Clonal origin of human tumors

Biochimica et Biophysica Acta, 458 (1976) 283-321 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - P ri nt e d in The N e t h e r l a n d s BBA 87028

CLONAL

ORIGIN

OF

HUMAN

TUMORS

P H I L I P J. F I A L K O W

Medical Service, Veterans Administration Hospital, Seattle, and Departments of Medicine and Genetics, University of Washington, Seattle, Wash. (U.S,A.) (Received F e b r u a r y 24th, 1976)

CONTENTS I. 11.

I II.

IV.

V.

VI.

VII.

VIII.

IX.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X-Chromosome inactivation mosaicism . . . . . . . . . . . . . . . . . . . . A. X - C h r o m o s o m e i n a c t i v a t i o n . . . . . . . . . . . . . . . . . . . . . . . . B. G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e enzymes . . . . . . . . . . . . . . . . C. I n a c t i v a t i o n at the glucose-6-phosphate d e h y d r o g e n a s e locus . . . . . . . . . . D. I n t e r p r e t a t i o n of d o u b l e - e n z y m e p h e n o t y p e s (bot h B a n d A enzymes) . . . . . . E. T u m o r s with single e n z y m e p h e n o t y p e s (B or A enzyme) . . . . . . . . . . . . C h r o n i c myelocytic l e u k e m i a . . . . . . . . . . . . . . . . . . . . . . . . . A. C l o n a l origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Stem cell origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. R e m i s s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. N o r m a l stem cells in c h r o n i c myelocytic l e u k e m i a . . . . . . . . . . . . . . E. Blastic t r a n s f o r m a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . F. E t i o l o g y a n d p a t h o g e n e s i s . . . . . . . . . . . . . . . . . . . . . . . . Other m y e l o g e n o u s stem cell diseases . . . . . . . . . . . . . . . . . . . . . A. P o l y c y t h e m i a vera . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. I d i o p a t h i c myelofibrosis . . . . . . . . . . . . . . . . . . . . . . . . . C. P a r o x y s m a l n o c t u r n a l h e m o g l o b i n u r i a . . . . . . . . . . . . . . . . . . . C h r o n i c l y m p h o p r o l i f e r a t i r e diseases . . . . . . . . . . . . . . . . . . . . . A. I m m u n o g l o b u l i n m o s a i c i s m . . . . . . . . . . . . . . . . . . . . . . . . B. C h r o n i c l y m p h o c y t i c l e u k e m i a . . . . . . . . . . . . . . . . . . . . . . . C, Multiple m y e l o m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. W a l d e n s t r 6 m ' s m a c r o g l o b u l i n e m i a . . . . . . . . . . . . . . . . . . . . . E. C h r o n i c cold a g g l u t i n a t i o n disease . . . . . . . . . . . . . . . . . . . . . F. L y m p h o m a s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hereditary tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. M u l t i p l e n e u r o f i b r o m a t o s i s . . . . . . . . . . . . . . . . . . . . . . . . B. O t h e r hereditary t u m o r s . . . . . . . . . . . . . . . . . . . . . . . . . Viral a n d p u t a t i v e viral t u m o r s . . . . . . . . . . . . . . . . . . . . . . . . A. W a r t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Burkitt l y m p h o m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Acu te l y m p h o b l a s t i c l e u k e m i a . . . . . . . . . . . . . . . . . . . . . . . D. A c u t e m y e l o b l a s t i c l e u k e m i a . . . . . . . . . . . . . . . . . . . . . . . . Endocrine-influenced t u m o r s . . . . . . . . . . . . . . . . . . . . . . . . . A. T h y r o i d t u m o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Breast c a r c i n o m a . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. M u l t i p l e " h i t " a n d t u m o r p r o g r e s s i o n theories . . . . . . . . . . . . . . . .

284 285 285 286 287 288 289 290 291 292 292 292 293 294 295 295 295 296 296 296 297 297 298 298 298 300 300 301 301 301 302 306 308 308 308 309 309 309

284 B. Embryonal tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Carcinoma of the uterine cervix . . . . . . . . . . . . . . . . . . . . . . D. Carcinoma of the colon . . . . . . . . . . . . . . . . . . . . . . . . . . E. Anaplastic carcinoma of the nasopharynx . . . . . . . . . . . . . . . . . . F. Metastases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Leiomyomas of the uterus . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Parthogenic origin of ovarian teratomas . . . . . . . . . . . . . . . . . . . . XII. Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIlI. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

309 310 311 31 I 313 313 314 315 315 317 317

Abbreviations: GIc-6-P dehydrogenase, glucose-6-phosphate dehydrogenase; Ig, immunoglobulin; CML, chronic myelocytic leukemia; EBV, Epstein-Barr virus; PNH, paroxysmal noctumal hemoglobinuria.

I. INTRODUCTION The necessary proscription against direct investigation o f h u m a n tumorigenesis limits the types o f studies that can be done. Consequently most investigations o f h u m a n t u m o r development necessarily bear only indirectly on causative factors. One such indirect a p p r o a c h involves determining the n u m b e r of cells f r o m which tumors arise, thereby providing important clues to their mode o f origin. F o r example, a neoplasm that results f r o m a rare, more or less random, event like "spontaneous" somatic mutation would be expected to arise in a single cell. On the other hand, multicellular origin might be seen for a t u m o r caused by cell-to-cell spread o f a virus with transformation o f infected cells. The n u m b e r o f cells from which tumors arise and several related questions can be investigated in subjects with at least two genetically distinct types o f cells, i.e., in subjects with cellular mosaicism. To illustrate this point, assume that a person has two types o f cells, A and B. If the event which initiates a t u m o r occurs in only one cell (e.g., in an A cell), all the neoplastic cells will be o f one type (A), since they are all descended from the one progenitor cell. On the other hand, if the oncogenic event occurs in m a n y cells, it is likely that both A and B types will be affected and the t u m o r could then contain descendants o f both cell types. Several mosaic systems have been employed in studies o f h u m a n tumorigenesis. F o r example, c h r o m o s o m a l mosaicism, a condition in which some cells contain a readily identifiable c h r o m o s o m a l abnormality while other cells o f the same tissue are normal, has been utilized. However, this system has limited applicability. A second type o f marker system is dependent u p o n immunoglobulin synthesis and therefore is restricted to cells o f the immune system. The most generally applicable cell marker system is dependent upon X - c h r o m o s o m e inactivation mosaicism. In this c o m m u nication studies done using c h r o m o s o m a l and immunoglobulin cell markers are

285 r e v i e w e d briefly, b u t t h e m a i n e m p h a s i s is o n s t u d i e s u t i l i z i n g X - c h r o m o s o m e activation

mosaicism

with the gluocose-6-phosphate

dehydrogenase

in-

l o c u s as t h e

X-linked marker.

11. X - C H R O M O S O M E INACTIVATION MOSAICISM

HA. X-Chromosome inactivation In accordance with the inactive X (Lyon) hypothesis, only one of the two Xchromosomes

in e a c h s o m a t i c cell is g e n e t i c a l l y a c t i v e .

T h i s is i l l u s t r a t e d i n Fig. 1.

