated from data published by Gsell, 1951, and Nielsen and Clernmesen, 1954), (4) Holland, (5) U.S.A., ( 6 ) Australia (death rate for 1951-52), (7) Denmark, (8) Canada, (9) Sweden, and (10) Norway. Coescient of correlation between death rate and tobacco consumption, 0.10 k 0.31.

Wright (1955), in particular, exposed 80 mice of the “Strong A ” strain t o an average concentration of 0.08 mg. of smoke per liter for 20 hours a day for 5 days a week. There were many deaths in the early weeks of exposure, and Wright doubts if a stronger concentration could be used successfully. The average number of tumors in the 34 mice which survived

ETIOLOGY OF LUNG CANCER

25

from 3 to 15 months exposure was higher in the experimental group than in a control group of paired 1it)ter mates killed a t the same ages (1.15 against 0.76), but the number of tumor-bearing mice was less (44% against 56%). Neither difference is statistically significant. According to Moore

t

400

Annual cigarette consumption c i g e r e t t e s per person(1930)

FIG.7. Crude male death rate from lung cancer in 1950 and per capita consumption of cigarettes in 1930 in various countries: key as in Fig. 6, with the addition of (11) Iceland (death rate estimated from cancer notification rate). Coeflcient of correlation between death rate and cigarette consumption, 0.73 f 0.30.

(1953) Graham produced “what appeared to be one small papilloma in the bronchus of a dog” by painting tar from cigarette smoke through a fistula onto the bronchial mucosa; but the report proved to be erroneous and no tumor has appeared after three years (Graham, personal communication). Tumors comparable to bronchial carcinoma as it appears in man have not been produced by tobacco products by any method.

26

RICHARD DOLL

Occasional tumors on the skin of mice and rabbits have, on the other hand, been produced by several workers. The literature is reviewed by Wynder, Graham, and Croninger (1953), who have themselves been able to produce tumors in a high proportion of treated animals. They obtained tar from cigarettes smoked mechanically under conditions which approximated to the physical conditions of normal smoking and applied it three times a week to the skin of mice, until a carcinoma appeared or the animal died. To avoid toxic reactions from the nicotine content of the tar, the dose given initially was small and it was increased gradually over the following two months. The first papilloma appeared after eight months of painting: the first carcinoma, after one year. Of 81 mice initially included in the series, 62 survived for a year or more and 36 of them (Le., 58 %) finally developed cancer. Passey (personal communication) has, however, pointed out that with the method of combustion used “Temperatures up to 966°C. were obtained” and that this is appreciably higher than the combustion temperatures in normal smoking (see page 27). Tar obtained from cigarettes burnt at temperatures not exceeding 750°C. has, in the hands of Passey et al. (1955) not reproduced Wynder, Graham, and Croninger’s results. Three potentially carcinogenic substances have been distihguished in tobacco smoke : arsenic, benzpyrene, and radioactive potassium. Arsenic is present in many tobaccos, probably because of its use as an insecticide. It is present in greatest amounts in tobacco of American origin and is completely, or almost completely, absent from Oriental types. Daff and Kennaway (1950) estimated that an ordinary “Virginian” cigarette as smoked in England, contains about 50 pg. expressed as AszOa and that approximately 15% is volatilized in smoking. Smoking 10 cigarettes a day means, therefore, that as much arsenic as is present in one maximum official dose of Fowler’s solution is volatilized in 10 weeks. Arsenic is believed to be capable of inducing bronchial carcinoma in man (see page 33) but it is unlikely to be the responsible agent in tobacco smoke, since (1) the amount to which smokers are exposed is very small compared with the amounts encountered industrially, and (2) bronchial carcinoma forms a high proportion of cancer cases found at necropsy in Istanbul (Schwartz, reported by Daff, Doll, and Kennaway, 1951) and arsenic is almost completely absent from Turkish tobacco. The presence of polycyclic hydrocarbons in tobacco smoke has long been suspected, but no individual substances were identified until Cooper and Lindsey (1953) and Commins, Cooper, and Lindsey (1954) reported the presence of anthracene and pyrene in tar obtained from cigarette smoke. Subsequently Cooper, Lindsey, and Waller (1954) reported that they had also distinguished the presence of 3,4-benzpyrene. To obtain

ETIOLOGY OF L U N G CANCER

27

the tar, the cigarettes were smoked mechanically but care was taken to make the physical conditions of combustion correspond closely to those which occur in normal smoking. The substances were detected by means of chromatography followed by absorption spectrophotometry; 10.2 pg. of anthracene, 9.0 pg. of pyrene, and 1.0 pg. of 3,4-benzpyrene were estimated to be present in the tar collected from the smoke of 100 cigarettes. The amount of benzpyrene is small and it is necessary to smoke 200 cigarettes t o obtain enough to produce, by local injection, a sarcoma in a mouse. Quantitatively it is less important than the amount in town air, since i t would be necessary to smoke 50 cigarettes in order t o inspire as much as is inspired from the air in one day by a “standard man” in a n average English industrial town (Waller, 1952; Blacklock et al., 1954). On the other hand, the benzpyrene in cigarette smoke, being dissolved in the form of a fine suspension in a solvent material, may well be more active than the atmospheric benzpyrene, which is largely adsorbed on carbon particles and is, in this state, relatively inactive (Steiner, 1954). Part of the atmospheric benzpyrene is also likely to be filtered off by the nose. Benzpyrene has also been identified in the tar collected from the smoke of “cigarettes” made entirely of paper (Cooper and Lindsey, 1954; Lefemine, 1954). The quantity present was, however, such th a t the combustion of the paper could account for only about 5% of the benzpyrene present in the smoke from ordinary tobacco cigarettes. Lindsey (1954 and personal communication) has, moreover, also found that benzpyrene is present in the smoke from tobacco burnt in a pipe in the same order of quantity, weight for weight of tobacco, as was obtained from cigarette smoke. Commins, Cooper, and Lindsey (1954) suggest that the polycyclic hydrocarbons may be formed by the pyrolysis of acetylene. Kennaway (1924, 1925) has shown that strong heating of acetylene and other unsaturated materials produces carcinogenic tars a t temperatures of 700°C. and above, and Fishel and Haskins (1949) showed that acetylene was present in tobacco smoke. The temperature of combustion in ordinary cigarettes, in paper “cigarettes,” in a pipe, and in a cigar, were, according to Lindsey (1954 and personal communication), as follows:

Ordinary cigarette Paper “cigarette ” Pipe Cigar (Havana)

Quiescent Combustion Temperature 650°C. Varies Varies 400”-500”C.

Suction Combustion Temperature 700°C. 655°C. 470°C. 560°C.

Surface Temperature 900°C. 900°C. 700°C. 800°C.

+ + + +

28

RICHARD DOLL

Closely similar temperatures in burning cigarettes have been recorded by Wynder, Graham, and Croninger (1953) and by Hamer (1954), and a similar temperature for combustion in pipes was recorded by Cooper et al. (1932). If Lindsey’s observations on the presence of benzpyrene in the smoke from tobacco burnt in pipes are confirmed, it would seem likely that polycyclic compounds can be formed a t lower temperatures than have, hitherto, been thought to be necessary. If benzpyrene is the active agent responsible for tobacco cancer, the findidg of its presence in pipe smoke will accord with the high incidence of cancer of the lip and buccal cavity, known to occur among pipe smokers. I n this case it will be necessary to explain the considerable difference in the incidence of lung cancer which is found between pipe and cigarette smokers on the basis of differences in the physical dispersal of the smoke (and of particles and droplets in the smoke), associated with the two methods of smokingfor example, in the proportion of smokers who inhale. It will, however, be recalled th at although benzpyrene is a strong carcinogen t o which man and animals are both susceptible, it has yet to be directly established that it has any such action on the bronchial mucosa. It is possible that the active agent is, in fact, some substance hitherto not recognized as being carcinogenic. On the other hand, the high mortality from lung cancer among gasworkers, who are specifically exposed to large quantities of benzpyrene in the course of their work (see page 3P), supports the hypothesis th at benzpyrene is also carcinogenic in the human bronchus. Mulvaney (1953) suggested that radioactive potassium, present in tobacco as a naturally occurring isotope, might be an effective carcinogen. Swinbank (personal communication) found that a typical cigarette contained 24 mg. of potassium and that the radioactivity corresponded to 2 f 1.7 mg. more. The errors of the experiment were likely to have been greater than the statistical error, so that there was probably no excess activity a t all. Even if there were, and it were all due to the presence of the most dangerous substance, radium, the amount estimated would not be biologically important. Swinbank estimates that, if the whole potassium content of cigarettes were inspired and if 20 cigarettes were smoked per day for 50 years, it is unlikely that the total dose would amount t o more than the maximum permissible weekly dose recommended by the International Commission on Radiological Protection. Spiers (1954) found, however, that such radioactivity as is present in cigarettes remains almost entirely in the ash, and he was able to detect only the equivalent of 6 fig. of potassium in the smoke. It is, therefore, not possible to attribute any significant carcinogenic effect to radioactivity in tobacco.

ETIOLOGY O F L U N G CANCER

29

The further possibility remains that cigarette smoke is not in itself carcinogenic but that it acts as an activator or a co-carcinogen to substances already present in the air from other sources. This hypothesis is less attractive than it was, now that benzpyrene has been found t o be present in appreciable quantities in cigarette smoke, but it is possible that the effect of tobacco is enhanced by the presence of solvents in the smoke such as pyridine and pyrrole which could, theoretically, elute the benzpyrene adsorbed on to carbon particles in the inspired town air and so render the atmospheric benzpyrene more active. Such a secondary effect can readily be envisaged as being responsible for some of the differences in mortality in urban and rural areas referred to below. H. Various Criticisms. The conclusion that cigarette smoking is a cause of lung cancer has not been uniformly accepted. It has not, to the author’s knowledge, been argued that the basic data are factually erroneous (this would hardly be possible, since all who have investigated the subject have found the same general trends) but it has been suggested that the wrong interpretation has been put on the results-an interpretation incompatible with all the known facts. At first the principal objections were (I) that the retrospective study of patients’ histories provided too many opportunities of bias for the results to be relied on (Hammond and Horn, 1953; Shapiro, 1954) and (2) that no known carcinogen had been identified in tobacco smoke. I n the light of the follow-up studies on men of known smoking habits and of the recent biological and chemical studies on cigarette smoke, these criticisms are now only of historical interest. The current objections may be considered under five main heads. 1. That the evidence i s purely “statistical” and that it has not been possible to produce the disease in laboratory animals by means of tobacco smoke (Shapiro, 1954). A similar type of objection could have been made to Snow’s conclusion that cholera was a water-borne disease or to Pott’s conclusion that employment as a chimney sweep in childhood led t o cancer of the scrotum. It is difficult t o see what can be more relevant to the etiology of human cancer than observations on the extent of human mortality under different environmental conditions and, in this instance, nature has performed the appropriate experiment, in which the amount smoked has been varied while, so far as can be seen, other variables have been kept constant. I n view of the variation in animal susceptibility and the impracticability of reproducing the exact conditions of human smoking in animal experiments, the failure to reproduce the human type of bronchial carcinoma by exposing animals to tobacco smoke cannot outweigh the extensive positive evidence obtained from direct observations on men.

30

RICHARD DOLL

2. That the data recorded in the various investigations are not wholly consistent. Although the general trend of all the results has been consistent, there have been inconsistencies in the reports of the relative risks attached t o cigarette, cigar, and pipe smoking and in regard to the significance of inhaling (Hueper, 1954). The inconsistencies reported in relation to the different methods of smoking have already been considered (see page 16), but no reference has yet been made t o inhaling. It would commonly be expected that any effect of cigarette smoking would be most noticeable among persons who inhaled the smoke, and both Lickint (1953) and Breslow and his co-workers (1954) found that a higher proportion of patients with lung cancer than of control patients said they inhaled. On the other hand, Doll and Hill (1952) found no difference in the proportion of inhalers in the two groups, although there was a suggestion that inhaling might be commoner among men with peripheral growths and even less common than among the controls in men with central growths. The explanation of these conflicting reports is unknown. It may, perhaps, derive from a failure on the part of the patients t o understand correctly the import of the questions. 3. That the interpretation put on the results i s incompatible with the evidence from vital statistics. Several of the principal criticisms of this type have already been discussed and they will, therefore, only be listed here. a. That although the consumption of tobacco has undoubtedly increased, it has not yet been shown satisfactorily that there has been any real increase in the incidence of the disease (Rigdon and Kirchoff, 1953; Shapiro, 1954). See page 4. b. That (on the contrary) the increase in tobacco consumption has not been great enough to account for the increase in the incidence of the disease (Todd, 1954). See page 22. c. That the correlation between mortality and cigarette consumption as recorded in a number of countries is imperfect (Hueper, 1954) and that the data from Britain and the United States are, in particular, incompatible (Russ, 1954). See page 23. d. That the male predominance among cases of the disease persists despite the fact that the increase in cigarette smoking has been relatively greater in women (Hueper, 1954). See page 22.

Other objections which have not been referred to previously are:

e. According to Hueper (1954) the fact that the sex ratio of cases of the disease was practically equal in Norway until about 1930, and that since then an increase has begun t o be recorded which is most marked in men and in towns, weighs against the view that

ETIOLOGY O F LUNG CANCER

31

smoking is an important cause of the disease. He considers that special factors must have influenced the epidemiological behavior of lung cancer in Norway, whereas Norwegians smoke the same type of cigarette as that smoked in the United States. He omits, however, to take into consideration the very small quantity of cigarettes which were consumed in Norway before 1930, and the Norwegian experience may, with greater justification, be cited in support of the view that cigarettes are one of the principal causes of the disease. f . Several authors have pointed out that the mucosa of the lip, mouth, and larynx also comes into contact with cigarette smoke and yet the mortality from cancer of these sites, in contrast with the mortality from lung cancer, has remained stationary or has fallen (Hueper, 1954; Passey, 1954; Maxwell, 1955). Many investigators have, however, shown that cancer of the lip and mouth is, if anything, associated with the smoking of cigars and pipes (Levin et al., 1950; Sadowsky et al., 1953), so that a reduction in mortality from cancer a t these sites would have been expected t o occur as smoking habits were switched from cigars and pipes t o cigarettes. The evidence with regard t o laryngeal cancer is more conflicting. There is no a priori reason t o suppose that it is necessarily produced by the same factors as produce lung cancer, and, in fact, none of the known industrial causes of lung cancer are known to cause laryngeal cancer. On the other hand, there is evidence that laryngeal cancer is associated with cigarette smoking (Levin et al., 1950; Sadowsky et al., 1953). It is possible that the lack of any marked increase in the recorded mortality from laryngeal cancer results partly from improvements in the treatment of intrinsic cancer of the larynx and partly from confusion by the classification under one head of cancer of the extrinsic and intrinsic larnyx. 4. That a n association i s not necessarily causal and that both lung cancer and smoking may be the end results of a third common factor. It is not possible to give any conclusiye answer to this type of criticism and, indeed, a similar hypothesis always provides an alternative explanation to any scientific theory. The principle of Occam’s razor has, however, proved of value to the development of scientific thought in the past, and it would seem reasonable to adhere to it now, and to work on the basis of the most economical hypothesis-unless or until some conclusive reason is shown for abandoning it. The possibility that lung cancer and smoking may both be end results of a third common cause is as much applicable to the results

32

RICHARD DOLL

of the prospective studies on mortality among smokers and nonsmokers as it was t o the results of the retrospective studies among patients. There is, however, no evidence to suggest that it is the explanation of them. Some of the theoretically possible common factors have been considered above (see page 14) ; another which has been suggested is that persons of a particular physical constitution might be prone to lung cancer and t o heavy smoking (Parnell, 1951). There is no evidence of such a physical constitution characteristic of patients with lung cancer; and if one did exist, we should still have to find some environmental factor to account for the increase in the incidence of the disease. 5. T h a t the e$ect of smoking i s limited to determining the site of the growth in persons previously destined to develop cancer (Fairweather, 1954 ; Loxton, 1954). This objection derives from Cramer’s (1936) hypothesis that the total incidence of cancer in a population is constant and that environmental and hormonal factors exert their effect by determining the site a t which the cancers develop. I n this general form, it can readily be demonstrated to be not true (Case, 1954). I n the particular case of cigarette smoking it can also be shown to be untrue; for, if it were true, it wouId follow that cancer of sites other than the lung would have t o be relatively more common among nonsmokers and light smokers than among heavy smokers. Several reports of the smoking habits of persons with cancer in other sites have been made. Except for a report b y Gilliam (1954) none has, in fact, shown a negative association between smoking and the type of cancer investigated, though several have suggested the possibility of other positive associations, e.g., between cigarette smoking and cancer of the larynx and between pipe smoking and cancer of the lip (Levin et al., 1950; Doll and Hill, 1950, 1954a,b; Sadowsky et al., 1953; Hammond and Horn, 1954). Gilliam added details of the data on cancer of the skin t o the data previously reported in conjunction with Sadowsky and Cornfield, and these showed a greater prevalence among nonsmokers than among cigarette smokers. The excess prevalence of skin cancer among nonsmokers could conceivably result if it were found that there were fewer smokers in the South of the United States than in the North. I . Conclusion. I n a review of the evidence relating lung cancer to smoking, Gilliam (1954) concludes that: “Proof in the mathematical sense is unobtainable in dealing with medical problems. Direct experimental verification in humans ‘is possible to conceive but impossible to conduct.’ Indirect experimental verification in humans, through country-wide discontinuance of smoking and subsequent determination of trends of the disease, could be practically accomplished only by informing the public that the disease is caused by cigarettes. If this is true, the procedure is unnecessary as an experiment.