T h e f e m a l e z y g o t e a t t h e o n e cell s t a g e h a s i n h e r i t e d o n e X - c h r o m o s o m e

\ ~

from the

/ )

/

/\

Zygote

\

/\ Embryo

Inactive X m / ~ . . . ~ / ' ~

(Barr body)

^"

... ~V'f

^-

vo ~ V ' ~ v n ~ " ~

A~

^

XP Inactive

(Ban" body)

Fig. 1. Diagrammatic illustration of X chromosome inactivation. At birth the female zygote has inherited one X chromosome from the mother (X'") and the other is of paternal origin (X0. At some time early in embryogenesis, the two Xs in each somatic cell behave differently - only one is genetically active, and the other is inactive. In interphase the inactive X becomes condensed and forms the nuclear chromatin or Barr Body. The factors that determine in each cell which X chromosome will be active are unknown, but this is probably a random process. Consequently, on the average, half the somatic cells have an active X m, and half have an active X p. Once it is determined which X is to be active in a given cell, that X not only remains active for the life-time of the cell but also is active in all the cell's progeny. All descendants of a cell with an active X p will have an active X p. Thus. the adult female is a mosaic of two cell types - those with an active X m and those with an active X p, A tumor with a clonal origin will consist entirely of X m or X p cells whereas a tumor with multicellular origin may contain both X m and X p cells. Reprinted, by permission from the New England Journal of Medicine (Vol. 291 ; p. 27, 1974).

286 mother (X M) while the other is of paternal origin (XP). At some time early in embryogenesis, the two X - c h r o m o s o m e s in each somatic cell behave differently. Only one of them is genetically active and the other is inactive. When the cell is not dividing, the inactive X is condensed and forms the nuclear chromatin or Barr body. It is not known what tells a cell which of its two X - c h r o m o s o m e s is to be active, but presumably this choice is made more or less at random. Therefore, on the average, one-half of the e m b r y o ' s somatic cells will have an active X M and one-half will have an active X P. While the initial choice of which X - c h r o m o s o m e will be active in a given cell is probably made at random, once it is made, it is stable to cell division and therefore is fixed not only for that cell, but for all its descendants. Thus, the adult female is a mosaic of two cell types - those with an active X M and those with an active X P. A t u m o r with unicellular origin will begin either in an X M or an X P cell, and, therefore, the mature neoplasm will consist entirely of X M or X P cells. In contrast, a neoplasm with multicellular origin may contain both X M and X P cells. To apply this system to the study o f tumors, it is necessary to distinguish X M from X P cells. This can be done if the female is a heterozygote for an X-linked trait and if the phenotype produced by the gene on X M can be distinguished at the cellular level from the phenotype produced by the allele on X P. The X-linked glucose-6-phosphate dehydrogenase locus fulfills these criteria and is particularly useful as a marker in t u m o r studies. l l B . Glucose-6-phosphate dehydrogenase

The usual starch gel electrophoretic type o f glucose-6-phosphate dehydrogenase and some c o m m o n variants are shown in Fig. 2. Females h o m o z y g o u s for the B gene

+

IV". . . . . .

(1)

C2)

C5)

(4)

C5)

Fig. 2. Starch gel electrophoretic phenotypes of GIc-6-P dehydrogenase obtained from females. The enzyme was derived from blood cells from (1) and (2) GdB/Gd B homozygotes, (3) Gda/Gd A heterozygote, (4) GdA/Gd A homozygote, (5) GdA/Gd A heterozygote. The preparations were not standardized for hemoglobin concentration.

287 (Gd B) have type B enzyme. This phenotype is seen in the vast majority of whites, but

in many black populations 30-40 ~,/, of males have a variant enzyme, either type A or A-. The A enzyme has faster electrophoretic mobility and only slightly less activity than the B type. The two enzymes, B and A, differ by a single amino acid substitution [1]. A - enzyme has the same electrophoretic mobility as A, but its activity in red cells is only 8-20 ~/oof normal. The underlying defect is a structural gene mutation resulting in an abnormal enzyme which initially has normal activity, but is more susceptible than the B or A types to loss of activity during cell ageing [2,3]. Because red cells lack nuclei, they do not have continued synthesis of nascent enzyme, and they manifest the A - enzyme deficiency. However, nucleated cells from A - subjects have almost normal activity; therefore, these cells cannot be used to distinguish A from A- by measurement of enzyme activity. The electrophoretic variants are particularly well suited for studies of mosaicism since the minor component in a mixture of B and A can be detected even if it constitutes as little as 5-10 ~/o of the total activity [4]. Other Glc-6-P dehydrogenase variants are prevalent in Mediterranean areas, but they cannot conveniently be distinguished from type B enzyme with electrophoretic techniques. In general, the Mediterranean type of Glc-6-P dehydrogenase deficiency is more severe and affected males have less than 10 o/ o/ o f normal activity in their red cells. Furthermore, in contrast to the A - type of Glc-6-P dehydrogenase deficiency, enzyme activity is also markedly decreased in nucleated cells. In about one-third of females who are heterozygous for the Mediterranean variant, Glc-6-P dehydrogenase activity levels are in the homozygous-normal range and in 3 ~,, of heterozygotes, levels are in the homozygous-deficient range (ref. 5 and Stamatoyannopoulos, G., personal communication). This overlapping somewhat limits that usefulness of the quantitative Mediterranean-type variants in tumor studies. IIC. Inactivation at the glucose-6-pho~phate dehydrogenase locus

A subject heterozygous at the Glc-6-P dehydrogenase locus for Gd a and a variant allele such as Gd a has both B and A enzymes, but a given cell or clone of cells shows only one of the two enzyme types seen in a mixture of cells [6-8]. This was conclusively demonstrated by cloning experiments [7,8]. The electrophoretic pattern of an extract made from cultured skin fibroblasts from a GdB/Gd * heterozygote contains both types of enzymes, but clones derived from the culture by single cell platings exhibit only type A or type B enzyme. Similarly, neoplasms arising from a single cell should exhibit only one enzyme type, while those with multicellular origin might contain both enzymes. About 40 ~o of black females are GdB/GdA or Gda/Gdaheterozygotes; consequently, the GIc-6-P dehydrogenase approach can be applied to many tumors in black populations. The potential use of this system for the study of human tumors was suggested in 1964 [9,10], and since then it has been used to study many neoplasms. Before these investigations are reviewed, possible sources of error in interpretation of Glc-6-P dehydrogenase phenotypes are considered briefly.

288 liD. Interpretation of double-enzyme phenotypes (both B and A enzymes) 1. Normal cell admixture. Before concluding that a tumor in a Glc-6-P dehydrogenase heterozygote contains both enzyme types (B and A) and thus has a multiple cell origin, the possibility must be considered that the actual tumor cells have a singleenzyme phenotype, and that the second enzyme type is due to the presence of nonneoplastic cells admixed with the tumor cells in the specimens subjected to electrophoretic analysis. This is especially important for invasive "solid" malignancies since they often contain non-tumor cells (e.g., stroma, normal epithelial cells, inflammatory ceils, etc.) in sufficient quantitities to make firm interpretation of double-enzyme phenotypes extremely difficult. For example, a tumor with 20 ~ B and 80 ~/o A that has 40~o non-tumor cell admixture, is probably clonal, rather than multicellular in origin despite the double-enzyme phenotype (i.e., the normal cells contribute the second enzyme). Consequently, careful histological examination of tumor samples is imperative. One method used is to divide tumor biopsies into several small samples, each measuring 3-8 mm in diameter [l l]. Individual samples are then divided into three equal portions; the two outer pieces are combined and analyzed for Glc-6-P dehydrogenase, and the center piece is examined histologically. 2. Activity of both X chromosomes in a single cell. Another possibility which conceivably could underlie a double-enzyme phenotype is activity of both X chromosomes in a single cell. This occurs in oocytes but no somatic cells are known to have two active X-chromosomes [12]. GIc-6-P dehydrogenase is a dimer and when both genes are active in a single cell, a hybrid molecule of B and A subunits is formed. This molecule which has electrophoretic migration intermediate between B and A [12], has not been reported thus far in any human tumor with double-enzyme phenotypes. Activity of both X-chromosomes in single cells can also be tested by cytological examination of neoplastic cells for nuclear chromatin (Barr) bodies and/or latereplicating X-chromosomes. Since the nuclear chromatin body is an expression of the inactive X chromosome, its presence in a tumor provides evidence that only one of the two X-chromosomes is active. 3. Ratio of B to A enzyme activity in normal tissues. One other important factor in assessing the significance of a double-enzyme phenotype is the relative activity of B and A enzyme in the normal tissue from which the neoplasm originates. For example, in one case of carcinoma of the nasopharynx, all samples of the tumor biopsy had both B and A enzymes [1 l]. Without further study it might have been concluded that this tumor had a multiple cell origin. However, normal tissue adjacent to the tumor displayed 20 ~ B and 80 ~ A enzyme. In contrast, samples consisting predominantly of tumor cells contained chiefly type B enzyme. These data suggest that the tumor actually originated in a single cell of type B and that the A enzyme present in the biopsy specimen was contributed by non-neoplastic cells admixed with the tumor cells.

289

liE. Tumors with single-enzyme phenotypes (B or A enzyme) Several possibilities other than clonal origin could theoretically explain the occurrence of single enzyme phenotypes in tumors. 1. Patch size. One possibility is that the tumor arises from several cells which by chance have the same type of Glc-6-P dehydrogenase. The probability that this will occur depends upon the growth pattern of the tissue from which the tumor arises. If, after cell division, daughter cells tend to remain adjacent to one another there will be patches of cells with like Glc-6-P dehydrogenase phenotypes. When this occurs extensively during tissue formation, the patches will be large and a tumor with singleenzyme ' phenotype may develop from many cells which by chance have the same enzyme type. Patch size can be estimated for a given tissue by statistically analyzing the results obtained from study of multiple small samples [13]. The estimates are based on the fact that the degree of heterogeneity in B:A composition is inversely related to the number of patches per sample. Estimates of patch sizes are 10 000 cells for normal adult uterine tissue [13], 950 to 3500 cells for scalp [14] and 1500 cells for skin epithelium overlying the vulva [15]. When the patch size for the tissue in which the tumor arises is known, the probability that a single-enzyme phenotype tumor has arisen from two or more nearby cells with the same enzyme type can be calculated. If complete random cell migration occurs in a tissue composed of equal numbers of B and A cells, the chance that two particular cells will be alike is 0.5. The probability that all of five neoplasms arising from a tissue in which complete random migration occurs will have singleenzyme phenotypes because the neoplasms happen to arise from two or more adjacent cells with the same enzyme type is less than 1 in 32. The possibility that neoplasms with single-enzyme phenotypes arose from adjacent cells of the same enzyme type decreases with increasing degrees of cell mixture in the tissue of origin. Presumably, considerable degrees of cell mixture occur in hematopoietic tissues. In other tissues, the larger the patch size, the larger the number of tumors with single-enzyme phenotypes that are required before it can be firmly concluded that the tumor has single cell origin. 2. Repetitive sampling. Another possibility other than clonal origin whereby neoplasms may display single-enzyme phenotypes is repetitive "sampling" during tumorigenesis. If a considerable proportion of cells die during the growth of a neoplasm so that cell production only slightly exceeds cell death, even tumors with multicellular origin will eventually have only one enzyme type [16]. The time required for complete loss of one cell type presumably must be very long, and the predicted double-enzyme phenotypes in small (presumably young) tumors and in the cores of old tumors have not been observed. Consequently, it is unlikely that this explanation applies to many, if any, human tumors with single-enzyme phenotypes. 3. Seleetion. The most difficult problem to deal with in the interpretation of single-enzyme phenotypes is selection. One cell type may predominate through selective overgrowth even though the tumor originated from many cells. This possibility can be evaluated by taking multiple small samples from "young" tumors,

290 especially from their cores; both cell types may be found in the early stages of tumor development. Selection based solely on Glc-6-P dehydrogenase type is unlikely if it can be demonstrated that the proportions of tumors with B phenotypes and A phenotypes are similar. Another way to evaluate selection is through study of multiple tumors from the same patient. If selection for genes on the X-chromosome is an important force, all tumors in a patient should show the same single-enzyme phenotype. This has not been found in studies of multiple tumors arising independently in the same subject (e.g., leiomyomas of the uterus). The fact that in tumors with multicellular origin induced in animals, both cell types can be demonstrated throughout the disease, indicates that selective overgrowth in mosaics is not a universal phenomenon (e.g., Herpesvirus saimiri induced lymphoma and leukemia in marmoset monkeys, [17,18]). Furthermore, very rapidly proliferating growths in man such as "venereal" warts retain their double-enzyme phenotypes [15] as do non-neoplastic cell proliferations such as are seen in hyperthyroidism (Fialkow, P. J., unpublished findings). These observations suggest that the single-enzyme phenotypes observed in the many benign and malignant neoplasms studied to date are unlikely to be due simply to chance overgrowth of one cell type by the other. 4. Miscellaneous. Other possibilities such as X-chromosome loss and non-specific disturbances in gene expression can easily be excluded by appropriate techniques and have not been observed in any tumors found to have single-enzyme phenotypes.

ili. C H R O N I C M Y E L O C Y T I C L E U K E M I A

Chronic myelocytic leukemia (CML) is characterized by an overabundance in marrow and blood of white cells, predominantly myelocytes and more mature granulocytic cells. Approximately 85 oL of patients have a very characteristic and specific chromosomal abnormality, the Philadelphia (Ph 1) chromosome; the other 15 ~o lack Ph 1. These Phi-negative patients have notably poorer prognoses and show other differences from Phi-positive cases [19,20] indicating important biologic distinctions between the two types of CML. Ph ~, first described by Nowell and Hungerford [2l], is a chromosome 22 [refs. 22,23] lacking about 40~,, of its DNA. Rowley has shown recently than in most patients the missing DNA is not lost but is translocated onto a number 9 chromosome [24]. Occasionally, it is translocated onto another chromosome (e.g., refs. 25-27), but even when other chromosomes are involved, the number 9 may be part of the rearrangement [28]. In the usual case of CML, Ph 1 is detected in 90 to 100~ of dividing marrow cells and is present throughout the course of the disease. It has even been observed in some marrow cells several years before the onset of overt leukemia [29]. Occasionally Ph ~ may be found in other myeloproliferative disorders, but it is rarely, if ever, present in nonmyelogenous diseases or in normal subjects. It is the most specific cytogenetic abnormality yet detected in a human neoplasm.

293 of four patients with CML in relapse, marrow-derived colonies were found to be either Phi-positive or Phi-negative [44]. No colonies were observed to contain a mixture of Phi-positive and Phi-negative cells, but one of eight colonies from one patient and three of six from another, were Phi-negative suggesting that some normal stem cells persist in at least a proportion of patients. However, other investigators have found such colonies to be uniformly Phi-positive [46-48]. Further evidence in favor of the presence of some normal stem cells is provided by the occasional reports of CML patients with unusual sensitivity to busulfan therapy who, after recovery from profound marrow hypoplasia, manifest a Phi-negative population [49,50] and by the preliminary report of the temporary appearance of such cells in some patients treated with radical, intensive chemotherapy [51]. This question can be further investigated through Glc-6-P dehydrogenase studies of single stem cell colonies grown from heterozygotes with CML.