ETIOLOGY OF LUNG CANCER

33

Production of the disease in experimental animals, under conditions simulating human smoking, would strengthen though not establish the hypothesis, but inability to do so could in no circumferences justify its rejection. . . ‘ I We are left for ‘proof,’ therefore, with indirect and circumstantial evidence derived largely from considerations of the pathogenesis of the disease in individuals and its observed distribution in human populations: in short, with epidemiological evidence. It is a matter of opinion how many and what facts must be consistent before this hypothesis may justifiably be accepted or rejected.”

.

I n the author’s opinion, taking into consideration the philosophical principle of Occam’s razor which has already been referred to, the facts are such t ha t the hypothesis that cigarette smoking is a cause of the main histological types of lung cancer should be accepted. They also, in his opinion, justify a strong presumption that the smoking of pipes and cigars is, in this respect, relatively innocuous. The discovery that a known and powerful carcinogen is present in tobacco smoke in significant quantity strengthens the credibility of the conclusion, but it has yet to be shown experimentally that the substance concerned has a direct action on the bronchial mucosa. The great majority of the observed facts accord with the hypothesis, but the picture is not yet complete. We need to know, in particular, why the mortality from the disease in the United States is so low relative to the past consumption of cigarettes; and why the association which appears t o exist between cancer of the larynx and cigarette smoking has not been reflected in an increase in the incidence of cancer of the larynx comparable t o that believed to have occurred with cancer of the lung. The data on the significance of inhaling are also conflicting, and it is uncertain whether the difference between the effects of smoking tobacco in the form of cigarettes and in a pipe can be attributed to differences in the extent to which the smoke is usually inhaled or whether it is necessary to postulate some other mechanism. These fields of uncertainty are, however, small in relation to the extent of established knowledge and do not justify throwing doubt on the main conclusion. 2. Industrial Hazards

When Smith (1953) presented his report to the Louvain symposium, five industrial processes (the mining of certain radioactive ores, the refining of nickel, and the manufacture of asbestos, chromates, and coal gas) had been recognized as involving a special risk of lung cancer; and there was fairly strong evidence to suggest that exposure t o heavy concentrations of arsenic in the air-of up to 1000 pg. per cubic metermight also produce the disease (Hill and Faning, 1948; Perry et al., 1948). Bonser (1955) has now suggested that hematite miners should be added t o the list. During the last 20 years, 17 cases of lung cancer were

34

RICHARD DOLL

observed a t autopsy among 192 hematite miners (8.9 %), whereas the same pathologist found only 44 lung cancers among 2378 autopsies on men over 20 years old from the same area (1.9%). The evidence is suggestive, but it is not conclusive, since it is possible that miners with chest symptoms may have been more likely to come to autopsy than men with similar symptoms in nondusty occupations. The extent of the risk has now been defined more closely in the case of asbestos, chromates, and coal gas. Brinton, Frasier, and Koven (1952) extended the initial observations of Machle and Gregorius (1948) and compared the sickness and mortality experience of insured workers in the seven chromate-producing plants in the United States with the whole sickness data obtained by the U.S. Public Health Service and the death rates for the U.S. population. They found that, over the period 1940 to 1948, the mortality from lung cancer among white males employed by the plants was 14 times the expected; and among colored males it was 80 times the expected. I n view of the small number of cases, these estimates must be liable t o considerable error, but it is clear that employees of the industry were exposed to a risk which was many times the normal. The physical conditions to which the workers were exposed have been investigated by the Division of Occupational Health of the U.S. Public Health Service (Federal Security Agency, 1953), but it has not, as yet, been possible to define which of the substances involved in the manufacturing process are carcinogenic. The authors suggest that acid-solublewater-insoluble compounds found principally in the residue from the leaching tanks may be responsible. If this were so, it might explain why the hazard has been less apparent in British factories, where the residue is discarded (Bidstrup, 1951). The British industry has, however, only recently come under observation and the possibility that a considerable risk exists has not yet been excluded. Doll (1952) studied the causes of death among 2071 male pensioners of a London gas company and found that the number of deaths from lung cancer was approximately double that expected by comparison with male inhabitants of London of the same age distribution (25 deaths against 13.8)-that is, showed practically the same excess as had been estimated by Kennaway and Kennaway (1947) from study of the national mortality statistics. In both cases the gasworkers covered a multiplicity of occupations, and it is possible that the risk may have been greater for those most closely concerned with the production process. I n a more detailed study, Sutherland (personal communication) found an incidence of respiratory cancer among the employees and pensioners of a Canadian gas company who had worked in the retort house, which was several times higher than that recorded in the general population of the district. The excess was

ETIOLOGY OF LUNG CANCER

35

apparent only among men who had worked a t a particular station where the gas was manufactured in horizontal retorts. No excess was observed among employees who had never worked in the retort houses. According to the earlier reports of Kuroda and Kawahata (1936) the risk experienced by men employed in generator gas plants of a Japanese steel mill is likely t o have been greater still (21 cases of lung cancer occurred in a 6-year period among 100 workers who had been employed for more than 10 years). The risk to which asbestos workers were exposed has been defined more fully by a study of the mortality among 113 men who had been exposed to the dust for 20 or more years (Doll, 1955). Eleven of the thirtynine men who had died were found to have asbestosis and cancer of the lung a t autopsy, whereas the number of deaths expected to have been due to lung cancer was estimated as less than one (0.8). Since the mortality was considerably less among men who had been employed for less than 10 years in the conditions which existed before 1932, when measures were taken t o reduce the amount of dust in the atmosphere, it must be presumed that the risk had a t one time been appreciably greater than the average estimated over the whole period. The occurrence of lung cancer in 14 out of 72 subjects found to have asbestosis a t autopsy has been reported by Bonser (1955). The number of men employed in all these occupations taken together constitutes only a small fraction of the total number of men employed in industry, and the number of cases of lung cancer due to these special hazards can have contributed only a very small proportion t o the total number of cases. Some of the occupations are, however, of particular interest since the carcinogenic agents which are presumed to be responsible for the added risks also have a more general distribution, i.e., radioactive substances and benzpyrene. It is also of interest, as was pointed out by Smith (1953), that the majority of the specific industrial risks appear to be related to inorganic substances, whereas occupational tumors of other organs have usually been traced to organic compounds. Numerous other occupations have been suggested as possibly giving rise t o specific risks. The evidence has been fully reviewed by Hueper (1951, 1952), but in no other instance is it adequate to justify a positive conclusion.* Recent studies by Wynder and Graham (1951), Doll (1953a), and Breslow et al. (1954) have compared the occupational histories of men with lung cancer with the histories of comparable groups of men with other diseases, and Kreyberg (1954a) has compared the occupational histories of patients with his “group I ” tumors with the histories of

* Nor is the recent evidence adduced by Dunner and Hicks (1953) with regard to boiler scalers and grain dockers.

36

RICHARD DOLL

patients with ‘ I group I1 ” tumors and with the occupational distribution of the population of Norway, as shown by the Census. The most interesting finding has been the negative one, that workers who were particularly exposed t o the fumes of motor exhausts (road transport drivers, etc.) were not disproportionately represented among the lung cancer patients in any of the series. 3. Atmospheric Pollution

A. Mortality in T o w n and Country. The principal reason for thinking that atmospheric pollution may be responsible for some cases of lung 4000

r

3000-

-I

c 9

o-

-

-. 1

--

Men

Women

E L

W

n 01 c

2 2000-

>100,000popn.

5

01 73

m

< 100,000popn.

S S

a I

1000

c

0 15

:

i

25

35

#++-

*+edc 45

55

65

Rural districts

Conurbations /Other towns />100,000 popn.

,r-Towns

’I=&=-*

75

arcinogenduring the first week of the esperiment had larger and more iiunierous adrnomas up to the 64th week than did animals receiving only the goitrogen, but after 18 months of CH,TU treatment, the thyroids of both groups appeared similar. Metastases, invasion of neighboring structures, and penetration of the capsule were the morphological criteria used t o determine malignancy. The presence of cancers in both groups at about the same time indicated that the change t o malignancy occurred independently of the initiating effect of the carcinogen, Paschkis

FIG.2. Thr r~lationshipbetneen the length of T U treatment and the percentage of rats developing thyroid gland tumors (Money et al., 1953).

ct af. (194S), on the other hand, found that the simultaneous administration of thioiiracil and 2--hA\Fto rats not only hastened the development of tumors of the thyroid gland but increased their incidencc. Tn T,ong-Evans strain rats fed a basic stock diet containing 3 pg. of iodine per gram and 0.2% PTT' for 18 to 32 months, progressive increase in thyroid weight with time was noted by Goldberg and Chaikoff (1952). Some thyroids increased t o 50 times the size of the glands of control animals not receiving PTC, yet none of the goitrous animals appeared t o develop neoplastic lesions of the thyroid other than adenomas. d refractoriness of thyroid cells in rats treated with 2-AAF and C'H3TL- was noted by Bielschoivsky and Griesbach (1950) who found that rats injected with 5.0 pg. of DL-thyroxine per 100 g. body weight daily for the last three weeks of the experiment reacted to the hormone

79

DEVELOPMENT AND METABOLISM O F THYROID TUMORS

treatment with complete reversal of thyroid hyperplasia and pituitary basophilia. A dose of 2.5 pg. of DL-thyroxine equivalent to the normal daily requirement of their rats, on the other hand, resulted in significant differences in response between animals receiving 2-AAF plus CH3TU and those on CHaTU alone. Most of the 2-AAF-treated females receiving 2.5 pg. DL-thyroxine daily had active thyroids in contrast to the controls, but the pituitaries appeared normal. Many of the animals still had large goiters in which the epithelium was almost as high as in animals not receiving thyroxine, indicating that the pituitary had not been inhibited in most of the 2-AAF-CH3TU-2.5 pg. daily-thyroxine-treated rats. The 2-AAF, therefore, seemed to have effected some change in the thyroid not restored by the usual maintenance amounts of DL-thyroxine. The nature of the change is a t present unknown. The prolonged ingestion of PTU combined with a relatively large amount of iodide (1.5 mg. NaI per rat per day) reduced the goitrogenic effect of the drug (Sellers et al., 1953). The average metabolic rate of the I group than in rats after 15 months was decreased more in the PTU the P T U groups. Thyroid powder almost completely restored the metabolic rate. The plasma protein-bound iodide, however, in the group given PTU I was above the value of the group receiving only PTU, and the T h was slightly below protein-bound iodide of the group given P T U the PTU-treated controls. This supports the view of Sellers et al. (1953), that the amount of thyroid given was inadequate t o replace that normally supplied by the normal gland. The 24-hour uptake of in rats treated with PTU for 15 months, as expected, was diminished per unit of thyroid tissue. The uptake was less in animals fed P T U T h and least for animals fed PTU I, but when calculated on the total weight of thyroid tissue no significant difference existed. Twenty-four-hour radioautographs of the thyroids showed little or no concentration of 1131 in the three PTU-treated groups and no apparent concentration of iodine in the deeply stained colloid of the adenomas. The thyroids of all treated groups varied greatly not only in weight, as will be noted in Table IV, but also in morphology. Large areas comprising most of the gland showed hypertrophy and hyperplasia of the follicular cells and mitotic figures t o such an extent that the normal architecture of the gland was lost. Glands which weighed double or more than control glands had frequent adenomas resembling papillary cystadenomas of human thyroids. Metastases in the lungs were found in a few PTU- and PTU Th-treated animals. Thyroid follicles were found invading the capsule of the gland and muscle venules adjacent t o the gland; they were also found in the submucosa of the esophagus, in the

+

+

+

+

+

+

80

HAROLD P. MORRIS

pulmonary artery, and in numerous alveolar vessels. The metastasizing thyroid tissue resemliled the normal gland in some iiistaiiws, whereas sometimes hyperchromatism, stratification, and frequent mitotic figures Thwere seen. The tremendous enlargement of the thyroid in PTU treated rats was unexplained, but the suggestion was made that during prolonged treatment the amount of thyroxine present in the dried thyroid powder was insufficient t o inhibit the pituitary. The thyroid powder contained considerably more iodine than the maximum allowed

+

TABLE IV Observation on Rats after Prolonged Treatment with PTZi (from Sellers et al., 1953) Thyroid \\-eight

i

Pituitary (mg.)

(I%.)

Group I----

Medim

Range

1

1

.\rediari

Range

dian

Range

47.4! 11.8 9.2- 18.0 130,4 88,4-196.4 39.0; 14.5 10.8- 35.7

36.0 28.5

27.420.2-

PTU

97.7 79.8

44.6- 395.2 12.2 7.8- 17.0 40.2- 568.0 15.0 9.0- 22.4

PTU

50.1 48.6

27.6- 143.4 1 1 . 6 8.2- 15.6 32.0- 94.0' 15.3 7.8-500.0

+ Th

bg./100 mg. urct wt.)