HIE. Blastic transformation CML is a chronic disorder and often is associated with prolonged survival unless myelofibrosis or acute blastic transformation occurs. Thus, in one sense the disease can be regarded as a pre-leukemic condition. Blastic crisis is the most common cause of death in patients with CM L. This event is characterized by increasing failure of leukocyte maturation with continued overproduction of very immature cells, generally myeloblasts and promyelocytes. Additional chromosome abnormalities are very frequently superimposed on Ph ~ during this malignant transition. In several respects, blastic crisis in CML is similar to actue myelobastic leukemia. What factors govern the progression of CM L to malignant leukemia? During blastic transformation Ph ~ and the single-enzyme phenotypes persist indicating that the acute malignancy occurs in preexisting CML cells. One possibility is that a single leukemic cell undergoes transformation and gives rise to the acute-leukemia-like clone. Alternatively, the blast cells could originate from multiple CML cells. It is difficult to distinguish between these possibilites by means of Ph ~ and GIc-6-P dehydrogenase markers since by the time blastic transformation occurs, all or most of the hematopoietic cells are Phi-positive and have a single-enzyme phenotype. However, studies with other chromosomal markers strongly suggest that a single CML subclone evolves to the blastic transformation [52-54]. Occasionally blastic transformation in CML seems to be characterized by the presence of cells with morphology and sensitivity to steroid hormones reminiscent of lymphoblasts rather than myeloblasts [55]. Furthermore, the proliferating cells in some patients with blastic transformation have high activity levels of terminal deoxynucleotidyl transferase, an enzyme thought to be specific for thymus-derived cells and found in increased amounts in many patients with acute lymphoblastic leukemia [56,57]. One possibility to explain these findings is that CML involves a pluripotential stem cell common to the lymphocyte as well as the myelocyte. Another possibility is that patients with CML are predisposed by virtue of the disease to its therapy to develop acute lymphoblastic leukemia. Were this the case, lymphoblastic cells in

294 GIc-6-P dehydrogenase heterozygotes might be expected sometimes to manifest phenotypes different from those observed in the C M L cells.

IIIF. Etiology and pathogenesis The genetic data indicate that C M L arises in stem cells, but on morphologic grounds this form of leukemia is not considered to be a stem cell leukemia. The predominant picture in the marrow is an overabundance of myelocytes and more mature granulocytic cells with little evidence of increased stem cell proliferation. The morphologic and genetic observations are reconciled by the assumption that the normal stem cell population is replaced by leukemic stem cells, which in turn give rise to the myelocytic cells exempt from normal growth-control mechanisms. Although normoblasts and megakaryocytes also originate from this stem cell and have the Ph 1 chromosome, generally red cells and platelets are not greatly increased in number. Thus, it appears that in most cases the factors determining the development of leukemia operate primarily in the differentiated environment of the granulocytic series. A small number of phi-positive patients with C M L have an important proportion of dividing marrow cells which lack the abnormal chromosome even late in the course of the disease. One possible explanation for this unusual situation is that Ph ~ occurred in a stem cell that had already undergone differentiation toward the granulocytic line, so that phi-negative marrow cells are normoblasts. Alternatively, the disease may always involve a multipotential marrow stem cell, but the extent to which the ensuing Phi-positive clone replaces the normal marrow stem cells is variable and in a few patients there is a noteworthy persistence of normal stem cells. Since Ph ~ is generally detected in 90 to 100 ~/o of dividing marrow cells, is present throughout the course of the disease even before overt leukemia [29] and is so characteristic and specific, it is probable that the Ph ~ chromosome rearrangement is closely involved in the pathogenesis of C M L and may be its immediate cause (perhaps by virtue of a "position effect"*). However, the primary etiologic agents, the factors that presumably induce the Ph ~ anomaly, remain largely unknown. Among the possibilities are rare "spontaneous" or induced genetic accidents, viruses, radiation and physiologic defects in marrow homeostasis. The "spontaneous" mutation hypothesis predicts unicellular origin and the genetic marker observations are in accord with this suggestion. The defective homeostasis hypothesis implies that the basic abnormality is not intrinsic to the marrow cells themselves, but is found in the mechanisms which regulate their proliferation and maturation. In so far as this hypothesis predicts multicellular origin, the genetic marker data make it very unlikely to be correct. Clonal origin also virtually excludes any hypothesis of pathogenesis based on continuous cell recruitment (i.e., the ability of a cell that has undergone leukemic transformation to induce continuously other cells to become a part of the neoplasm). * Position effect refers to the observation in some experimental organisms that a gene may exert a different effect when placed in a new chromosomal region through, for example, translocation.

295 Viral or radiation-induced origin could be from one or from many cells. Clonal origin for example could be found if the Ph ~ rearrangement occurred spontaneously in a rare cell and were a necessary precondition for viral transformation or if the oncogenic change induced by a virus were rare (e.g., the putative virus might induce chromosome abnormalities in many cells, but only the rare cell with Ph ~ would evolve into CML). Alternatively, the putative oncogenic virus might have specific affinity for the DNA in the involved regions on chromosome 22 and 9, in which case Ph ~ could be induced in multiple cells. The latter mechanism is rendered less likely by the genetic marker data which strongly suggest clonal origin. This discussion assumes that the development of CML is dependent upon one step. If, however, multiple steps are involved and one of these occurs in a single cell, it is not possible with one marker system to determine if previous or subsequent steps in leukemogenesis affect one or multiple cells.

IV. OTHER MYELOGENOUS STEM CELL DISEASES IVA. Polycythemia vera This chronic disease is characterized by increased numbers of marrow erythroblasts and red blood cells with consequent high blood viscosity, enlargement of the spleen and cyanosis. Single-enzyme phenotypes have been found in red cells from two Gda/Gd A patients suggesting that polycythemia vera has clonal origin [58]. The same singleenzyme phenotypes were found in granulocytes and platelets indicating that the disease is a stem cell disorder. Since blood lymphocytes displayed a normal doubleenzyme phenotype, at least a major lymphocyte population does not emanate from the abnormal polycythemia vera stem cells. Detailed analyses of single erythroid colonies grown in semi-solid medium indicate that there is a small number of residual, presumably normal stem cells [59]. The number of colonies derived from normal progenitor stem cells was increased by erythropoietin treatment indicating that these residual normal cells retain their sensitivity to that hormone. The data also suggest that progenitors of the polycythemia cells respond to erythropoietin in vitro [59]. IVB. Idiopathic myelofibrosis This disorder is characterized by varying degrees of fibrosis in the marrow with blood formation in the spleen and enlargement of that organ. Myelofibrosis may develop during the course of chronic myelocytic leukemia or polycythemia vera, but many cases occur without obvious predisposition. Acute and chronic forms are found. The nature of the fundamental disturbance is unknown, but many feel that it is a neoplasia and there has been much debate about the identity of the neoplastic cell. Thus far, only one GIc-6-P dehydrogenase heterozygote with this disease is known to have been studied [60]. One and the same enzyme type was found in the

296 blood granulocytes, red cells and platelets indicating that a basic abnormality resides in hematopoietic stem cells. The single-enzyme phenotype suggests that these cells are clonally derived and by inference, that the disease is "neoplastic". CIonal origin argues strongly against an altered microenvironment hypothesis previously advanced to explain the hematopoietic cell proliferation and fibrosis. According to this suggestion, idiopathic myelofibrosis results from damage to the marrow and spleen reticuloendothelial stroma (microenvironment) which directs stem cells into one or another line of hematopoiesis. In contrast to blood cells, cultured marrow fibroblasts displayed a normal double-enzyme phenotype suggesting that the marrow fibrosis which so predominates the clinical picture may be a secondary phenomenon. Similar conclusions have been reached using chromosomal markers [60,61]. IVC. Paroxysmal nocturnal hemoglobinuria This rare chronic disease is characterized by hemolytic anemia and by nocturnal excretion of hemoglobin in the urine. Affected patients have two populations of red cells, one very sensitive to acid hemolysis, and the other resistant to that treatment. One explanation for this finding of two different cell types is that the paroxysmal nocturnal hemoglobinuria (PNH) cells represent a clone arising from a somatic mutation [62-64]. This hypothesis has been tested by determining the GIc-6-P dehydrogenase phenotype of the acid-sensitive PNH cells from a GdB/Gd A heterozygote [65]. In contrast to the double-enzyme phenotype of the normal erythrocytes, the acid-sensitive PNH red cells had a single-enzyme phenotype strongly suggesting their clonal origin. Since over 25~,~ of red cells usually bear the PNH abnormality, to reconcile this observation with the somatic mutation theory, it must be assumed that the mutant cells have a selective advantage over normal cells. In fact, even before the Glc-6-P dehydrogenase studies, it had been suggested that PNH was a neoplastic disorder [64,66]. Since abnormalities are also found in granulocytes and platelets [67,68], the putative somatic mutation giving rise to the PNH clone may have occurred in a stem cell which would then be the site of the selective advantage. This possibility can be explored by studying Glc-6-P dehydrogenase phenotypes in granulocytes and platelets from heterozygotes with PNH.

v. CHRONIC LYMPHOPROLIFERATIVE DISEASES Included in this category are chronic lymphocytic leukemia, multiple myeloma, Waldenstr6m's macroglobulinemia, idiopathic cold agglutinin disease and lymphomas. For the most part, these disorders have been studied with immunoglobulin markers. VA. Immunoglobulin mosaicism lmmunoglobulin (Ig) mosaicism was the first mosaic system employed for the

297 purpose of determining the number of cells from which human tumors arise [69]. A given mature Ig-producing cell is committed to the synthesis of molecules with only one light chain and only one variable region (idiotypic specificity). Since there are many different kinds of Ig molecules, normal lymphoid tissue displays considerable cellular mosaicism. Tumors with clonal origin will contain cells synthesizing only one Ig molecule, whereas those with multicellular origin may contain cells synthesizing both types of light chains and many variable regions. The primary antibody-secreting cells are plasma cells and their precursors are B-lymphocytes derived from the marrow and thought to develop independently of the thymus (as opposed to thymus-dependent, T-cells). B-lymphocytes synthesize Ig molecules and although the latter are generally not secreted, they are easily detected on the cells' surfaces where they presumably act as antigen receptors. When stimulated by antigens, B-lymphocytes become "immunoblasts" and differentiate into plasma cells that secrete the appropriate antibodies. A cell intermediate between the B-lymphocyte and the plasma cell may be the so-called plasmacytoid-lymphocyte, a cell which not only secretes lg, but also has it on its surface. B-cell neoplasia may involve any of the cells along the pathway from the B-lymphocyte (chronic lymphocytic leukemia) to the plasma cell (multiple myeloma) (e.g., see ref. 70). Both secreted and cell-surface Ig can be used as markers to determine the quantitative cellular origin of these tumors.

VB. Chronic lymphocytic leukemia This disease is a malignant proliferation of small lymphocytes. The leukemia cell surfaces in almost every patient bear "monoclonal" Ig. Thus, the cell-surface immunoglobulin is restricted to one light chain class and one heavy-chain class* [71-74] and to one variable region (idiotypic specificity) [77,78] (the latter characterizes the Ig molecule which is the product of a single cell or clone of cells). Circulating monoclonal Ig is not usually found, suggesting that generally there is a maturationblock in the proliferating lymphocyte clone. VC. Multiple myeloma This malignant proliferation of antibody-secreting plasma cells is characterized by multiple bone tumors and marrow infiltration with consequent anemia, bone pain and fractures and by high blood levels of "monoclonal" Ig, usually IgG. In a typical case, there are billions of myeloma cells, but they each secrete the same Ig [79,80] indicating that the disease is clonal in origin [69]. Although blood lymphocyte counts are not increased, the majority of circulating B-lymphocytes bear on their surfaces the same monoclonal lg as is found free in the blood [81,82]. Thus, multiple myeloma reflects proliferation of a clone of immunocytes that ultimately differentiate into lg-secreting plasma cells. This is in contrast to chronic lymphocytic leukemia in which there is a block in the clone's maturation. * An exception is lgD which is found on leukemia cells bearing lgM [75, 76]. However, the lgM and IgD share the same idiotypic specificityand therefore have the same variable region.

298

VD. Waldenstr6m's macroglobulinemia Small amounts of macroglobulin (IgM) are normally present in the serum but when the level excedes 5-10 ~ of the total protein content, macroglobulinemia is said to be present. "Secondary" macroglobulinemia occurs in association with other diseases such as carcinoma, connective tissue disorders, chronic infection, and cirrhosis of the liver, etc. Idiopathic or "primary" macroglobulinemia bears the name of Waldenstr6m, the man who first described it [83]. This disease has some features of multiple myeloma and others of chronic lymphocytic leukemia. It is characterized by monoclonal IgM in the serum and by proliferation in the marrow of pleomorphic cells in a spectrum from lymphocytes and plasmacytoid-lymphocytes to plasma cells. The surfaces of these cells have the same monoclonal IgM as is found in the serum [84,85]. Furthermore, although the blood lymphocyte count is not increased in most patients, many circulating lymphocytes, like their marrow precursors, have membrane-bound monoclonal IgM [84]. In this sense, Waldenstr6m's macroblogulinemia and multiple myeloma may be regarded as forms of leukemia. They resemble chronic lymphocytic leukemia in that they are clonal proliferations of lymphocytes, but in chronic lymphocytic leukemia there is a block in maturation of the lymphocyte clone, whereas in Waldenstr6m's macroglobulinemia and multiple myeloma, the clones continue to mature from the small lymphocyte to the plasma cell. VE. Chronic cold agglutinin disease Cold agglutinins are Ig molecules which induce agglutination of red blood cells in the cold (0 ° to 5 °C). Small amounts are present normally in the serum. Excessive levels may occur secondarily in association with diseases such as mycoplasma pneumonia or "primarily" as in one form of acquired, idiopathic hemolytic anemia. This form, chronic cold agglutinin disease, is characterized by cold-induced blotchy skin and cyanosis, etc. The antibodies have specificity for the I and i antigens on human erythrocytes [86,87]. As in Waldenstr6m's macroglobulinemia, the serum Ig molecules are of the IgM class, they are monoclonal [88] and in each case, the same Ig molecule is found on a proportion of the patient's blood lymphocytes [89]. The clonal origin of chronic cold agglutinin disease suggests that it might be classified as a B-cell neoplasia, similar to Waldenstr6m's macroglobulinemia. VF. Lymphomas Lymphomas are characterized by progressive enlargement of lymphoid-tissuecontaining organs such as lymph nodes and spleen. Burkitt lymphoma has been studied extensively with both Glc-6-P dehydrogenase and Ig markers and is discussed in detail below (Section VIIB). Single-enzyme phenotypes have been found in the few studied non-Burkitt lymphomas arising in GIc-6-P dehydrogenase heterozygotes (Table I). Similarly, lymphomas of B cell origin synthesize monoclonal lg (Table I) strongly suggesting unicellular origin. As for the chronic hematopoietic diseases, the data suggests clonal origin for

299 TABLE 1 SINGLE OR MULTIPLE CELL O R I G I N OF T U M O R S D E T E R M I N E D WITH I M M U N O G L O B U L I N (Ig) A N D GLUCOSE-6-PHOSPHATE D E H Y D R O G E N A S E (GIc-6-PD)MARKERS Marker

Myeloproliferative disorders Chronic myelocytic leukemia Polycythemia vera Idiopathic myelofibrosis Paroxysmal nocturnal hemoglobinuria Lymphoproliferative disorders Acute "Burkitt-type" leukemia "Hairy-cell" leukemia Chronic lymphosarcoma leukemia Reticulum cell sarcoma Burkitt lymphoma Non-Hodgkin lymphoma Chronic lymphocytic leukemia Waldenstr6m's macroglobulinemia Cold agglutinin disease Multiple myeloma

Single cell (No. of specimens)

Multiple cell References (No. of specimens)

GIc-6-PD GIc-6-PD GIc-6-PD

8t 2 I

0 0 0

[30,31,*] [58] [60]

GIc-6-PD

2

0

[65]

lg Ig

6 2

0 0

[ 165] [166]