-~

Control

PTU

Iodine Content of Thyroid

I

517.1 363.8- 6 7 4 . 1 20.9 10.0-164.0

2 , 4 o,4- 11.4 23.7

5,6

8.3- 39,9

2,6-

PTU = Proyyl tliiouracil. I = KI. T h = Thyroid r,owder.

by the T7.S.P.for such preparations. The great enlargement of the thyroid n-it h the development of metastasizing aderiomas makes it appear likely that such adenomas, if not actually malignant, were potentially so, and probably the important causal factor in their formation was the continued stimulation of the thyroid by the pituitary. A relative increase in the ehromophobic cells of the pituitary of treated rats was noted by Sellers et al. (1933), who thought such cells might have originated from chromophil cells, but in the large hemorrhagic tumors chromophobic cells were present interspered with mast cells. The pituiTh-treated rats were also significantly heavier (Table taries of P T U

+

DEVELOPMENT AND METhBOLISM OF THYROID TUMORS

81

IV) than those of glands of other treated or control groups. The development of chromophobe adenomas after prolonged PTU treatment in both rats (Sellers et al., 1953) and mice (Moore et d.,1953) suggests the prolonged depression of the circulating hormone of the thyroid as the likely causative factor. The thyroid powder used by Sellers et al. (1953) had a higher iodine content than that of the U.S.P. product, and their experimental evidence would suggest that one explanation of the perplexing observations was that an insufficient amount of thyroid hormone(s) was administered in the thyroid powder to inhibit the TSH of the pituitary. The additional iodine intake of the animal via the thyroid powder further complicated an interpretation of the effects observed. These complex results need further clarification. Although rats treated with 2-AAF alone for periods up t o seven months do not develop tumors of the thyroid gland (Bielschowsky, 1949), it would appear from the observations of Hall (1948) that neoplastic cells of the thyroid gland were created earlier by exposure t o this carcinogen, and that such neoplastic cells remained latent for considerable periods. The subsequent stimulation of the gland by the use of a goitrogen after 2-AAF treatment appears to cause thyroid gland tumors to develop more rapidly than they do by the effects of goitrogen stimulation alone. Partial thyroidectomy was found by Bielschowsky (1949) also t o be an effective means of providing sufficient stimulation from the pituitary t o result in the induction of thyroid adenomas in the thyroid remnants of rats treated for several weeks with 2-AAF. XII. TRANSPLANTABILITY AND METABOLISM OF RAT THYROID GLANDTUMORS 1. Transplantability The transplantability of tumor tissues constitutes one useful criterion for determining the degree of malignancy of a neoplasm. Unsuccessful attempts made by Bielschowsky et al. (1949) to transplant thyroid gland tumors into normal rats regardless of whether the original tumor was induced by CH3TU alone, or in combination with 2-AAF, or whether it had metastasized were good indications of the low degree of malignancy of such tumors. The tumors could be transplanted successfully, however, to thyroid-deficient hosts where hormonal conditions were similar to those existing during the development of the original neoplasms. Even though these rat thyroid tumors (Bielschowsky et al., 1949) showed evidences of invasive growth, ability to metastasize, and a close histological resemblance to malignant thyroid neoplasms in man, they still appeared

82

HAROLD P. MORRIS

to have a low degree of malignancy aiid were still dependent tumors, lacking complete autonomy. The de\.elopment of a transplantable tumor of the thyroid gland from a rat \\-hich had ingested C'HxTU for 23 mouths was obtained by Purves el al. (1!)51). Part of the same inoculum placed in each of two rats rereiving C'HS'I"' comprised the first transplant generation. Histological examination of the first generation grafts indicated the tumor to be an adenoma with well-developed variable acini filled with colloid. The tumor was g r o w for several transplant generations, and by the fourth generation three different histological types were observed; namely, (1) microfollirular adenoma, (2) a lightly staining tissue having a well-developed aciiiar struvture closely resembling normal hyperplastic thyroid and surrounded by adenomatoiis tissue, aiid (3) anaplastic carcinomatous tissue devoid of acinar structure. The anaplastic carcinoma, (3) above (designated as TB'L), varied widely in rate of growth in different animals, but growth of the graft was much more rapid than the growth of the original transplantable tumor. Giant cells with variable numbers of nuclei were a constant feature of this tumor, which Purves et al. (1951) classed as a giant cell carcinoma of the thyroid. This tumor usually killed its host within three ivecks, although some tumor-hearing animals survived several months. These transplantable tumors attained weights of 30 to 50 g., whereas the host weight was less after removal of these enormous tumors than before inoculation, suggesting that withdrawal of nutrient from the host had omirrcd for the use of the tumor. TZe transplantable tumors were found to grow equally well in normal animals as in animals ingesting CI-I sTI', shoning that they were 110 longer dependent on increased amounts of endogenous TSH for continued growth. 2. 7'imor Depeiidericg

The transplantable tumors of the thyroid gland of the rat which devcJloped after proloiiged C'H,TU intake were dependent upon high thyrotropic hormone levels for their continued growth. Such tumors either in the thyroid gland or as grafted tumors disappeared upon the withdrawal of the C'H3TI- aiid could not be made to reappear by thc subsequent readmiiiistration of the goitrogen (Purves et al., 1931). The view was held that the goitrogeri had no influence on the formation of the ncoplastic cells which Purves el al. (1951) believed arose spontaneously in the nornial rat thyroid. Yisible neoplasms occurred only when conditions were suitable for the growth of such neoplastic cells. The suggestion was made (Purves et a!., 1951) th a t the carcinogen 2-AAF acted by accelerating the formation of neoplastic cells of the thyroid

DEVELOPMENT AND METABOLISM OF THYROID TUMORS

83

gland, and the almost total absence of spontaneous tumors of the gland in normal rats could then be explained by the usual amounts of secretion of thyrotropic hormone being insufficient for the growth of the preformed cells. The opinion was advanced (Purves et al., 1951) that the continuous growth of thyroid gland tumors or their propagation by transplantation occurs by the natural selection of fast-growing types which would lead to an increase in the malignancy of such tumors. The slow-growing benign type of structure by virtue of its slow growth and low metastasizing power could not be selectively propagated by transplantation. The truly invasive and metastasizing carcinoma once it appears (Purves et al., 1951) would invariably supplant the benign type entirely after a few transfers. 3. Hormonal Imbalance and Pituitary Cytologg

The existence of a hormonal imbalance for a prolonged period could provide the stimulus (Purves et al., 1951) for the growth of a primary benign tumor from which a malignant variant could be derived possessing complete independence of the original hormonal stimulus causing its development. Such a condition, if it exists, might have considerable importance in human thyroid gland cancer, provided it became possible to recognize precancerous states which could be altered by variation in hormonal levels during the precancerous period. Would i t be possible to develop a TSH assay procedure sufficiently sensitive to detect dangerous levels of circulating thyrotropic hormone which could be used to recognize the existence of potentially precancerous states? Additional work needs to be done on assaying circulating levels of TSH in order to establish its role in the thyroid precancerous and cancerous processes. Some studies of “ thyroidectomy ” cells (Griesbach, 1951) which develop in the center of the pituitary in thyroxine-deficient rats and are derived from the “thyrotropic” cells may help to clarify our knowledge in this area. This cytological method of distinguishing sugar-containing hormones of the pituitary by means of the periodic acid-leocofuchsin test in the rat, dog, sheep, and rabbit has been used by Griesbach (1951) to show that pituitary cells which lose their hormone content and staining properties in thyroxine overdosage are the cells which hypertrophy in response to thyroxine deficiency. The basophil cells (Griesbach, 1951) are the only cell units in the pituitary which contain glycoprotein. The Gomori (Gomori, 1950) elastin tissue stain has been shown by Purves and Griesbach (1951) t o stain cells in the rat pituitary which are identical to those “basophils” t o which thyrotropic function has been ascribed on the basis of correlation of glycoprotein content with content of thyrotropic hormone. The Gomori positive granules are discharged during thyroxine

8-1

H.4ROLD 1’.

MORRIS

defic*ienr.y,and according to Purves and Griesbach (1951) such stained granules are the storage form of thyrotropic hormone.

4. dletabolism of Goitrogen-Produced Tumors in Rats Jlctaholism studies with transplantable thyroid gland tumor Tzz (Purvcs et al., 1951) showed that the tumor concentrated only half as much IIs’ as did the plasma 24 hours after administration. The iodine metabolism of dependent transplantable thyroid gland tumor TI in the 211d transplant generation was found to be approximately one-fifth that of normal rat thyroid. Tltumors did not grow continually but seemed to reach a maximum weight of about 1 g. Second generation TI tumors also appeared to secrete almost sufficient amounts of thyroxine to meet the requirements of the host, although the tumor growth appeared to he depc~rideiiton the presence of unnaturally large aniouiits of TSH. The fact that such tumors reached a growth stasis when wvighing about 1 g. Inflriencc. of

Sormal Thyroidectoniy Thyroiclrrtomy

TI

TAkBI,E V Rat Thyroid Tuntor Sccrction 011 Pituitary Crll Composition (i’nrvcs el nf., 16351)

+ turnor graft

Siiiiihcr

.Xcidophils

of .ktimnls

(70)

narophils

(%‘I

Chromophohcs

(%I

10 4 3

51 2 0 1 e3 5

8 i 13 3 11 I

30 1 86 7 25 4

suggests that an equilibrium may have heen cstahlishcd twtween the tumor thyroxine elaboration and the host thyrotropic hormone secretion. The increase in acidophil and decrease in chromophobe cells of the pituitary in thyroidectomized rats bearing Tl tumor grafts (Table V) supports such a view. Functional activity indicated by radioautography in one of three types of tumors of the thyroid gland in rats was also observed by Money and Rawson (1950) after prolonged treatment with thiouracil. There appeared to be no gross morphological correlation between follivular diameter, cell height, or colloid staining and concentration of I l o r in tumors of the thyroid gland in rats after prolonged ingestion of the thiouracil (Money et al., 1933). Marked differences in concentration rvcrc frequently observed in identical-appearing adjacent follicles. TTndifferentiated masses of embryonic-looking cells containing a few follicles and surrounded by typical goitrogen-stimulated cells were not observed to take up in radioautographic sections. Money et al. (1953) were unable to state whether this type of tissue collects I’31.

DEVELOPMENT AND METABOLISM O F THYROID TUMORS

85

Tumors comprised of cordlike tubular arrangement of follicles with relatively little colloid present in the lumen of the follicles were observed radioautographically by Money et al. (1953) in a few instances to take up some Ii31within the cells that make up the tubular follicles, but in no case were they found to concentrate radioactivity to the extent th a t macrofollicular areas did. A somewhat similar condition was noted in a transplantable malignant thyroid gland tumor, subline 1, of the mouse, which was characterized histologically by a tortuous cordlike arrangement in 5 of cells (Morris et al., 1951) and took up only n \ a t h as much hours after administration of as was taken up by the host's thyroid gland (Wollman et al., 1951). Radioautographs of involuted follicles scattered throughout areas of hyperplasia were found by Money et al. (1953) to develop intense blackened areas indicative of concentration, which was roughly proportional t o the size of the colloid follicle even though some of the follicles appeared to be completely surrounded by hyperplastic tissue. The greatest uptake of 113' in tumors indicated by radioautographs were in lesions showing abundant colloid (Money et al., 1953). Heavy concenbration of isotope was roughly correlated t o the duration of goitrogen administration. Dalton et al. (1948) noted after more than 12 months of thiouracil treatment in C3H mice a tendency for colloid-containing follicles t o reappear in the hyperplastic thyroid gland. Their observation may have some similarity to the abundant colloid-containing lesions of rats given thiouracil for 13 t o 21 months (Money et al., 1953). Massive follicular areas of tumors, however, were found unable to collect 1131, although small follicles containing colloid within the same lobe showed some functional activity. The opinion was advanced (Money et al., 1953) that many areas including single follicles and obvious primary tumors differed from normal thyroids in their response to thiouracil because such areas concentrated significant amounts of I'31as indicated by radioautographs. The inorganic should have been almost completely removed during histological sectioning. The measurements of collected I'3124 hours after administration as presented at the bottom of Table VI for three rats show in one of three animals the uptake per milligram thyroid to be five times that of rats on thiouracil for only ten days (Money et al., 1953). The other two animals, although showing a high uptake per gland, did not have any greater uptake per unit of thyroid tissue than controls treated with thiouracil for ten days (last column, 3rd line from top). 1131 in thyroid gland tumors and abnormal follicles in goitrogentreated rats, possibly occurring as organically bound radioactive iodine, may be present in much higher concentration in localized areas than is

86

HAROLD P. MORRIS

possible in the gland as a whole. If such were the case, the possibility arises that some parts of the tissue may have escaped the thiouracil block. Some explanations for such a condition (Money et aZ., 1953; Purves et al., 1931) might include: (1) that the amount of thiouracil necessary to block 1 l 3 l concentration may differ in long treated compared to normal glands, (2) that thiouracil may not reach the cells in abnormal areasthis possibility seems unlikely because of the great vascularity of thyroid glands of rats ingesting thiouracil, and (3) th at the iodinating mechanism of such thyroid glands is abnormal. Nuch additional work will have to be (wried out to adequately explain the mechanisms of the observed changes. TABLE VI by Rat Thyroids 24-Hour Collection of (Money el al., 1053) Thyroid Kcight Trestmen t

Controls Low-iodine dirt Thioriracil

Long-term thiouracil

(mg.) 11 4 12 0

Per Cent 1 1 3 1 / Gland 10 67 40 00 0 67 0 Bi 59 80 16 88

Per Cent 1131/1ng.

0 3 0 0 0 0

98 38 03 01 16 01

XIII. BIOCHEMISTRY O F THYROID GLAXDTCMORS I N MICE 1 . Inferrelalionships between Thyroid Hormone ( T H ) and Thyroid-Stimulating Hormone ( T S H )

X more detailed knowledge of the metabolism of the normal thyroid gland and thyroid gland neoplasms is needed to clarify the marly factors which are involved in the biosynthesis of TH by t’w normal thyroid gland and the partial or complete loss of avidity for iodine which occurs in many thyroid gland tumors. The variety of procedures available today for the production of thyroid gland tumors in animals provides a unique opportunity t o study merhanisms of thyroid gland carcinogenesis. T h e direct chemical incorporation of radioiodine into the secretion(s) of the thyroid gland provides a precise isotopic label for following the functional activities of that gland during a sequence of gradual morphological changes which occur usually ill portions of the thyroid gland during the development of thyroid gland neoplasms in rats and mice. These gradual changes may best be described as a sequence of changes from the normal

DEVELOPMENT AND METABOLISM O F THYROID TUMORS

87

through hyperplasia to conditioned or dependent neoplasia and finally to autonomous or independent growth. The reciprocal relationship between T H and TSH of the pituitary, as formulated by Salter (1940); Stanley and Astwood (1949); D’Angelo and Gordon (1949); D’Angelo et al. (1951); and others, is illustrated diagrammatically in Fig. 3 (Kracht, 1952; Furth, 1953). Although this relationship has been quite generally accepted, Goldberg (1954) suggested that the level of plasma protein-bound iodine may not be the only factor governing the output of TSH by the pituitary gland. Blood thyroid hormone (TH) (thyroxine and/or triiodothyronine)

i 1 Low I

High 1. Anterior pituitary:

J

Anterior pituitary:

1

1

Augmented TSH output

Inhibition of TSH output

1

JThyroid:

Thyroid:

Increased protease activity

Decreased protease activity ,-Increased

1

1

Hydrolysis of thyroglogulin

thyroblogulin storage

-Release Diminished iodide uptake Diminished T H synthesis

--

1

of T H (thyroxine) (triiodo thyronine) Increased iodide uptake Increased synthesis of thyroglobulin

J

Declining Blood TH ----+ Rising FIG.3. Influence of blood thyroid hormone level on thyroid and pituitary gland activity.

The most convenient experimental methods now available for the production of thyroid gland tumors exert their effect by goitrogenic interference with the hormonal balance between the thyroid and pituitary glands which seemingly is initiated by a low circulating level of thyroid hormone(s). Figure 3 shows some of the events which take place when either a low or a high blood TH level occurs. The maintenance of a n imbalance for prolonged periods appears necessary to implement thyroid tumor development. Pituitary hyperplasia, which may be followed later by pituitary adenomas and eventually pituitary tumors, also appears to result from the prolonged imbalance. It appears that this thyroid-pituitary imbalance in mice can be produced in different ways, (1) surgical thyroidectomy (Dent et d.,1955),

88

HAROLD P. hfORRIS

(2) prolonged treatment with goitrogens (Dalton et al., 1948; Moore et al., 1953), (3) irradiation (Gorbman, 1949; Gadsden and Furth, 1953; Furth and Burnett, 1952; Dent et al., 1955). Evidence th a t surgical thyroidectomy in mice was about as effective in the induction of pituitary tumors as a single injection of 50 gc. of was noted b y Dent et al. (1955) when they found micro tumors and other pretumorous changes in the pituitaries of surgically thyroidectomized mice which had received neither Il3' nor pituitary tumor implants. Most of the thyroid remnants of surgically thyroidectomized mice including several bearing transplantable pituitary tumors were composed of a few typically formed follicles \vith greatly thickened epithelium containing much intracellular colloid. Other remnants were described by Dent et al. (1955) as sharply circumscribed nodular structures containing irregularly shaped follicles and considerable numbers of interstitial cells which were held t o be thyroid adenomas not unlike those described by Morris et al. (1951) for the mouse and Money and Rawson (1950) for the rat. Surgical depression of the thyroid was also shown by Dent et al. (1955) to be as effective as radiological depression in the conditioning of hosts to carry grafts of dependent pituitary tumors. The development of pituitary tumors in surgically thyroidectomized mice retaining thyroid remnants as well as in mice injected n ith single doses of 25 or 50 pc. of (Dent al., 1955) clearly indicate complete destruction of the thyroid is unneressary for their deyelopment or growth. The precise level of thyroid function, howewr, in mice or other species to prevent pituitary tumor formation is unknown. A long latent period follou-ing depression of thyroid actix ity is essential preceding tumor formation. Some support was gireii (Dent et al., 1955) that the latent period varies inversely with the extent of thyroid depression, but insufficient information was obtained to express such a relation in quantitative terms. Although the precise conditions required for the induct,ion of thyroid and pituitary tumors by goitrogens in mice are undetermined (Dent et al., 1955; Moore ct nl., 1953), the deciding factor appears to be the degree of block of synthesis of TSH-a partial block yielding thyroid tumors; a more complete block pituitary and thyroid tumors; and the complete destruction of the thyroid epithelium, pituitary tumors only (Furth, 1%4). Tumorigenesis by derangement of the usual hormonal equilibrium between thyroid and pituitary might serve as a n attractive model for research 011 neoplastic growths of other cells because of the relative ease and precision of quantitatively assaying the two opposing stimulating agents, TH and TSH. Autonomous pituitary tumors produced in mice, radiothyroidwto(71

DEVELOPMENT A N D METABOLISM OF THYROID TUMORS

89

mized by large doses of when grafted into normal mice cause thyroid stimulation (Furth, 1954). The degree of thyroid enlargement and of thyroid adenoma formation was directly related to the length of the tumor-bearing period, and indicated excessive thyrotropin production by the pituitary graft (Furth, 1954). The stimulated thyroid glands of pituitary tumor-bearing mice were characterized microscopically by resorption of colloid, enlargement of epithelial cells, cytoplasmic colloid masses, vacuolization of the cells, and, after prolonged periods of stimulation, formation of increasing numbers of adenomas some of which were papillary. Radioautographs of such glands (Furth, 1954) indicated a great variability of 113’ uptake in stimulated follicles, but an almost complete lack of uptake in the thyroid adenomas. Invasion of blood vessels by thyroid tissue and metastasis of thyroid adenomas in regional lymph nodes were rare findings, but no special search was made by Furth (1954) to detect the spread of thyroid adenomas. Pulmonary metastases, however, of the thyroid adenomas were not observed. The adenomas were grafted in muscle of mice bearing autonomous pituitary tumors or by intravenous injection of minute thyroid particles. Disseminated “tumor ’’ nodules developed in the lungs which exhibited the same degree of stimulation as the animal’s own thyroid gland, but Furth (1954) did not observe malignant transformation thus indicating that these thyroid adenomas are still dependent tumors. If the observations of Morris et al. (1951) that successive grafts of thiouracil-induced thyroid adenomas become carcinomas can be used as a criterion the malignant transformation of thyroid adenomas induced by pituitary grafts might also be achieved by successive grafts of such adenomas in mice bearing autonomous thyrotropin-secreting tumors. Should such dependent growths (thyroid and pituitary) be called true neoplasms? Any answer it is believed t o this question will remain academic until more complete knowledge has been obtained of the alterations in the hosts and in the cells which accompany formation of dependent and autonomous growths. 2. Biochemical and Histological Comparisons

Transplantable thyroid gland tumors of the mouse (Morris and Green, 1951) possess a number of important biochemical as well as histological characteristics. Some of these characteristics as investigated by Wollman, Morris, and Green (1951) have been summarized in Table VII. The histological characteristics of the generation (Table VII) of transplanted grafts immediately preceding the uptake measurements were characterized as follows:

TABLE VLI Summary of Characteristics of Four Transplantable Mouse Thyroid Gland Tumor Sublines (From Wollman el al., 1051)

--

T irn TurnorIhring IIost SllhGiven linc TlJ-Containing Diet (days) t b

Growth of Tumor Off I)cTU prndent (days) on Goitrogrri

1181

Time aftcr

kvg. Wt Avg. 1131 Gener- Thyroid Wt' Admn. ation Gland Tumor and (mg.1 (mg.) Sacrifice (hours)

I131 I131

Admn. Uosc in Thyroid Gland (avg. %)

Admn. Dose in Tumor (avg.

Ratio in Thyroid to 1 1 8 ' in Tumor

%J)

Conccntrating Power Rclativc to Thyroid Gland

% 1'31 as Thyroxine in

Tumor Relative to Thymid Gland

r

U

?'

53 E

m 1

2 3 4

-

57 86 98 98

14 20 20 20

X V

NO

KO Yes

11 5 5 7

9.9 10.2 17.6 8.8

131 140 50 71

5 17 18

18

15.7 f 2 . f 1.21 5 0 . 1 2 202 f 75 0 . 6 f 6.1 b.06 f 1 . 8 0.1 15 18 3 f 3 . 1 0 . 8 f 0.35 ..55 k 0 . 2 3 2 . 1 k 2.8 4 . 4 f 6 . 5 . 3 1 f 0.08:

1

-

TBh

0.14 0.57 1.10

T3pa 1/10.3 112.16

DEVELOPMENT AND METABOLISM O F THYROID TUMORS

91

Subline 1.-Adenocarcinoma-invasion of muscle, long tortuous tubules, absence of colloid. Subline 2.-Adenomatous, colloid in tissue space. Subline 3.-Adenocarcinoma, lung metastases, variable-sized follicles, ligh t-s taining colloid. Subline 4.-Adenoma, numerous small follicles. These four tumor sublines were functionally different, as shown by the data in Table VII. The most rapidly growing tumor, subline 1, had practically no ability to concentrate I'31.The uptake of tumor sublines 2 and 3 was & to as effective as that of the thyroid glands of the host, whereas subline 4 tumors possessed 8 the ability to concentrate 1131 as their host's thyroid. The per cent of 113'measured as the thyroxinelike fraction in tumors of sublines 2, 3, and 4 was 0.14, 0.57, and 1.1 that found in the respective thyroid glands of the tumor-bearing host. The thyroid glands of the tumor-bearing mice were intermediate in size, compared with those of non-tumor-bearing mice given thiouracil (TU) for three months (Dalton et al., 1948). This smaller size may in part have been due t o the fact that the mice were deprived of goitrogen for two or three weeks before sacrifice, but the tumors showed progressive growth averaging from 3 to 14 times the size of the thyroid glands together with less ability to concentrate Sublines 2 and 3 derived from the same initial thyroid tissue appeared strikingly different after five generations of transplantation-an observation not in accord with the view of Purves et al. (1951) that the propagation of rat thyroid gland tumors occurred through the natural selection of fast-growing types which were more malignant than the slower growing types. The mouse thyroid gland tumor sublines 2 and 3 grew a t approximately equal rates, i.e., 5th generation in both cases, but they differed in both functional and histological characteristics. 3. Competition of Host Thyroid and Transplantable Tumor for

Radioactive Iodine A later and more extensive investigation of the radioiodine uptake in transplantable tumors of the thyroid gland after 9 to 11 generations of serial transplantation was carried out by Wollman et al. (1953a). Two groups, descendants of the original subline 4, were studied in the 10th and 11th transplant generations. One of these sublines, 4F, had become independent, whereas the other subline, 4HJK, was still dependent on thiouracil ingestion by the host for continued growth. Subline 3C, an independent subline-descendant of the original subline 3, was examined in the 9th and 10th transplant generations. The 10th transplant genera-

02

HAROLD P. MORRIS

tion of subline G was examined for ability to concentrate for the first time. Among the first of the many problems considered was whether any of these transplantable mouse thyroid gland tumors actively competed with the thyroid gland for radioactive iodine. A high negative correlation existed in uptake of 113' between the thyroid gland of the tumor-bearing host and that of the tumor in dependent tumor subline 4HJIC. This correlation is illustrated by Fig. 4, which shows that a high uptake of IL3'by the tumor depressed uptake by the thyroid gland, although there appears to be no correlation between the weight of the host's thyroid and the size of the tumor in this subline (Fig. 5 ) .

I

I

I

I

I

I

I

I

I

I

FIG.4. T h r relation bet\\ ern tltc pcr cent of adniinistcrcd IL3'in thtt thyroid gland and that in the dcpmdcnt thyroid gland tumor, subline 4HJK, in thc same host at Levera1 intwvals after 113' administration (\Vollnian el nl., 1953a).

The influence of thyroid gland tumor subline 4 H J K in depressing uptake by the thyroid may he due in part to the higher avidity of this tumor for radioiodide. The removal of a tumor as active as this one could be expected to affect both the iodide calearance by the thyroid gland and the host's blood 1 1 3 ' concentration (Wollman, 19.53). The observed results as shown in Fig. 4 for the subline 4HJIC tumors suggest that removal of a tumor collecting 70% of an administered dose of I'31would ultimately increase thyroid uptake from the low value of 5% to 70";, provided all the administered became freely available to the thyroid gland and kidney. However, according to the kinetics of clearance as described by Wollman (19.53), the expected thyroid uptake would be

93

DEVELOPMENT AND METABOLISM O F THYROID TUMORS

+

3 = 3.3. This obvious discrepancy is not increased by a factor of 1 clearly explained by the experimental observations (Wollman et al., 1953a) and must await further study for clarification. On the other hand, an inactive independent tumor, subline 6, showed no correlation in 113' uptake between the thyroid gland of the tumorbearing host and the tumor, as shown a t intervals of 2, 6, and 25 hours after administration of a tracer dose of (Fig. 6) (Wollman et al., 1953a). The variation in clearance of in one organ would not be expected to affect uptake of the other as noted (Fig. 6) where the clearance I

I

*

c

I

*

I a

I (3

a

w

3 a

I

I

I

1

TUMOR LINE 4 H J K Chronic T h i o u r o c i l * O f 1 Thiourocil 5 Days 0011 Thiouracil I 9 Days

**

0

2 I-

0;

2b0

A0

6bO

A 0

l,bO

l,bO

,100

'

TOTAL TUMOR WEIGHT MG. FIG.5. The relation between the weight of the thyroid gland and the weight of the dependent transplantable thyroid gland tumor, subline 4HJK, in the same host (Wollman et al., 1953a).

by thyroid gland and tumor are both small with respect to kidney clearance (Wollman, 1953). The uptake of expressed as the percentage of the administered dose, however, increased in each transplantable tumor line studied (Wollman et al., 1953a) with increasing weight of the tumor. This increase occurred either with or without thiouracil administration to the host, as illustrated by Figs. 7, 8, and 9. This correlation appeared to hold for periods of 2,6, and 25 hours after was given. One dependent tumor line investigated, 4HJK, had a proportionately greater uptake in the smaller tumors than in the larger tumors. This is illustrated by Fig. 9. This decrease in uptake per milligram tumor when uptakes were above 30% has been described by Wollman (1953) as a kind of saturation phenomenon.

94

HAROLD P. MORRIS

r 25

r

I

r

1

I

1

I

TUMOR L I N E 6 No T h i ~ u i ~ c ~ l

-

m

bJ0 COZ

o a 0-J

I

a

0

2 noVr%qtt.r 6

A2S

20-

.

'

A

-

'

* *

(3

LL

A

gz 2 : c+

w

A

a 10

5 -

A

.

A

O f l I 5

21 +tour,

6 Houri

a

.

-

a

c

S

0

I

I

I

2 nour*

I

I

I

I

i

FIG.6 . The relation between the per eent of administered 113' in the thyroid gland and that in an inactive independent transplantalile thyroid gland tumor, siibline 6, in the same host at several intervals after administration (\VolIman et al., 1953a).

0

200

400

600

800

1000

1200

1400

1600

TUMOR WEIGHT MG.

FIG.7 . The relation between 1131content and the weight of transplantable inactive independent thyroid gland tumor, subline 6, in a low-iodine diet containing TU. The arabic number near some of the points indicates the per cent of the administered dose of I l a l in the thyroid gland of the host hearing the tumor (Wollman et al., 1953a).

95

DEVELOPMENT AND METABOLISM O F THYROID TUMORS I

I

I

I

I

I

I

I

I

-

e0

-

4 TUMOR LINE 6 No Thlourasll e Hour$ oiter I"'

m 6 A25.

- . .-

-

*

-

1.

0

200

800

600

400

1000

1200

1400

I I600

I

I800

TUMOR WEIGHT MG. FIG.8. The relation between the content and the tumor weight of inactive transplantable thyroid gland tumor, subline 6, in mice fed a diet adequate in iodine (Wollman et al., 1953a). I

I

I

I

I

I

TUMOR LINE 4HJK Chronic T h i w r o c I I

rn I naUr 0

5

1'''

"

- :

"

*

m25" 049

,

I

200

I 400

I

I

600

800

I 1000

I

1200

1400

TUMOR WEIGHT MG.

FIG.9. The relation between the 1 1 8 1 content and the tumor weight in a dependent transplantable thyroid gland tumor, subline 4HJK (Wollman et al., 1953a).

96

HAROLD P. MORRIS

4.

Radioactive Ioditie I'ptake per U n i t of Tissue

Much of the variation in uptake of 1 1 3 1 in these transplantable thyroid gland tumors was removed when the uptake of radioactive iodine was expressed in terms of unit weight of tissue (Wollman et al., 1953a) (Fig. 10). I t was also rioted that the Ii31uptake per milligram of thyroid gland usually continued to increase after administration of the isotope, but the uptake in animals ingesting T U was a t a lower level. It was further I

I

I

I

1

1

TUMOR LINE 6

0

1 I0

I 20

I

I

I

1

30

0

10

20

HOURS AFTER

Ib3'ADMINISTRATION

FIG.10. The per rent of acln1iIiistert.d T l a ' per milligram tissue in thyroid gland and in transplantahle thyroid gland turnor, subline 6, a t several time intervals after administration. The wrtical line through each point represents the standard error. The scale For thyroid gland is on ttir Irft; the srnlc f o r t h r tumor is on the right (Wollman r t a!., 1953a).

observed that the maximum thyroid content of TI--treated animals was reached prior to 6 hours after administration, whereas untreated mice uptake per had not reavhed maximum I i 3 I content by 24 hours. The milligram of tissue in the more active thyroid gland tumors, 4H,JIC, and 3C', more nearly paralleled the corresponding values for the thyroid gland but at a lowered concentration (Fig. 11). Some evidences of binding of 1 1 3 ' by tumor, subline 6, was shown by the increase in the coilcentration of the radioiodirie up to 24 hours after administration (Fig. 10). Further indication of binding in subline 6 n-as shown 25 hours after administration of the isotope by the 40-fold greater Ii31content per unit of tissue in the tumor compared to the liver. A suggestion indicating failure of bind-

DEVELOPMENT A N D METABOLISM O F THYROID TUMORS

97

ing, however, was the insignificant increase in concentration per milligram of tumor compared to the concentration in the host's liver which was noted in subline 4F for several time intervals. The uptake of was always less for the tumor than for the thyroid gland in each mouse when the uptake per milligram of thyroid tissue was compared to the uptake per milligram of thyroid tumor. The expression of the uptake values per milligram of tissue was found to be a more precise way of describing the ability of the tumors to take up than was the total uptake, even though the total uptake in the more active lines was many times greater than the total 113' uptake of the thyroid gland.