11 2 45 92 4 38

0 0 1 1 0 0

[84,166] ÷ [11,*] [121,*] [121] [141,*] [167,168] ÷

Rare

[84,166] +

lg GIc-6-PD GIc-6-PD lg GIc-6-PD Ig lg

~> 150

lg Ig GIc-6-PD lg Glc-6-PD

31 4 2 Many 1

1 0 0 Rare 0

[84] [89] [136,153]

Carcinoma Nasopharynx (anaplastic) Cervix, pre-invasive invasive Adrenal cortex Bladder Colon Kidney Ovary Palate Thyroid Vulva, Bowen's disease Melanoma Neuroblastoma Nephroblastoma

GIc-6-PD GIc-6-PD GIc-6-PD GIc-6-PD GIc-6-PD GIc-6-PD GIc-6-PD Glc-6-PD GIc-6-PD Glc-6-PD GIc-6-PD GIc-6-PD GIc-6-PD GIc-6-PD

7 9 8 1 1 0 1 3 4 5 I 2 1 2

0 0 2(?)~ 0 0 1 0 0 0 0 0 0 0 0

[11 ,*] [139,140] [139,140] [*] [*] [141] [*] [*] [11 ,*] [11 ,*] [137] [11 ,*] [11 ] [*]

Hereditary Neurofibroma Trichoepithelioma

GIc-6-PD GIc-6-PD

0 0

14 12(?)t

Viral Common wart "Venereal" wart

GIc-6-PD GIc-6-PD

6 0

Plasmacytoma

0 4

[11 ]

[90] [93] [94] [15] continued on next page

300 Table 1 continued Marker

Singlecell Multiple c e l l References (No. of specimens) (No. of specimens)

Endocrine Solitary thyroid adenoma

GIc-6-PD

22

0

[11,*]

GIc-6-PD 184 GIc-6-PD 6 GIc-6-PD 2 GIc-6-PD~ 39 GIc-6-PD 2

0 0 0 0 0

[150--153] [153,*] [11,*] [153,156,157] [*]

Miscellaneous benign Leiomyoma of uterus Lipoma Salivary gland adenoma Ovarian teratoma Neurofibroma (sporadic)

t Evidence for clonal origin of CML also derives from chromosome, 6-phosphogluconate dehydrogenase and Rh blood-group antigens. See text. * This table includes cases reported in literature (references given) and recently studied in the author's laboratory (indicated by an asterisk). + Only reterences for the two largest series given. t Contamination by normal tissue possible. Numerous other isoenzymeand chromosomal markers have been used. See text. most of the many other benign and malignant neoplasms that have been studied with cell markers (see Table I). What types of tumors might be expected to have multicellular origin? One possibility is a neoplasm that develops in a patient with an hereditary disease strongly associated with tumor formation. All of the cells in the target organ are susceptible to tumorigenesis by virtue of the inherited mutation. On the other hand, the nature of the external agent may be the major factor in determining the number of cells affected. For example, multicellular origin should be found for tumors caused by cell-to-cell spread and transformation by oncogenic viruses and might also be detected for neoplasms strongly influenced by hormones. Vl. HEREDITARY TUMORS

VIA. Multiple neurofibromatosis (yon Recklinghausen's disease) This disease which is inherited in an autosomal dominant fashion is characterized by skin spots with increased pigmentation (caf6-au-lait spots) and multiple neurofibromas. These tumors consist predominantly of one cell type, spindle-shaped cells resembling fibroblasts; they are presumed to arise from the nerve sheath (Schwann cells). Malignant changes occur in the hereditary neurofibromas, but the number of patients in whom this happens is unknown. Estimates of the per cent of patients who develop malignancy in a neurofibroma vary from fewer than 5 to more than 15. Tumors and the overlying skin from 7 non-contiguous sites were studied from each of two unrelated Gda/Gd g heterozygotes with typical neurofibromatosis [90]. Double-enzyme phenotypes were found in each of the 14 tumors. Possibilities other than multicellular origin to explain the double-enzyme phenotypes, such as admixture with non-neoplastic cells or activation of both X-chromosomes in individual neuro-

301 fibroma cells, have been excluded [90,91]. It was estimated that the minimum number of cells from which a neurofibroma develops is 150, but the figure could be in the order of several thousand. The initial tumorigenic step in this disease is a mutation inherited through the germ line; subsequent steps involving many cells could come about in at least two ways. In one, a relatively large number of cells may be simultaneously affected by the tumorigenic process. This might be seen with neoplasms induced by hormonal changes. Increased levels of nerve-growth factor have been described in patients with hereditary neurofibromatosis [92]. Alternatively, origin of a tumor from hundreds of ceils could be seen if the oncogenic mechanism initially altered only a single cell and if this alteration subsequently influenced the pattern of growth in neighboring cells. In contrast to these hereditary tumors, single-enzyme phenotypes indicating clonal origin are found in sporadically occurring neurofibromas (Fialkow, P. J. and Jacobson, R., unpublished observations). Thus, although the biological and histological features of the hereditary and sporadic varieties are similar, there is a fundamental difference in the tumorigenic mechanisms. VIB. Other hereditary tumors Double-enzyme phenotypes have also been reported in the dominantly inherited disease, multiple trichoepithelioma [93]. Well-differentiated forms of this tumor display the structure of hair follicles while the more poorly differentiated forms resemble basal cell carcinomas in histological appearance. In addition to the neoplastic epithelium, these tumors contain fibroblasts thereby making the GIc-6-P dehydrogenase data difficult to interpret (i.e., the double-enzyme phenotypes do not necessarily exclude clonal origin of the neoplastic epithelial cells). The concept of multicellular origin of hereditary neurofibromas cannot be generalized to apply to all hereditary tumors. For example, from what is known about the molecular abnormality in xeroderma pigmentosa, one probable mechanism of carcinogenesis, somatic mutation secondary to defective DNA repair, predicts that each of the multiple tumors that arises in a patient with this disease will have single cell origin (see also discussion on embryonal tumors, Section IXB).

VII. VIRAL AND PUTATIVE VIRAL TUMORS The only neoplasms in man with proven viral etiology are warts. VIIA. Warts 1. Condyloma aeuminata (+'venereal" warts). These rapidly growing infectious "'neoplasms" which occur at mucocutaneous junctions such as the vulva are caused by a papova virus. Each growth consists of cauliflower-like clumps of small verrucous subunits. The latter have a central core of connective tissue covered with epithelium. Double-enzyme phenotypes were found in each of four warts from two Glc-6-P

302 dehydrogenase heterozygotes [15]. Epithelial remnants were teased free of connective tissue and even the smallest verrucal subunits have multicellular origin. Detailed analysis suggests that a wart probably develops from about 4000-5000 cells. Thus, it is likely that the tumorigenic virus is highly infectious on a cellular level, but the factors which limit the growth of warts and prevent transformation to malignant neoplasms are unknown. One possibility is that growth of the condyloma accuminatum is limited by immunological surveillance, perhaps against viral antigens. Malignant outgrowth would only occur if this control were breeched by a clone of cells. Another possibility is that a malignant tumor will develop only if a virallyinfected cell is the site of one or more additional events such as genetic or epigenetic changes. 2. Common wart (verruea vulgaris). This growth is also caused by a papova virus, but in contrast to the double-enzyme phenotypes found in the "venereal" warts, single-enzyme phenotypes were found in six common warts from as many patients [94]. These findings are compatible with clonal origin. An alternative explanation is that the common wart arises from several cells, but in each of the growths studied, all the transformed cells happened to have the same GIc-6-P dehyrodgenase phenotype. If this interpretation were correct, further study of common warts should reveal some with double-enzyme phenotypes. If, in fact, common warts have unicellular origin whereas "venereal" warts have multicellular origin and both are caused by the same papova virus, there may be a significant effect of initial inoculum size and/or of local environmental conditions on the facility of viral spread and the pattern of tumorigenesis. VIIB. Burkitt Lymphoma There is much circumstantial evidence of a viral cause for Burkitt lymphoma, a lymphoblastic malignancy that occurs with notable frequency in children throughout certain areas of Africa and New Guinea. Affected patients usually have multiple tumors, most commonly occurring in the jaw, orbit, gonads, kidneys, mesentery, retroperitoneal and paravertebral structures, liver and spleen [95]. 1. Puatative viral cause. The putative agent in this disease is Epstein-Barr virus (EBV), a ubiquitous lymphotropic DNA herpesvirus first isolated in a cell culture derived from a patient with Burkitt lymphoma [96]. Data implicating this virus include the consistently elevated titers of EBV-related antibodies in African children with Burkitt lymphoma [97,98] and the presence of EBV genomes in Burkitt-tumor cells [99,100], which also have a very characteristic, virally determined nuclear antigen [101]. EBV genomes have not been detected in the cells of any other lymphoma. The capability of EBV to cause lymphoblastoid proliferation is shown by its roles as an agent that causes infectious mononucleosis (a self-limited lymphoproliferative disease) [102,103]; that induces lymphocytes which ordinarily have a finite life-span to form lymphoblastoid cell lines that proliferate indefinitely in vitro [104,105]; and that induces fatal lymphomas when injected into susceptible primates [106-109]. These data are suggestive, but there is no direct evidence that EBV is a cause of