-

z

TUMOR

3.0

I

I

LINE 4 H J K

-

-

- 3 . 0 lo-' ~

0

LL-

I)

c

- 2.0 -

1.0

n 0

I

I

D m;o aJ0

D m

OThyroid *Tumor

C n J

00

I

Chrome Thiouraol

w a

n

I

I

a

I

I

I

IT

zz 94

23 q 00 ;o

g m

0

FIG.11. The per cent of administered 1 1 3 1 per milligram tissue in the thyroid gland and in dependent transplantable thyroid gland tumors, subline 4HJK, a t several time intervals after I L 3 1 administration. The vertical line through each point represents the standard error (Wollrnan et al., 1953a).

25 The ability of a dependent tumor subline, 4HJK, to take up hours after administration of the isotope was also studied after the removal of T U from the diet of the host. There was a relative increase of the content per milligram tumor at 19 days compared to 5 days, yet during the same time interval there was a decreased activity per milligram of thyroid so that the ratio uptake per milligram tumor/uptake per milligram thyroid had increased from 0.091 f 0.006 at 5 days to 0.193 f 0.022 at 19 days. 5. Factors Altering the ThyroidlSerum Radioiodide Ratio ( T / S ) of the Thyroid Gland and Thyroid Gland Tumors

Two functions of the thyroid gland, (1) ability to concentrate iodide, and (2) ability to bind the collected iodide, can be measured independently; first, by carrying out the tests under conditions in which

98

HAROLD Y. MORRIS

organic binding of radioiodide is blocked by the administration of a n appropriate goitrogen such as P T C , and secondly, by permitting binding to occur. The first type of test measures the iodide-concentrating capacity of the t>issue over that of the blood, whereas the second measures the capacity of the tissue both to concentrate and organically bind iodide. If the radioiodide concentration of the thyroid gland tumors to that of serum (T/S) is greater than 1, then that tissue possesses ability to concentrate radioiodide. The above procedure thus becomes a useful means of studying two separate processes of thyroid function. As pointed out elsewhere in this review, many factors affecting the concentrating or trapping proclivities of the thyroid gland or thyroid gland tumors require evaluation so that a more complete picture of thyroid gland tumor development and metabolism may be obtained. A beginning of such an evaluation has been made (Wollman et al., 1953a) in the study of the radioiodide-concentrating ability of several different sublines of transplantable tumors of the thyroid gland in mice. The T/S of thyroid glands in tumor-bearing animals measured when organic binding was Mocked varied betn-een 90 and 250. The factors causing this wide variation were not completely resolved. The T/’S obtained for tumors varied from 0.4 to 60. The ratio was always significantly lower for the tumor than for the thyroid gland in the same animal. The T/S was significantly less in the independent tumor lines than in dependent lines, and the concentration of radioiodide was not much higher in the independent tumors studied than it was in the serum. Transplantable thyroid gland tumor tissue of the mouse, when it becomes independent, apparently loses much of its ability to perform biochemical functions characteristic of normal thyroid gland tissue, and types of tissue may arise which possess different biochemical functions. Three such types of tumor tissue of the mouse have been noted (Wollman et al., 1953b) which possess (1) ability tlo eoucentrate and organically bind iodide, (2) inability to perform either of the functions under ( l ) , and (3) inability to concentrate iodide, but ability to organirally bind iodine. Tumor tissues possessing ability to concentrate and bind iodide are the most similar qualitatively to thyroid gland tissue. T ha t the loss in ability to concentrate iodide may occur during the transition from a dependent to an independent tumor received some support (Wollman et al., 1953b) when i t was observed th a t the T S ratio of the dependent tumor was higher than that of the independent tumor. The iiiterference tvith the binding mechanism in the biosynthesis of T H by the prolonged ingestion of goitrogens would be expected to reduce the blood T H to a minimum value. The feeding of the tumorbearing animal with a low-iodine diet following such prolonged periods of TV ingestion was found after 21 days to significantly lower the T/S

DEVELOPMENT A N D METABOLISM O F THYROID TUMORS

99

ratio of the thyroid gland below that observed in animals given a lowiodine diet for only 7 days, but the T/S ratio of the dependent tumor, subline 4J, was unaffected by the low dietary iodine regimen. Since one of the principal regulating factors governing the biosynthesis of TH is TSH elaborated by the anterior lobe of the hypophysis, the surgical ablation of the latter gland would also be expected to have a profound effect on the concentration of iodide by the thyroid gland. It has been shown that hypophysectomy reduced the T/S ratio of the thyroid in the tumor-free mouse to a value somewhat above 50 (Wollman and Scow, 1953a,b). An almost equal depression of the T/S of the thyroid occurs, however, by administration of small amounts of iodide to mice (Lipner et al., 1954). More attention, therefore, needs to be given to the iodine intake of the hypophysectomized animal. Goldberg et al. (1953) in studying the effect of iodine intake after hypophysectomy have noted increased efficiency of extraction of iodide from the blood by the thyroid gland of the rat. Although all the factors which affect the T/S ratio are not known, it has been found (Wollman et al., 1953a) that depression of the T/S ratio occurred in a dependent thyroid gland tumor, subline 4H, after hypophysectomy of the host. The host mice had ingested TU for a prolonged period before hypophysectomy, and were fed a low-iodine diet preceding and following the operation. Although a doubling of the T/S value has been noted for the thyroid gland of tumor-bearing mice after ingestion of PTU in a low-iodine diet for 14 days, the T/S ratio of a thyroid gland tumor, subline 3C, no longer possessing ability to concentrate iodide, was unaffected. Neither was there a significant alteration in the T/S ratio value of the thyroid gland of mice ingesting TU for long periods with or without transplantable dependent tumors of the thyroid gland. It appears that the presence of some dependent transplantable tumors of the thyroid gland in mice does not affect the T/S ratio of the thyroid gland of the tumor-bearing animal. The T/S of hyperplastic thyroids of tumor-bearing mice in which organic binding of iodide could take place was 15 to 20. The T/S of 12.8 of the transplantable tumor in one dependent tumor, subline 4H, was almost equal to the T/S ratio of 15 for the thyroid gland in the same host. The T/S ratio for several independent thyroid gland tumor sublines was significantIy less for the tumor than for the host’s thyroid when organic binding of iodide could occur. The inability to maintain a concentration of iodide above that in the serum, as found in two independent tumor sublines 3C and 6 (Wollman et al., 1953b), suggests that the iodide in such tumors is largely extracellular. Several nonthyroid tissues also lack ability to concentrate iodide (Stevens et al., 1950). Although it was also found that six different transplantable tumor sublines in mice in the ninth

100

HAROLD P. MORRIS

to eleventh transplant generations incorporated iodide into organic combination, the amount of bound per milligram of tissue was in each subline less than that in the thyroid gland. The concentration of I 1 3 l in the thyroid gland tumors of dependent tumor line 4H, when binding could occur was approximately equal to that occurring in the thyroid gland, but the organic binding in the tumor of this line was less than in the thyroid per unit 15 eight of tissue. This could conceivably indicate an impairment in the binding mechanism. The lower concentration in the tumor compared to the thyroid in several other tumor sublines investigated may esplain a t least a part of the impairment in binding. 6.

E$ect

of l'hiozcracil and Tkyrotropic Hormone on T u m o r Weights

Several different factors may affect the weight of transplantable thyroid gland tumors of the mouse (Morris, 1952; Wollman et a,?., 1953a; Lipner el al., 1954). Some of the data are presented in Table VIII. Tumor grafts of sublines 4 and 4F grew to a larger size in mice ingesting thiouracil than they did in control groups not ingesting the drug. The weight of subline 6 tumors, however, did not appear to be influenced by thiouracil ingestion. These observations raised the question as to whether or not the growth of some tumor sublines was increased by TSH. Obviously, although exogenous TSH obtained from another animal species was not the best material to use to test such an effect, it was the only way presently available and, therefore, was used by Lipner et al. (1954) to study the effect of the hormone in two sublines. The results as shown in Table VIII mere equivocal. When the thyroid gland was partially ablated by 1 1 3 1 prior t o tumor inoculation and then followed by exogenous TSH administration, similar results for the same sublines were obtained (Table VIII). The results to date suggest that some transplantable thyroid gland tumors i n mice are stimulated, while others are not, by the pituitary thyrotropic hormone. Additional work must be done on factors affecting the growth of thyroid gland tumors before arriving a t final conclusions. 7 . rlliscellaneoids Observations The frequently observed decreased ability of thyroid gland tumors iii man and ariinials to concentrate iodide could mean that the pathway of synthesis had been altered in the tumors. One test on transplanted thyroid gland tumors in mice has been reported (Morris, 1952) in which the same compounds were qualitatively identified from the mouse thyroid gland tumors that were present in the host's thyroid glands. Th;s was true in one dependent and one independent subline. It is still too early to predict, however, t hat the pathway of synthesis of iodinated compounds in the thyroid and the thyroid gland tumors may qualitatively be the same.

DEVELOPMENT AND METABOLISM O F THYROID TUMORS

101

TABLE VIII Average Weight of Transplantable Mouse Thyroid Gland Tumors after Various Treatments

Tumor Subline

Average Weight of Tumors (mg.) No. of Tumors 30 15 10

6 4F 4

Thiouracil 405 k 373 188 f 10 641 & 115

No. of Source Tumors No Thiouracil 41 554 f 460 Wollman et al. (1953a) Wollman et al. (1953a) 14 87 f 92 Morris (1952) 15 260 f 38

~

4JW 3c

7 7

TSH Injections for 33 days 10.4 f 3 . 1 23.7 f 6 . 7

7 7

Partial Thyroidectomy TSH - 33 days 14.2 2.5 20.3 f 4 . 8

7 7

+ 4JW 3c

7 7

No TSH 19.4 f 5.4 10.6 f 2 . 1

Lipner et al. (1954) Lipner et al. (1954)

-

Partial Thyroidectomy No TSH 30.1 f 11.9 Lipner et al. (1954) 10.0 f 2 . 0 Lipner et al. (1954)

Does the removal of thyroid gland tissue from its normal site by autotransplantation alone alter the iodide-concentrating properties of the transplanted tissue? This question was studied by Wollman and Scow (Wollman et al., 1953a), who report the uptake in a subcutaneous autotransplanted lobe of the thyroid gland of the rat to be only 70% of that present in the intact lobe. Lipner et al. (1954), on the other hand, found no significant difference in uptake in the mouse between the intact lobe and that portion of the lobe transplanted to a subcutaneous site for various periods up to three months after transplantation (Table IX). The mice received PTU in a low-iodine diet during the abovementioned periods so that the experimental conditions were similar to those in which thyroid gland tumors develop (Morris and Green, 1951). In a subsequent report Wollman and Scow (1955) found no significant difference in the radioiodide clearance from the blood by thyroid lobes of the rat in situ and autotransplanted. Neither did they find any significant difference in response of such thyroid tissue to hypophysectomy or to ingestion of PTU. The decreased ability of thyroid gland tumors to concentrate iodide compared to the capacity of the thyroid gland to concentrate iodide may represent an actual loss in the capability of the tumor to maintain a concentration gradient much higher than that of the serum iodide. If such a

102

HAROLD P. MORRIS

TABLE I S Effect of Xutotransplantation on t h e Functional Status of Thyroid Tissue in the hIouse ?iumtwr Period after IVeight Intact Weight of of Transplantation Lobe Autotransplant Animals (months) (w.1 (mg. 1

4 5 !I

1 2 3

12.8 k 2.0 1 4 . 4 rf: 1 . G 17.2 rf: 2 . 3

1 . 6 rf: 0.9 3.0 C 0 . 8 8 . 8 k 1.5

T/S Intact Lobe

T/S Autotransplant

179 i- 11 198 k 10 195 i-G

170 f 18 177 rf: 23 212 & 9

condition exists, it might also decrease the rate of organic binding of iodine. These few studies on the metabolism of transplantable thyroid gland tumors in mire serve to emphasize the great biochemical differences in surh tumor tissues. Further elucidation of the causes of these biochemical differences must await future investigations.

xI17.THYROID GL.i XD

CANCER I N n%AN

1 , Freqiwncy

Cancer of the thyroid gland, although comprising a relatively small percentage of human cancer, occupies a unique position for the study of mechanisms of carcinogenesis hot h in man and animals. The enormous capacity of the normal thyroid gland tissue to concentrate iodine and to utilize that iodine in metabolic functions provides a tissue wherein not only anatomical but also extremely sensitive functional changes can be detected and studied simultaneously. Many of these functional changes may he studied by the use of tracer amounts of radioactive iodine. Larger or caiiceroriclal doses of irradiation, on the other hand, can be delivered to those thyroid gland cancers in man which possess sufficient avidity for iodine. Such large doses of 113* provide a means of administering internal ionizing radiations to specific areas with what is thought to be minimal danger of injury to other body tissues. In a study of frequency of cancer in man, it should be pointed out that as a c’ause of morbidity and mortality, the position of cancer has changed greatly during the first half of the twentieth century and is still changing. The ratio of cancer of a given site to those of all sites has changed relatively little, however (Sokal, 1953). It is possible by the use of such a ratio, therefore, t o compare statistics of different years and t o some extent for diverse population groups, whereas incidence and mortality rates are less suitable (Table X). The conclusions arrived a t by the study of the

DEVELOPMENT A N D METABOLISM O F THYROID TUMORS

103

TABLE X Frequency of Thyroid Cancer in Man as a Percentage of All Cancer Summary by Sokal (1953) Clinical Frequency

Clinical data Mortality statistics Autopsy statistics Aggregate

Range (%)

Average

0.3-0.8

0.56

(%I

Cause of Death Range Average (%) (%I

0.4-0.6 0.0-1.3 0.56

0.41 0.47 0.44

statistics of the occurrence of cancer of the thyroid gland in terms of what might be expected in a “typical” community of the United States comprising a population of one million afford a convenient illustration. In such a community according to Sokal (1953) there would be 25 patients with thyroid cancer with one new case appearing every month; there would be one death bimonthly; one or two cases out of six would be studied at autopsy. Certain surgical statistics indicate a high incidence of nontoxic nodular goiters to be malignant a t operation. Such statistics appear to be in disagreement with figures presented by Sokal, but they may be explained by the fact that nontoxic goiters reaching the operating table comprise a highly selected group not representative of the population a t large-a point emphasized by Crile and Dempsey (1949). According to Sokal less than 1% of unselected nodular goiters are malignant. 2. Histological Patterns in Benign and Malignant Thyroid Gland Tumors

Meissner and McManus (1952) have classified histologically 500 thyroid gland adenomas and 200 thyroid gland cancers. It will be noted from their tabulations (Table XI) that the follicular group of benign tumors is common, whereas the group having a papillary structure is rare (ratio 17/1). The reverse is true in the malignant tumors (ratio 1/2). The age a t which benign and malignant tumors arise was not statistically significantly different. There is no demonstratable relation between nodular goiter and the common papillary cancer of the thyroid (Crile, 1953); this supports the idea expressed by Crile and Dempsey (1949) and by Crile (1953) that in man most thyroid gland cancers are malignant from their onset and do not arise from a preexisting benign phase. Such a view was not held by earlier investigators (Wegelin, 1928; Dunhill, 1931-1932) and is a t variance with most animal studies on the experimental induction of thyroid gland tumors.

104

HAROLD P. MORRIS

T.IRI,E XI Benign and Malignant Thyroid Tumors in M a n

Description Follicular Embryonal Fetal Simple Colloid Hurthle-cell Papillary Unclassified Total

Benign Adenomas (%)

Carcinomas (%)

86.6 (13.0) (52.3) (12.2) (3.8) (5.2) 4.8 8.6 100

27.0 (3.5) (1 6 . 0 ) (2.5) (0) (5.0) 50.0 23.0 100

3 . )Come lllrfabolic Characteristics of H u m a n Thyroid Gland Cancer Tissue

Little correlation was found between malignancy and transaminating activity of human thyroid tumors by Awapara (1952), who noted in the same hpecimen a tendency for tumor tissue to have more transaminating activity than normal tissue. The number and concentration of free amino acids as well as the transaminating enzyme were higher in samples having more ~ 1 1 s . S o hiJtologica1 changes in the thyroid tissue of man were noted by Freedberg el nl. (1932) 7 days after doses of 17 and 20 mc. of Central destrurtion of the gland was noted 14 and 24 days, respectively, after the administration of 39 and 26 mc. of I131. Up to periods of one year thyroid glands showed increasing amounts of fibrous tissue after dosage. The amount of fibrodis depended on the length of time following treatment. I t is b e l i e i d more studies on thc metabolic rharacteristics of human thyroid gland cancer tissue would give valuable aid in treating the disease in man. $. I'se of Radioactive Iodine and Goitrogens C'nncw of the thyroid gland in man as noted above is not high in comparison with other types of caticer with which man is afflicted, and only a small proportion of those patients with malignant thyroid cancer benefit from treatment with ionizing radiations from radioactive iodine. Although the effert thus far produced by the introduction of this method of treatment on the general problem of the management of patients with cancer is probably insignificant (Smithers, 19531, nevertheless it has a n interest out of all proportion to its success as a tool in rancer treatment because it permits achievements in therapy never before possible. Those few

DEVELOPMENT .4ND METABOLISM O F THYROID TUMORS

105

patients who are now alive, and apparently well, who would otherwise have died are living testimonials to the importance of the use of radioactive iodine in treating the disease. Radioactive iodine is not needed for the treatment of the less malignant localized tumors whose structures and function approach more nearly that of the normal thyroid. It has been found ineffective for the more malignant undifferentiated types (Smithers, 1953). The present value of radioiodine in the treatment of cancer of the thyroid gland lies in its use in treating a few patients having well-differentiated tumors which have advanced locally to the point of inoperability or which have metastasized. The future use of this new weapon in the fight against cancer in man would appear to depend on the discovery of ways of increasing the avidity of thyroid gland tumors for I'31.Removal of functional tissue has been tried by Dobyns and Maloof (1951); Freedberg et al. (1951); Seidlin et al. (1948); Rawson et al. (1948); Rawson et al. (1951); and others in attempts to increase collection by thyroid uptake tumors in man. Dobyns and Maloof (1951) tried to increase by thiouracil; Rall ef al. (1951) by a combination of total thyroidectomy and thiouracil or by thyrotropic hormone administration (Trunnell et al., 1949). These attempts have, more often than not, been unsuccessful in significantly increasing the number of cases materially benefited. The theories underlying the use of thiouracil were: (1) that the patient may be reduced to a state of iodine deficiency so that the avidity for iodine of any tissue remotely capable of functioning as thyroid tissue would be greatly enhanced; (2) the goitrogen may augment endogenous TSH elaboration, which would increase functional activity of the cancerous thyroid tissue; and (3) the action of endogenous TSH on the tumor may be augmented. The thyroid gland in man appears to function like that of the rat when thyroid hormone synthesis is inhibited by antithyroid metabolites, since the human thyroid gland is able to collect appreciable quantities of radioactive iodine after virtually complete inhibition of thyroid hormone synthesis by antithyroid medication (Stanley and Astwood, 1948). It is further believed that the collected iodide is in the form of iodide ion because the administration of thiocyanate ion or large doses of iodide promptly discharges the collected iodide from the thyroid gland. was injected into rats and mice point Animal experiments in which to the possibility that the use of in treating thyrotoxicosis in man could eventually prove carcinogenic. Treated human patients probably would need to be followed for 15 to 25 years, however, to substantiate the danger and to determine whether or not a higher proportion of such patients receiving 1 1 3 1 develop thyroid carcinoma.

106

H.4ROLD P. MORRIS

Precautions need to be taken in the use of radioactive iodine in the treatment of thyroid gland cancer in man. Some of the precautions advocated (Pochin, 1932; Wayne, 1932; Doniach, 1953; Ramson et al., 1953) inclutle: (1) treatment of thyrotoxic patients under 45 years of age with 1 1 3 ' only when other methods of treatment are contraindicated; (2) the use of a minimal dose of to produce remission only after a period of 1015- iodine intake; (3) treatment only of cases in which tracer doses of I131 indicate tumor uptakes sufficient to produce therapeutic effects; (4) iiistitution and maintenance of thyroxine medication after thyrotoxic symptoms have been relieved; (5) avoidance of doses so large that serious body irradiation occurs; and ( G ) avoidance of use of goitrogenic drugs a t any time after radioiodine therapy. It is suggested that a number of procedures to improve the uptake of 1 ' 3 1 by tlie thyroid might he explored with the intention of decreasing the minimal effective dose of II3' required to obtain a therapeutic effect. .imong these are drastic reduction of iodine intake accomplished by low food iodine lei-el for several weeks followed by a 24-hour fast just prior t o administration, the use of cyanate or chlorate ions to discharge inorganic iodide, and the use of hesperidine methyl chalcone (Reilly el al., 19.52). It frequently happens (Egmark et aZ., 1953) after total thyroidectomy, without any suhstitution therapy, that a hormonal balance takes place in man which sometimes lasts for several years with no evidence of mysedenia. I t is presumed that the metastatic lesions in such cases take over the functional activity of the thyroid, and in some subjects appear capable not only of taking up iodine but of converting it to thyroxine. One of the first steps followed by Rawson et al. (1953) in treating patients irhose tumors exhibit a minimal or no thyroidal function is to subject suc*hpatients to total thyroidectomy either surgically or with l I 3 ' . .ipprosimately 5 0 7 of their patients after such treatment were observed t o develop a significant capacity to concentrate radioactive iodine in one or more metastases within six months. Some patients after total thyroidectomy have been treated with thiouracil, and the subsequent increase in thyroidal function observed has been attributed (Rawson et aZ., 1953) t o a n increased secretion and availability of thyrotropic hormone. One third of 1.3 patients treated with exogenous TSH have been observed to increase or develop significant function in one or more metastatic lesions. After more than ten years experience with the use of I I 3 l in the treatment of thyroid canter in man, Rawson et al. (1953) recommended its use only in those patients whose lesions cannot be removed indihy a competent surgeon, and then only when a tracer dose of cates the cancer would concentrate a cancerocidal amount of without

DEVELOPMENT AND METABOLISM OF THYROID TUMORS

107

serious damage to extrathyroidal tissues. Patients with metastatic carcinoma of the thyroid who are accepted in some clinics (Rawson et al., 1953) have their tumors evaluated for ability to concentrate radioactive iodine by use of tracer doses of The amount of the tracer dose excreted after 48 and 96 hours is measured, in vivo, over the gland and over the metastatic lesions. The normal gland is ablated, followed by tracer doses every four to six weeks for three to six months. At that time thiouracil is administered in doses of 1to 1.5 g. daily. Radio-tracer studies are made every six to eight weeks 48 hours after stopping thiouracil administration. If less than 40% of the administered dose is excreted in 96 hours, the patient becomes a candidate for radioiodine treatment,. A second but larger test dose of I I 3 ' is given. Daily urinary excretion and TABLE XI1 Some Results of Therapy in Man (Rawson et al., 1953) No. Patients 10 10 8

7 Total 35

% 28.5 28.5 23.0 20.0 100.0

Comment Showing sustained objective improvement Showing transient objective improvement Failures Treatment too recent for evaluation

blood levels are determined, as well as in vivo measurements of radioactivity a t 48 hours over the known tumor area. From a graphic plot of blood levels and retained there is calculated the amount of radiation that will be delivered to the hemopoietic system and other extrathyroidal tissues. Attempts are made to deliver less than 400 rep to the blood (using a factor of 10.2 rep per gram per day). If the calculations and estimated size of the tumor indicate that a cancerocidal dose of 1 1 3 1 can be delivered to the tumor without causing the patient serious harm, the maximum safe dose of radioactive iodine is administered. The same determinations of urinary, blood, and tumor radioactivity are made after a therapeutic dose as before. Careful checks are made on the peripheral blood for three weeks after 113' therapy for evidences of injury. Using the above rather rigid criteria in selecting and treating cancer of the thyroid with radioactive iodine, Rawson et al. (1953) report that (Table XII) about one half of these cases showed sustained or transient improvement-an indication that certain cancers of the thyroid

108

HAROLD P . MORRIS

gland can be a t least partially destroyed with The opinion was expressed (Rawson ~1 al., 1953) that a large percciitage of the thyroid gland ('anrers treated by the procedures described above had been made to assume some functions of normal thyroid tissue. 5. Possible Consequeiices of Thyroid Hormone Deficiency

The report of IIerrmann (19.51) of two patients with exophthalmic goiter who had received methylthiouracil medication over a long period (3 to 4 years) arid who developed aderiopapillomatous carcinoma and hemarigioenciothelioma, respectively, deserves careful consideration. The two patients observed by Herrmann came from an endemic goiter area. and therefore, may have lived for many years on insufficient iodine. which probably resulted in a prolonged thyroxine deficit,. The thyroxine deficit would also have continued during goitrogen administration. Herrmaiin (1951) was of the opinion that the occurrence of the adenomas resulted from excess elaboration of thyrotropic hormone. Two rases also of neoplastic changes in the thyroid glands in man after prolonged treatment with thiouracil \yere reported by Money and Rawson (1947). The histological examination of the thyroid of one patient after ten months of the thiouracil treatment for Graves' disease showed a discrete nodule which proved to have localized changes similar to some of those observed in thiouracil-treated rats. The changes observed consisted of intrafollicular growth of undiff ereiitiated tissue projecting into the lumina of several follicles. Thyroxine deficiency may also occur if sufficiently large doses of 1 1 3 1 are given so as t o almost completely ablate the thyroid gland. Unless such treatment is followed by an adequate thyroxine medication, the thyroxine deficiency could possibly result in an excessive secretion of thyrotropic hormone. Since the effective dose range in rats appears to be comparable with the therapeutic dose range in the thyrotoxic thyroid gland in man ill so far as its biological effects in reducing thyroid size without actual gland destruction or gross decrease in thyroxine synthesis is concerned, if the dosage of 113*used in the treatment of exophthalmic goiter in man (Doiiiach, 1953) rendered the patient euthyroid without producing a n irreversible interference with thyroxine synthesis, the carcinogenic danger from its use would likely be very slight. If, on the other hand, future thyrotropic hormone studies show an augmented and persistent thyrotropiii secretion above normal after radioiodine treatment, the carcinogenic danger may be much greater. From the evidence available it would appear reasonable t o believe that any dosage of in man which sufficiently interferes with thyroxine secretion so as to lower the circulation

DEVELOPMENT AND METABOLISM O F THYROID TUMORS

109

level of this hormone below normal might well augment secretion of TSH unless followed by adequate thyroxine medication.

XV. SUMMARY AND CONCLUSIONS The occurrence of spontaneous tumors of the thyroid gland in rats and mice is exceedingly rare. Thyroid gland tumors, however, have been produced in these laboratory animals by several different methods. There is ample indirect proof that such tumors occur when the thyroid gland tissue is subjected to continuous prolonged stimulation by increased amounts of thyrotropin evoked by a deficiency of circulating thyroid hormone. Such neoplasms in rats and mice appear to develop through a series of gradual changes (Dubin, 1953) which include (1) hyperplasia, (2) local areas of proliferation of altered cells, (3) formation of adenomas, and finally (4) development of carcinomas. The amount of thyrotropin required to produce the necessary stimulation is unknown. The essential techniques used to produce experimental tumors of the thyroid gland include (1) chronic ingestion of a thiocarbamide type goitrogen by both rats and mice, (2) the direct irradiation of the thyroid gland (of rats only) by a single massive dose of radioiodine, (3) the chronic feeding of an iodine-deficient diet to rats, (4) a combination of a goitrogen with low doses of radiation in rats, and ( 5 ) a combination of the carcinogen 2-acetylaminofluorene with a goitrogen. The fifth procedure has been found unnecessary but may serve to accelerate the process in the earlier stages. The initial tumors produced by goitrogens depend on thyroid hormone deficiency for continued growth. After a variable number of serial transfers to TH-deficient hosts some, although not all, of the transplantable dependent tumors become independent of the stimulus causing their development. Numerous attempts to transplant experimentally induced thyroid gland neoplasms of the rat have frequently been unsuccessful except when made into thyroid-hormone-deficient hosts, although exceptions to this general experience were those obtained (Purves et al., 1951) in methylthiouracil-induced thyroid gland tumors. A partial explanation of the frequent failure to obtain independent transplantable tumors of the thyroid gland in rats may be due to the use of noninbred stocks of animals. The production of thyroid gland neoplasms in the mouse following prolonged chronic ingestion of goitrogens has received ample verification, although transplantation has been obtained only in the CSH strain. Nevertheless, it is believed other strains of inbred mice when adequately tested will also prove capable of carrying transplantable thyroid gland tumors. Evidence also has been obtained that thyroid

110

HAROLD P. MORRIS

gland tumors develop in mice from the direct stimulation from TSH p r o d u d by functional pituitary tumors. The conditions most farorahle to the development of independent or autoiromous thyroid gland tumors have not yet been completely apprehended. -1vailable studies support, the view that much variation occurs in experimentally produced thyroid tumorsjn both rats and mice. A long bu t8also 1-ariable induction period exists. Once developed, such tumors show progressive growth, ability to kill the host, rather low and variable metastastic activity, occasional invasion of surrounding tissues, and a reduced capacity to concentrate iodine compared to that of the normal thyroid gland tissue. Single doses of ionizing radiations from have produced malignant thyroid neoplasms in rats. 111such neoplasms the induction period was long, and thyroid gland epithelium did not appear unusually active, indicating the absence of unduly high levels of thyrotropic hormone secretion. 7'hc i o n i h g radiation from 1131, therefore, appeared to be the dire(-t initiating mrcinogenic agent responsible for the formation of the thyroid neoplasms. Dosages of ionizing radiation lower, higher, and equivalent on a weight basis to the cancerogenic dosages of ionizing radiation administered to rats did not induce thyroid gland tumors in mice. Administration of sufficient radiation to mice to destroy all or most of the thyroid gland tissue, however, did produce tumorous enlargements of the anterior lobe of the hypophysis. Low doscs of Ill1 (30 pc.) plus the goitrogen niethylthiouracil produced thyroid gland carcinomas in rats, whereas such neoplasms were absent in rats receiving the goitrogen alone. The stimulation to thyroid tissue by increased thyrotropic hormone secretion produced during goitrogen ingestion appears to have an additive carcinogenic effect on the rats' thyroid gland when combined with low doses of IL3'irradiation. Such a low range of irradiation has been found to interfere with thyroid function ; one explanation may be that if the interference is sufficient to lower circulating T H , the increased secretion of endogenous TSH which might he expected to occur would lead to stimulation of thyroid epithelium, thus resulting in conditions conducive to thyroid gland cwvinogcnesis. The direct chemical incorporation of radioiodine into the secretion of the thyroid gland or of functional thyroid gland tumors provides very precise labels adaptable to the correlation of the functional activities of the gland and of its tumors simultaneously with histological changes k n o ~ v nto take place during the transition of the thyroid gland to a thyroid gland neoplasm. Several transplantable thyroid gland tumors varied from practically no ability to concentrate radioiodine in some

DEVELOPMENT AND METABOLISM O F THYROID TUMORS

111

independent tumors to about one-half the concentrating capacity of the host's thyroid gland in some dependent tumors. A high uptake of in some dependent tumors depressed 113' uptake by the thyroid gland, although the thyroid gland weight in such instances was not related to tumor size. Some almost completely functionally inactive independent tumors showed no correlation in uptake between the thyroid gland of the host and the tumor. The uptake of 113' increased in several transplantable tumor lines with increasing weight of the tumor, and some dependent tumors had a proportionately greater uptake for smaller tumors than for larger ones. The uptake per unit of tumor tissue was much less variable than the total uptake because of the large differences in tumor size. The uptake per unit weight of tumor tissue of the most active tumors also more nearly paralleled uptake of the thyroid gland than did less active tumors, but all tumors studied in both rats and mice possessed less capacity to concentrate than did the thyroid gland. Ability of the thyroid gland and the tumor to concentrate and organically bind the collected iodide has been demonstrated in several transplantable tumors of the thyroid gland in mice. The thyroid to serum radioiodide ratio (T/S) was always significantly lower for the tumor than for'the thyroid gland in the same animal. The T/S was also significantly less in the independent tumor lines than!in dependent lines, and the radioiodide concentration in the tumor of several independent tumor lines was not much above that in the serum. The evidence a t present indicates that transplantable thyroid gland tumor tissue of the mouse and rat when it becomes independent loses much of its ability to perform certain biochemical functions characteristic of normal thyroid gland tissue. Some mouse tumors possess ability to both concentrate and organically bind radioiodide; other tumors could bind but lacked ability to concentrate the iodide; whereas still others lacked both functions. Organic binding of in several transplantable thyroid gland tumors was always less per milligram of tumor tissue than that in the thyroid gland. One dependent tumor possessed approximately equal ability with the thyroid gland to organically bind Of several factors studied hypophysectomy produced the greatest depression of the iodide-trapping capacity in both the thyroid gland and the tumor. Pituitary thyrotropic hormone stimulated the growth of some transplantable thyroid gland tumors of the mouse, whereas the growth of others was unaffected by the hormone. The pathway of synthesis of thyroid hormone intermediates appeared unchanged in some thyroid gland tumors from that occurring in the normal thyroid gland. The autotransplantation of thyroid gland tissue of the mouse did not per se

112

HAROLD P. MORRIS

aff wt its capacity t o concentrate I 1 3 * . Heterologous transplantation should not, therefore, be the primary cause for the loss in concentrating capacity observed in most transplantable neoplasms. Although the reasons for the decreased capacity t o collect and bind iodine which occurs in experimentally produced thyroid gland tumors remain largely unknown, and since most cancers of the thyroid gland in man have also lost much of their ability to collect iodine compared t o that of the normal thyroid gland, it seems reasonable to conclude that quite similiar explanations may exist for t,hese functional changes of thyroid gland neoplasms which occur during thyroid gland carcinogenesis in both animals and man. The experimental development of transplantable dependent and independent thyroid gland tumors in both the rat and the mouse, therefore, should contribute their share t o the solution of some of the many thyroid gland neoplasm enigmas. These problems pose unique and promising challenges for future solution.