303 Burkitt lymphoma in man. In fact, it has been suggested that the virus is merely a "passenger" in the malignant lymphoblastic cells. An argument against this suggestion is the failure to detect EBV-genomes in non-Burkitt lymphomas and in most of the rare cases of "Burkitt lymphoma" that arise in EBV-carriers in the United States and other non-endemic areas [110,111], despite the fact that the latter cases are often histologically and clinically indistinguishable from African Burkitt lymphoma [112]. 2. Co-tumorigenic factors. Although EBV may be an agent ofBurkitt lymphoma and has been established as a cause of infectious mononucleosis, a self-limited lymphoproliferative disease, most frequently EBV is associated merely with asymptomatic infection [113,114]. Why, on their first encounter with EBV, do some subjects have asymptomatic infection, others have infectious mononucleosis and still others have Burkitt lymphoma (if EBV is indeed an etiologic agent of that disease)? One possibility is that the different responses to infection are due to different sub-strains of EBV. This suggestion has not been excluded but thus far neither immunological nor molecular genetic differences have been identified between EBV samples isolated in association with Burkitt lymphoma or infectious mononucleosis [115-119]. Another possibility is that there are co-tumorigenic factors, one of which may be chronic holoendemic malaria. There is a strong epidemiologic association between the two diseases since Burkitt lymphoma occurs with high frequency only in areas with this type of malaria. Children with chronic holoendemic malaria not only have chronic infection but they are repeatedly exposed to fresh mosquito-borne infection. This condition might favor tumor development through continuous "preoccupation" and depression of the immune system, possibly allowing Burkitt-transformed cells to "'slip through" immunologic surveillance. In addition, malaria could further potentiate viral transformation by stimulating lymphoreticular proliferation. That there may be additional co-factors in Burkitt lymphoma is suggested by the observation that even among children with EBV infection and chronic holoendemic malaria, Burkitt lymphoma is a relatively rare occurrence (for further discussion of possible co-factors see Section VIIB, 4 below). 3. CIonal origin. Single enzyme phenotypes were observed in 45 of 46 Burkitt tumors obtained from 29 GIc-6-P dehydrogenase heterozygotes ascertained in Nairobi (refs. 120,121 and Fiaikow, P. J. et al., unpublished data). In contrast, normal double-enzyme phenotypes were found in the patient's unaffected tissues including blood lymphocytes, lymph nodes, ovaries, kidneys, etc. These observations strongly suggest that the great majority of Burkitt tumors have clonal origin. One of 46 tumors had approximately equal amounts of B and A enzymes. It is conceivable that there were non-tumor cells in the part of the specimen studied electrophoretically, but it is also possible that this one tumor had multicellular origin. Since on initial presentation, patients with Burkitt lymphoma generally have tumors at multiple anatomic sites, it is conceivable that this disease as a whole is multi-focal in origin even though individual tumors are clonal. If each tumor arises independently of the others, some neoplasms of type B and others of type A should

304 be found in the same patient. Conversely, if Burkitt lymphoma begins in a single cell at one site and then spreads to other parts of the body through some metastatic process, all tumors in the same patients should have the same single enzyme type. Two tumors were studied from each of 11 patients and in all cases the two tumors were concordant [121]. The probability that this degree of concordance occurred by chance alone is less than 0.01. Thus, on initial presentation, Burkitt lymphoma is a clonal disease beginning in one site and then spreading to other parts of the body. Since single cell origin of Burkitt lymphoma has important implications for its putative viral etiology, it is important to confirm the Glc-6-P dehydrogenase findings with another marker system. Burkitt cells frequently synthesize immunoglobulin which may be found on their surfaces; therefore, cell-surface immunoglobulin mosaicism can also be utilized to investigate the cellular origin of this disease. In a study of 93 Burkitt tumors, only one was found which had probable multicellular origin [121]. Cell-surface immunoglobulin markers were also determined on multiple tumors from the same patient on initial presentation (four tumors from one patient, three tumors from another patient and in two tumors from each of 11 patients), and with one possible exception, multiple tumors from the same patient were all concordant for the markers [121]. These data strongly support the conclusions reached with the Glc-6-P dehydrogenase system.

4. Implications of clonal origin of Burkitt lymphoma for its putative viral cause. Although viruses infect many cells, clonal origin of a neoplasm does not necessarily exclude viral cause. For example, single cell origin would occur if the oncogenic change induced by the virus were a relatively rare one such as a specific alteration in D N A (at the level of the gene or chromosome). Recently, a specific chromosomal rearrangement involving numbers 14 [122-124] and 8 [124] has been described in Burkitt iymphoma. Conceivably, the virus induces chromosomal changes in many cells, but only the one cell in which by chance this particular chromosomal rearrangement is induced will evolve into the Burkitt lymphoma clone, Another possibility is that the virus is a necessary, but not a sufficient causative factor and that one of the other tumorigenic co-factors is rare and affects only a single cell. For example, another way to look at the Burkitt lymphoma chromosome rearrangement is that its pre-existence in a cell from a "spontaneous" genetic accident is a necessary condition before a virus can transform the cell or before the virus-transformed cell can develop into clinical malignancy. Alternatively, the role of the virus may be to stimulate cell division in lymphocytes thereby increasing the likelihood for genetic changes (mutations and chromosomal rearrangements) to occur but only a rare cell will develop the specific alteration necessary for Burkitt tumor development. A third possibility is that the development of the tumor may be dependent upon the stepwise accumulation of several changes such that the required number and sequence will occur rarely and only in a single cell. And finally, many cells may be altered by the virus initially, but only a rare clone is able to escape surveillance mechanisms. No matter what the mechanism, the clonal origin of the malignant putative viral disease, Burkitt lymphoma, contrasts with the multicellular origin of the benign viral growth,