REFERESCES Albert, A. 1952. Ann. Rev. Physiol. 14, 481-498. Albcrt, A,, and Lorenz, h’. 1951. Proc. Soc. E x p l l . Bid. .?fed. 77, 201-205. hnapara, J. 1952. Texas Repts. B i d . and M e d . 10,933-936. Auelrnd, A,, and Leblond, C. P. 1 ! ) 3 . Proc. . I m .lssoc. Cancer Research 1, 2. Axelrad, A,, and Leblond, C. P. 1954 Proc. .1m. Assoc. Cancer Research 1, 2. Barkcr, M.EI. 1936. J . Am. Jfed. .Issoc. 106, 762-767. Barker, S. B. 1951. Physiol. Reiv. 31, 205-243. Bielschowsky, F. 1944. Brit. J . Erptl. Palhol. 26, 90-95. Bielschowsky, F. 1945. Brzt. J . Erpll. Pathol. 26, 270-275. Rielschowsky, F. 1949. &it. J. Cancer 3, 547-549. Bielschowsk>-,F. 1953. Brit. .I. Cancer 7, 203-213. Bielschowskv, F., and Griesbneh, W.E. 1950. Brit. J. Cancer 4, 133-138. Bielschoxvsky, F., Griesbach, \V. E., Hall, W. H., Kennedy, T. II., and Purves, H. D. 1949. Brit. J . Cancer 3, 541-546. Bullock, F. D., and Curtis, 31. 11. 1930. J. Cancer Research 14, 1-115. Bullock, F. D., and Rohdenburg, G. I,. 1917. J . Cancer Research 2, 39-60. Crile, G.. Jr.. 1953. N e w Engl. J. M e n . 249, 585-590. Crile, G , Jr., a n d Dernpsey, W. S. 1949. J . Am. M e d . Assoc. 139, 1247-1251. Dalton. A. J., Morris, 1%. P., and Dubnik, C. S. 1948. J. Natl. Cancer Inst. 9, 201-223, D’hngelo, S.A , , and Gordon, -4. S. 1949. Ttans. A t t i . Goiter Assoc., pp. 140-147. D’Xngrlo, S. A, l’nschkis, I005 246 I 07>P>OG 2610

~

_____

P 0.05 > P > 0.02 I' < 0.01 0 . 8 > I' > 0 . 7 P > 0.9 0 . 1 > P > 0.05 1' < 0.01 1' < 0.01 0.05 > P 0.3 > P

> 0.02 > 0.02

probability

disappears (the necrotic alterations may play a role). The greater heat stability of the investigated protein-nucleoprotein fraction may be due to the azo compound bound by the protein and so exerting protection against heat coagulation of the protein itself. Possibly the protection exerted by thymus nucleate on the heat coagulation of proteins (Carter and Creenstein, 1946) may be mentioned. If the administration of DAB is terminated, even after five months, the flocculation curve again resumes its normal character. On the other hand the administration of riboflavin (which is well known to be capable of retarding the carcinogenic action of D.4B) more or less inhibits the development of the flocculation change.

SOME ASPECTS OF CARCINOGENESIS

203

I n regenerating liver (after partial hepatectomy) a change in an opposite sense is observed 4 to 5 days after operation; higher turbidity curves are an expression of increased heat coagulability. It is difficult to assess the significance of this change in heat flocculation of a protein fraction, and especially to determine whether it is simply related to the presence of protein-bound azo compound or has some relation with the carcinogenic process itself. Several studies on the amino acid composition of tumor tissues have been recently undertaken; whereas Schweigert et al. (1949) found differences in comparison with normal tissues, the majority of investigators obtained no significant results, for example, Zamecnik et al. (1949), Sauberlich and Baumann (1951), and Biserte (1953), in accordance with the older work of Greenstein et al. (1941b). Similarly, Bassi and BernelliZazzera (1954) found (using bidimensional paper chromatography) no difference in amino acid composition between acidic hydrolyzates from normal rat livers and hydrolyzates from livers of rats fed DAB to the time of hepatoma development. On the whole, there is no clear evidence of a specific amino acid pattern in tumors. Some difference in the sequence of the amino acids along the peptide chains may be suspected (Biserte) ; but is obviously difficult to demonstrate. Much work has, however, indicated very active amino acid and synthetic protein metabolism in tumors, which have accordingly been defined as " nitrogen traps l f (Mider, 1951); Boulanger (1953), on the basis of experiments with radioactive glycine, advances the opinion that amino acids can penetrate the cellular membranes more readily in tumor cells, with, in addition, a more active interchange between different amino acids. An attempt to establish a specific pattern of amino acids and peptides in tumor tissue was made by A. Fischer (1950), using a biological method; embryonic chicken fibroblasts were grown in hydrolyzates from normal chicken muscles and in similarly prepared hydrolyzates from a methylcholanthrene-induced fowl sarcoma. The growth was more vigorous in the former medium than in the latter; and this author believes that the peptides from normal cells may be more easily utilized by the normal fibroblasts because of their physiological composition. It is, however, possible that the tumor hydrolyzates exert a toxic action because of those secondary necrotic changes which are seldom absent. Indeed, some slight deviations from the normal pattern of amino acids may be accounted for in malignant tumors by such degenerative and necrotic changes, which involve autolytic processes. The split products of proteins in the necrotic patches may be absorbed a t a different extent, blood plasma proteins may infiltrate the necrotic areas, and differing amounts of stroma may also hinder the comparison of tumor with normal tissue.

204

P. RONDON1

3. Infrared Absorption Spectra of Cancer Tissue

h study of proteins and of tissues by infrared absorption spectrography has been attempted by some investigators. The infrared absorption of proteins as well as of other organic compounds is due to the absorption of certain atomic groups a t characteristic frequencies, and the vibration and rotation spectra (Mellon, 1950) inform us of (1) the moment of inertia of the molecule about the axis of rotation, giving the molecular dimension; (2) the bond strength which is deduced from the fundamental frequency and the mass of the molecule; and (3) the configuration of the molecule. The study of protein solutions is particularly difficult as a result of molecular complexity and the infrared absorption of water. Nevertheless, much work of biological interest has already been conducted, as, for example, that of Lenormant and co-workers (1950-1953), who introduced the study of proteins treated with heavy water, so that the hydrogen of the imino-group in the peptide bond is substituted by deuterium ( ‘ I deuterisation des proteines ”). Lenormant (1952~)has also studied proteins in living cells and the denaturation process (1953). Further there are the important contributions of Darmon and Sutherland (1919), Ambrose et al. (1919), Mizoushima et al. (1950), and Elliot et al. (1950). As regards tumors, the paper of Blout and Mellors (1949) includes data on the infrared spectra of blood, normal tissues (e.g., breast gland), and neoplastic tissues (mammary cancer). Here the observations were made on fixed and paraffin-imbedded tissues and on dried and frozen tissues, differences being noted according to the method of treatment. An absorption spectrum was recorded in tumor material corresponding to an increased concentration of nucleic acid. Woernley (1952) gives many absorption curves of normal and neoplastic tissues, remarking that, owing to the complexity of the material under investigation, each absorption is probably a composite structure resulting from several different types of vibrations. The curves cover the spectral region of 1 to 8 p as well as the region of greater interest 8 to 15 p. The absorption in the former region depends on the distribution of some atomic groups; while above 8 p the skeletal vibrations generally mask the bond type. In a great number of tissues there is a more or less pronounced band at 8.1 p which may be assigned to rocking, wagging, deformation, bending, or twisting vibrations of CH, CHI, or CHa structures. For some tissues there is a band at approximately 9.2 p ; for others there are two or more bands a t 8.8 t o 10 p . Definite bands are also observed at about 10.2 p. Neoplastic tissues (human and mouse tumors) show a characteristic absorption pattern in the region between 8.0 and 11 p (bands approximately a t 8.1 p , 9.2 p, 10.25 p ) . In order to explain such findings absorption data

SOME ASPECTS O F CARCINOGENESIS

205

were obtained for tissue constituents, for nuclei and cytoplasm separately, in normal and neoplastic materials, these results being correlated with the chemical composition of the samples. It was concluded that the absorption in the region 8 to 11 p is mainly due t o nucleic acids; therefore highly cellular neoplastic and normal tissues, rich in nucleic acids (RNA as well as DNA), produce a strong and characteristic absorption in this region. More recently Ceselli and Guzd (1954) have investigated the infrared absorption spectra of the livers of 30 rats fed for 15 to 180 days on a ricecarrots-cod-liver oil diet with addition of dimethylaminoaxobenzene, as well as of livers of normal rats, using paraffin-imbedded tissues. They followed those changes occurring in the precancerous stages, of greatest significance for the malignant transformation. During treatment the tissues showed an increasing reduction of the bands between 8 p and 11 p , which are considered as due to the nucleic acids. Also, livers affected by precancerous lesions gave spectral curves with an evident diminution of absorption a t 9.25 and 9.70 p (the latter being more characteristic for DNA). Only in livers of some rats submitted to a more prolonged treatment with carcinogen did the band a t 9.70 p again appear evident. The findings of these authors are in contrast with those of others mentioned above, who studied human or animal tumors but not the earlier stages of the carcinogenic process. It may be that the DAB or some metabolic derivative combines with the tissue components and so modifies the spectra in the region 8 to 11 p ; or that in the initial phases a disorganization of high polymers, including NA, takes place, as a prelude t o a rearrangement corresponding t o definite malignancy, when the concentration of such compounds increases in association with vigorous cell multiplication. On the other hand, i t is also possible that in the initial stages the NA undergoes further polymerization or new combinations with different protein fractions, involving a shift of absorption towards other regions of the spectra where the enormous complexity of the tissue components prevents a clear evaluation. On the whole, the infrared absorption study demonstrates the occurrence of rather complex modifications of the NA behavior in tissues during carcinogenesis and in tumors. The opinion of a rearrangement a t the level of macromolecular constituents may be generally confirmed, but no evidence can be given about the distribution of atomic groups or their bond strength.

4. Ultrastructural and Immunological Considerations From the admittedly scanty data so far available, the opinion may nevertheless be held that cell transformation in malignancy affects either the architectural and folding specificity of certain protein constit-

206

P . RONDONI

iients or the state of protein aggregation. Hence, the change may reside a t a higher level than that of chemical composition and may consist of Some abnormal configuration in the three-dimensional molecular network of protoplasm, with distortion of peptide chains, new interrelations bettveen the chains, and the generation of a new field of polar forces. We have therefore t o deal with an abnormal tridimensional template henceforth dominating the synthetic processes. The change would fundamentally concern the u2trastructzrre of the protoplasm (Rondoni, 1946a,b), i.e., the structural level above that of purely chemical composition. The fundamental significance of an ultrastructural change in carcinogenesis has also been suggested by Powell (1944, 1946, 1947), who concludes that “ an irreversible or long persisting disorganization of protoplasm atid especially the inm-ordination of the components of the cytoskeleta1 framework results in an anaplastic transformation of the affected cells. It is possible that the majority of tumors arise in this way. I n such tumors the initiation of the disorganization or inco-ordination may be r e g a r d d as the proximate cause of the anaplastic state since it can produce most of the main features of tumor cells and is the root, cause of those properties which together constitute the facies of the anaplastic state. Protoplasmic disorganization or cytoskcletnl inco-ordination need iiot necessarily he the primary cause of anaplastic growth but could be I)rought about by, for example, viruses or gene mutations. On the other hand, inco-ordination of the protein microfibrils may be both a primary and proxiniltte intracellular cause in many instances. In view of the diversity of mrcinogenic agents, which also have other effects on cells, and the complexity of protoplasm, it seems that the competence t o respond t o such agents by the development of the anaplastic state is determined hy a (-ommoileffect on some ubiquitous sul)stratum in protoplasm. There is abundant evidence that carcinogenic agents affect the fibrous proteins of protoplasm; for example, hlottram (1942) observed that carcinogens changr the viscosity of cytoplasm, and cytological aberrations consequent upon alterations in viscosity occur in cells within a few hours of treatment (Pullinger, 1940). Summation experiments with different carcinogenic agents indicate that a common cellular substratum is involved in carcinogenesis.” ,J. Seedham (193Ga,b), who considers tumors as a product of abnormal embryonic organizers, also suggested a disorganization of the tridimensional structure of the protoplasm. If such hypothesis is correct, then carcinogenesis should occupy the same cellular level of organization as antibody formation, of course with a much more extensive involvement of cellular mechanisms. The carcinogenic agent in most cases is not a template in itself, as are antigens; but it induces that modification in cell constituents (proteins, nucleic

SOME ASPECTS OF CARCINOGENESIS

207

acids, and perhaps some lipoids) which brings about the generation of the abnormal template. Indeed, the most powerful chemical carcinogens (polycyclic hydrocarbons) are represented by large, rigid molecules, which, bound to a protein carrier, could exert antigenic activity (Creech, 1952). But there is no need for a carcinogen t o be an antigenic itself, and it may induce changes in proteins which may themselves be antagonized by the process of antibody formation, for example, in the experiments of Michle and Emde (1944). These authors found a decrease in the incidence of sarcomas in mice injected with benzpyrene and subjected t o parenteral treatment with various antigens. It is possible that antibody formation, as well as carcinogenesis, interferes with some similar aspect of protein metabolism. Pauling’s views (1948) concerning the self-duplication of viruses and genes and the formation of antibodies are relevant here; he believes that “it is molecular size and shape, on the atomic scale, that are of primary importance in these phenomena, rather than the ordinary chemical properties of the substances, involving their power of entering into reactions in which ordinary chemical bonds are broken and formed.” We know that the largest part of the cell proteins, perhaps nearly the whole, consists of enzyme proteins (Meyerhof, 1949) : “What was formerly regarded as protoplasm, containing the machinery of the life processes, must now be regarded as a multiple of distinct enzymes which are coordinated in their function by chemical cycles, by the influence of the cell structures and by the effect of hormones.” I n these words of Meyerhof the assumption is expressed that the proteins, some containing a dissociable effective group and some not, are in strict interrelation to each other, forming a complex system, where intermolecular association and ultrastructural conditions play an important role for enzymatic activity and consequently for all the processes of life. Villa et al. (1953) consider contraction of the chromosomes and an increase in the viscosity of DNA as characteristic features of leukemic leucocytes in comparison with normal ones. This finding might also indicate a higher degree of polymerization of DNA in leukemic cells, and therefore a n alteration chiefly concerning the ultrastructure. It may be recalled here that Boretti (1946) investigated the enzymes controlling the metabolism of RNA in extracts from human mammary gland and from breast cancer, finding those enzymes which catalyze the lower steps of RNA metabolism (nucleotidases) to be more active in cancer tissue, while the enzymes operating in the higher metabolic processes (polynucleotidases) are almost as active in the tumor as in the normal organ. This finding does not conflict with the results of Villa and others, since it concerns RNA only and is based on experiments in vitro.

208

P. RONDONI

An abnormal protein system (nucleoprotein or lipoprotein), characterized by a new intramolecular configuration or by some new intermolecular arrangement, could involve immunological specificity ; hence the search for antigens in tumors, more or less distinguishable from the species and normal organ antigens, is justified. There is a large number of papers on this subject; and for recent work we may refer to the review of Hauschka (1952). Others include the older work of Witebsky (1930) on a specific globulin in certain tumors; the observations of Maculla (1947-1948) on quantitative serological differences between adult and embryonic organs and tumors of mice; and the work of Malmgren et al. (1951), as well as of Weiler (1952), who investigated the particulate components of the protoplasm of normal liver and of hepatomas of rats and found clear serological quantitative differences in comparison with the normal organ. Fink et a l . (1953) present data which demonstrate th a t inbred mice are capable of producing antibodies t o a lyophilized preparation of their homologous tumor, as evidenced by the in vivo immunologic reaction of anaphylactic shock; the tail inoculation of a tumor produces an immunity to a subsequent inoculation. The immunological differences between cat,hepsins of normal and tumor tissue described by Maver and Barrett (1943-44) and Maver and Thomson (1944) are noteworthy in this respect. We may agree with the conclusions of Hauschka: '' . . . in retrospect, the serological studies of tumors, though extensive, were seldom controlled in a way which (except in the case of some viruses) would allow unequivocal isolation of specific neoplastic antigens. Where claims for specificity have been made, the genetic gap between tumor and host has generally been objectionable. The evidence for quantitative if not qualitative differences in the antigenic components of normal and malignant tissues is, however, convincing . . . " (see also Section V,l). 5.

rl

Comparison of the Neoplastic Transformation of the Cell with a Process of Protein Denaturation

It can be postulated th at the basic change responsible for malignancy is the constitution of a new and abnormal organization center overcoming the normal organizing system and inducing the formation of a low-grade protoplasm. Such protoplasm can be regarded as devoid of receptors for regulation and differentiation, and the word " despecialization '' used b y Powell (1946, 1947) may express the nature of the protoplasmic change. A very common in vilro phenomenon shows some similarity with the carcinogenic reaction (Rondoni, 1938a, 19-10, 1946a,b, 1949b), namely, that of protein denaturation. Fruton and Simmonds (1953) state th a t a denatured protein consists of disorganized peptide chains : the denatura-

SOME ASPECTS OF CARCINOGENESIS

209

tion brings about a change in the shape of the protein molecules, and there is generally an increase in asymmetry. There is also an increase of reactivity of certain groups, such as the sulfhydryl groups of cysteine, the disulfide groups of cystine, and the phenolic groups of tyrosine, owing to the unmasking of these groups by the uncoiling of the chains. The denatured proteins cannot be crystallized because of the disorganization of the internal structure. Here therefore are some of the changes, which may equally occur in the proteins of the cell subjected to malignant transformation. An increase of entropy takes place in the process of denaturation (Mirsky and Pauling, 1936; Anson, 1938) just as it most probably does in carcinogenesis (see Section 111). Indeed, the protein molecule from a specific, uniquely defined configuration passes after denaturation into a less orderly structure, i.e., from a less probable condition to a more probable one. The word “despecialization” may be applied here as also in the case of the neoplastic transformation. Some specific functions are lost in denaturation, but new functions might be acquired as a result of the molecular rearrangements permitting new bonds and new reactivities. Also, the sensitivity to enzymes is modified. Denaturation, involving the breaking of many weak bonds between the peptide chains, has a high value of heat of activation; the same seems to be true for the carcinogenic reaction, where an energy threshold must be overcome according to the considerations set forth in Section 111. Carcinogenesis is a new event in the life history of the cell, just as is denaturation in the in vitro condition of a protein; both processes are caused by many agents, physical and chemical, very different in nature and having a common issue in the despecialization of a protein system. Protein denaturation was first considered to be an irreversible process; but we now know that there are some types of denaturation which are reversible (Anson and Mirsky, 1931, 1934). It is, however, difficult to reproduce the folding specificity of a protein, i.e., to regenerate the normal protein configuration destroyed by a denaturing agent, and equally carcinogenesis itself appears to be an irreversible process. The work of A. Fischer (1936a,b) and of Rondoni (1938~)demonstrates that the heat denaturation of some protein systems is a partly transmissible process; a small amount of the already denatured protein induces an acceleration of heat denaturation in the homologous protein. The denaturation process seems to behave like a chain reaction. Of course the chain very soon breaks down in the in vitro reaction, whereas in vivo it may be easily perpetuated through the continuous turnover of the cell constituents. The protein molecules are rather rigid atomic structures, which notwithstanding can be very easily molded and submitted t o deformation. A given deformation can be transmitted from one molecule

210

P. RONDONI

to another; Astbury et al. (1935) spoke of “cannibalism” exerted amongst protein molecules. The abnormal organizer might be compared to a denaturation product able autocatalytically t o induce a further denaturation. The opinion of 0. Schmidt (1940, 1941a,b) is similar: carcinogenic agents react with some protein system of the protoplasm and induce the formation of a more stable, denaturation product, with release of energy which might be employed in the uncontrolled cell multiplication. Perhaps too much stress has been laid by rancer workers on the rapid growth of tumors and on the frequency of cell division. But such processes are in most tumors less pronounced than in the cmbryo or in the physiological regeneration of tissues. The outstanding characteristic of malignancy consists rather of a permanent error of differentiation, with reversion of the cell t o a kind of autonomy. If the proteins in cancer have a simplified structure. the synthesizing forces are very likely able t o build larger amounts of living matter than in the case of the more specialized and energetically more exacting proteins of normal cells. Whenever rapid protein synthesis takes place, a simplified protein type is usually found (histories and protamines in germinal tissues; histone combined with D X h in euchromatin). The presence in the cancer cell of a stabilized protein fraction acting as an abnormal template is not inconsistent with the well-known lability of such cells. The abnormal structural and ultrastructural substrate is largely masked by the normal constituents and therefore difficult t o identify even by serological procedures. The physical and chemical carcinogenic agents should be considered as denaturing factors, producing in some protein system related with the inner cell organization, and with differentiation, a particular type of deiiaturation or disorganization consistent with cell life and multiplication. Some viruses also should be able t o interfere in such a way with cell organization. Reference has been made to a concept of viruses as L 4 denatitrasea.” The introduction of heterologous proteins (or of proteins in any way foreign t o the blood) could bring about i n some cells, like those of the reticuloeiidothelial system, a derangement in protein synthesis and cause the formation of abnormal, immature cell strains; thus Pentimalli (1953) obtained, in rabbit blood and organs, pictures suggesting a leukemic or Ieukemia-like change, by means of repeated injections of foreign proteins, and pointed to a mechanism involving derangement of protein synthesis. Denaturation might be started by some masking process or by the binding of a chemical group (see Section IV), so inducing a shift in the dynamic equilibrium of the living system. A denaturation-like modification of the aggregation state arid of the stability of a protein system may

SOME ASPECTS OF CARCINOGENESIS

211

be produced by treatment with small quantities of hydrogen peroxide (Rondoni and Pozzi, 1933, 1935; Rondoni, 193%; Rondoni and Bassi, 1951). I n complex protein systems (e.g., serum or organ extracts) the addition of HzOz in certain concentrations brings about an increase of the trichloroacetic-acid-precipitable N-fraction. This phenomenon was attributed t o a physical aggregation of protein and peptide molecules induced by the oxidative destruction of peripheral polar hydrophilic groups, and t o oxidation of SH-groups with formation of intermolecular -S-Sbridges. It is also, very likely, the same phenomenon as was observed by Yamafuji and co-workers (see Paragraph 1 of this section), and considered by them to be a true polymerization with significance for the origin of virus particles. The “aggregation efect” of HzOZ.wassubstantially confirmed by Blagowestschenski and Korman (1934). (For a full discussion of this subject, see Rondoni, 1949b.) We know from the work of Greenstein et al. (1941a), and of v. Euler and Heller (1949) and others, that one of the most striking features of the enzymatic pattern of tumors is an enormous decrease of catalase. According to Greenstein the catalase activity of rat hepatomas and normal adult rat livers may be in the ratio 1/1000, whereas differences affecting other enzymes of the oxidative metabolism are much smaller. Regenerating liver has nearly the same catalase activity as normal liver. Rondoni (1952) advanced the hypothesis that the marked reduction in catalase activity might have some bearing on the protein rearrangement in carcinogenesis, producing an accumulation of H202 and consequently an aggregating effect on certain of the protein fractions. Indeed, Rondoni and Cudkowicz (1953) found a high content of HzOzin transplantable tumors of rats and mice, more or less approximating the content of organs with a highly active oxidative metabolism. It must be recognized that the chemical estimation of hydrogen peroxide in tissue is difficult; and the method then adopted (colorimetric estimation after reaction with titanium sulfate) was later modified by Cudkowicz (195410). The absolute values are therefore doubtful; however, the relative values may be justified. Cudkowicz (1955) reported with more reliable techniques the H20zcontent of the livers of rats fed for some months on a rice-carrotcod-liver oil diet with or without dimethylaminoazobenzene (DAB) (before appearance of hepatomas in the DAB series), and has found a regular increase in concentration in the livers of rats fed the special diet known to facilitate neoplastic degeneration, in comparison with the livers of rats fed a standard (Coward) diet: addition of the azo compound had no further effect on the HzOzcontent (see Table VI). Catalase activity also increased during administration of the cancer-promoting diet. The provisional opinion may theref ore be advanced that some cancer-pro-

212

P. RONDONI

TABLE TI per mg. Dry Weight) in 1,i.c.ersof Rats Fed Different Diets: H Z 0 2Content (g. I. Kormal (Coward diet) 11. Promoting diet (rice-carrot-cod-liver oil) 111. As in 11, plus p-dimethylaminoazobenzene (DAB) IV. A s in I plus DAB Duration of Treatment, Days

I1

I

I11

IV

6.5 7 5.5

10

9

4

8 7

8 5.5

6 6.5

30

7 1 8

12 11 10.5

15 12 12

5.5 6 5

60

5 7 1.5

13 12 8

12 15 12

6

16 14 12.5

10 12 12

120

15 14 9

13 10 11

150

12 13 13

12.5 14

15

90

6.5 5

9

The dejicieni diet i n II and I l l and the administration of D A B i n 111 snd ZV discontinued and normal (Coward) diet given to all groups of rats 165

8

7 7 180

6.5

7 5

7 6.5 8 6 7 7

SOME ASPECTS OF CARCINOGENESIS

213

moting or cancer-inducing agents can increase the formation of H 2 0 zin the exposed organ, and that an increase of the HzOz-splitting enzyme catalase may occur, as a regulatory attempt which later fails to operate when neoplastic degeneration sets in. Then there occurs a marked depression of activity and an increase in HzOz concentration. According to Cudkowicz (1952), the carcinogenic hydrocarbons 20methylcholanthrene and 3,4-benzpyrene when dissolved in caffeine solution exert a partial inhibition of liver catalase in vitro, whereas noncarcinogenic hydrocarbons are devoid of such action. However, aqueous suspensions of these hydrocarbons exert no such inhibition. That the accumulation of peroxide may play a role in the suggested protein rearrangement, interfering with the intermolecular relationship, remains of course a working hypothesis. The accumulation of H 2 0 f should bring about the abnormal protein arrangement or molecular deformation when it affects cells proliferating under influence of different stimulations, i.e., labilized protoplasmic systems. Such a hypothesis might, however, be supported by the fact that ionizing radiations, a well-known carcinogenic and mutagenic agent, operate by forming OH-radicals from tissue water, i.e., by a chemical mechanism involving oxidation reactions and very likely some kind of denaturation process in proteins. Abnormal growth of bacteria can also be associated with decrease of catalase activity, and Pontieri (1953) found such a decrease in the abnormal growth forms of Escherichia coli produced by urethane. The bacteriological literature contains many other observations on the production of atypical growth by accumulation of H202 and other peroxides in the culture medium under the influence of radiations. VII. SUMMARY Malignant tumors belong to the regressive processes of classical pathology, involving the growth of a more or less despecialized living matter. I n the neoplastic transformation of a normal tissue we see a transition of the living system from a less probable condition to a more probable one, i.e., an increase of entropy, as in all degenerative and prelethal changes. Some features of the oxidative metabolism of the cancer cell are the result and not the cause of neoplastic degeneration, and depend chiefly upon the necrobiotic changes in many malignant tumors. The carcinogenic aromatic hydrocarbons enter the cell through the lipoid constituents, and some disorganization of lipoids (lipoidolysis) is probably associated with the initial steps of the carcinogenic reaction chain; but the decisive attack affects some complex protein system. The binding or covering of the SH-groups of protoplasm by the carcinogenic hydrocarbons is discussed. The diamagnetic behavior of the hydrocarbons and of some

214

P. KONDONI

steroids may give support t o the electronic theory. Recent work on the electrophoresis of proteins from neoplastic tissues, on amino acids pattern, and on the heat coagulation of protein fractions, is reviewed and discussed, with studies on infrared absorption spectra. I n general, the purely chemical composition of proteins from cancer tissue does not seem t o differ from that of the normal parent tissue. It is therefore supposed that the main change concerns not the composition specificity but the Joldiny speci’icity, z.e., the shape and internal configuration of the molecules as well as their association. The change may reside chiefly at the ultrastructural level and concerns a faulty step in protein synthesis, occurring a t the same level (generation of shape specificity) as is involved in antibody formation. As a result of this interference with synthesis an abnormal tridimensional template is produced, which henceforth controls the synthetic processes and inducps the generation of a despecialized protoplasm. .\ comparison is made of neoplastic change with protein denaturation and the hypothesis is advanced that an abnormal accumulation of hydrogen peroside may play a role in denaturation, on the basis o f t h e hydrogen peroxide content of neoplastic and preneoplastic tissues.

I<EFEK EX C ES A q u a , C . 1945. d t t i accad. nazl. Lincei, Rend., Classe sci. 5s. mat. e nat. 21, 593-599. Ain1)rose. 1.;. J.. Elliot, A., and Temple, lt. B. 1949. :\iatrtre 163, 859-862. Anson, 11.L. 1938. In “Chemistry of the -\mino .kids and Proteins” (L. A. Schmidt, (4.).Charles C Thomas, Springfield, Illinois. Anson, 11.L., and Mirsky, A. E. 1931. J . Geti. Ph!y.sio(. 13, 133-143; ibirl. 14,725-732. :Inson, 11. L.. aiid 3firsk A. E. 1‘334. J . Gen. Physiol. 17,393-398; ibid. 17, 399-408. hsroli, 11. 1935. Klin. II hschr. 14, 1593-1597. Ascoli, 11.,d’.Uessantlro, G . , and Indovina, K. 1934. Klin. 11-ochschr. 13, $03. Xstbury, 1V. T., Dickinson, S., and Railly, I