305 "venereal" wart. This difference suggests that virus-infected cells give rise to a malignant clone only if one or more additional events occurs. This hypothesis would be supported by the demonstration that the squamous cell carcinomas which arise occasionally in venereal warts have clonal origin. Another herpesvirus, Herpesvirus saimiri, induces experimental lymphomas with multicellular origin in nonhuman primates (e.g., marmoset monkeys) [17,18]. This contrasts with the unicellular origin of the putative EBV-induced naturally occurring Burkitt lymphoma. One possibility which could simply explain this difference is that the size of the virus inoculum is larger in the experimental model than in natural infections. However, host factors are probably also important. Man is the natural host for EBV; the presumed EBV-induced Burkitt tumors are rare and clonal in origin. The marmoset is not the natural host for H. saimiri and lymphoid tumors have not been described in its natural host, the squirrel monkey. Obviously, for a ubiquitous virus to survive in evolution, it cannot have a high oncogenic potential for its natural host. In contrast, the experimental host has not evolved efficient defenses against H. saimiri; tumors are universally induced experimentally and they are multicellular in origin. 5. EBV-negative Burkitt lymphoma. EBV genomes are absent in about 3~/0 of cases of African Burkitt lymphoma [125]. The frequency of EBV-negative disease in Africa is probably similar to the frequency of Burkitt lymphoma in non-endemic areas such as the United States. Most of the latter cases are also EBV-genomenegative [110,111 ]. One interpretation of these findings is that agents other than E BV may be resposible for a rare disease that closely mimics the usual African Burkitt lymphoma. 6. Burkitt recurrences. The majority of patients have therapeutically-induced "complete" remissions, but tumors reappear in at least 60 ~ of cases [126,127]. Are these true recurrences of the old disease (i.e., re-emergence of the original malignant cell line) or are these new occurrences of disease in susceptible patients? The first studied relatively "late" recurrence was in a 6 year old girl who had ovarian and parotid tumors on initial presentation [128]. The ovarian neoplasm displayed only type B enzyme in contrast to the normal tissues which had double-enzyme phenotypes. The patient was treated and went into what was judged to be a total remission, but four months after the onset of therapy she had recurrence of disease in the left parotid gland (a previously involved site). Like the original tumor of the ovary, the recurrent parotid neoplasm was typed as B. Two months after a second remission, she had for the first time, a tumor in the left orbit. In market contrast to the first two tumors, it showed type A enzyme. Thus, in this patient, exacerbation of disease in a previously uninvolved site did not result from re-emergence of the originally detected malignant cell line. The two most likely origins for the "new" malignant clone are that it arose as a reinduction of malignancy in a hitherto normal host cell or that it was present, but undetected when the patient was initially studied because, for example, it did not grow sufficiently to form a clinically evident tumor. In two of eight other patients with relapses occurring after five months, discordant immunoglobulin phenotypes

306 were found in the recurrent tumors [121]. These observations contrast with those of 27 early relapses which are uniformly concordant with the phenotypes found in the initial tumors [121]. Thus, the data indicate that early relapses are re-emergences of the original malignant clones; but some late recurrences may be the result of newly induced malignant clones. Similar suggestions have been made on the basis of clinical observations [127]. Early relapses tend to appear at previously involved anatomic sites whereas late regrowths often occur in hitherto uninvolved organs. Furthermore, the prognosis for late recurrences is similar to that for initial presentations, whereas early relaspes respond very poorly to therapy [129]. The finding that some late relapses are due to the emergence of a second malignant clone is compatible with the existence of predisposing factors to Burkitt lymphoma. These may be endogenous changes or environmental factors. However, recurrences could be associated with therapy which is generally immunosuppressive and may favor the emergence of a new clone due to escape from immunologic surveillance. It is remarkable that even discordant late recurrences have clonal origin. Thus, continuous recruitment of hitherto normal cells to the malignancy rarely if ever occurs in Burkitt lymphoma despite its putative viral etiology.

VIIC. Acute lymphoblastie leukemia Another lymphoblastic disease with putative viral etiology is acute lymphoblastic leukemia. The question of whether this disease is clonal or the result of cell recruitment had been debated for many years. Thus far, Glc-6-P dehydrogenase studies in patients with acute lymphoblastic leukemia have not been reported. However, chromosome markers have been utilized to show that at least under very special circumstances in two patients, the disease developed in more than one cell line. The first such patient studied was a 16-year old girl with acute lymphoblastic leukemia who was engrafted with marrow from her histocompatibility-matched brother one day after she received 1000 rad whole body irradiation [130]. As expected, the supralethal dose of irradiation led to a decrease in white blood cell count to essentially zero, but by day 20 the count began to rise and regenerating islands of marrow tissue were evident. Thereafter, the white count increased to normal levels and on day 52 the patient was well enough to be discharged from the hospital. Unfortunately, on a return visit 10 days later, lymphoblasts were detected in the peripheral blood. This was followed by a precipitous development of florid leukemia so that by day 80 the white blood count was 50 000/mm 3 with 95% lymphoblasts. Sheets of these cells replaced the normal marrow. Despite intensive therapy, the patient died on day 102. Because the host was a female who had been transplanted with marrow from a donor male, it was possible to use sex chromosomes as markers of host and donor cells. Throughout the early period of marrow recovery we could find only XY cells in marrow preparations studied directly (without culture). These observations indicate that donor cells had repopulated the marrow. Was the leukemia recurrence in residual host cells or was it a new occurrence in the donor cells? At a

307 time when the white blood cell count was 50 000 with 95 ~ lymphoblasts and the marrow was replaced by sheets of these cells many of which were in division, only XY cells were found in direct studies of blood and marrow. That these cells were XY, was confirmed by fluorescent and DNA-replication studies. Thus, the acute lymphoblastic leukemia recurred in donor cells [130]. Subsequently, a similar case was observed [131]. This patient was a 7 year old girl who had only XX cells in normal tissues and marrow prior to receiving a graft from her histocompatibility-matched brother who had only XY cells. Leukemia recurred on day 135 and the malignant cells had XY sex chromosomes. Possible mechanisms underlying these recurrences in donor cells have been discussed [130,131 ]. The two donors are normal without evidence of leukemia several years after marrow donation. Although the donors do not have acute lymphoblastic leukemia, it has been argued that among the 10" 109 engrafted marrow cells, there was one cell that had already undergone leukemogenic transformation. Such rare variant cells may occur frequently in normal subjects with rapid elimination by surveillance systems. In the supralethally irradiated host, these systems are deficient; thus a clone arising from a single transformed donor cell could give rise to clinically detectable tumor (about 10" 109 cells) after approximately 33 cell divisions. The rapidity with which the leukemia recurred indicates that the hypothesis that recurrent leukemia is derived from a single transformed donor cells very unlikely to be correct. Furthermore, this suggestion assumes that the rare donor transformed cell in both patients happened to be one that gave rise to a neoplasm morphologically and biologically indistinguishable from the original pregraft host disease. Although there are other possible mechanisms [130,131], perhaps the most likely is that hitherto normal donor cells were recruited to form the recurrent leukemia by activation of a leukemogenic virus in donor cells or by transfer of such an agent from host to donor cells. Irradiation has been shown to activate leukemogenic virus in rodents [132] and possibly a similar activation occurred in the irradiated patients treated with marrow grafts. Fortunately, since the initial observations in these two patients, no firmly documented cases of leukemia relapse in donor cells have been reported. It is of considerable interest to know if untreated acute lymphoblastic leukemia has unicellular or multicellular origin. If on initial presentation, the disease has single cell origin, the findings of multicellular origin in donor cells in the two immunologically suppressed transplanted patients would not only support a viral cause for acute lymphoblastic leukemia, but would be consistent with an important role for immunological surveillance in controlling the development of this disease in normal persons. An alternative explanation would be that the irradiation activated leukemogenic virus in such vast quantities in the marrow recipients that even a rare oncogenic interaction with a susceptible donor lymphocyte might have occurred several times.

308

VlID. Acute myeloblastic leukemia GIc-6-P dehydrogenase heterozygotes with this disease have not been reported, but studies with chromosome markers indicate that this leukemia involves a stem cell common to erythrocyte precursors and myeloblasts. About 40 ~,, of patients with acute myeloblastic leukemia have chromosomal abnormalities in marrow cells. Earlier studies suggested that chromosomal aberrations are present in red cell precursors as well as myeloblasts [133]. This has been confirmed directly by showing that abnormal chromosomes characteristic of the patient's leukemic cells are present in radioactive-iron-incorporating red cell precursors [134].

VIII. ENDOCRINE-INFLUENCED TUMORS Hormones affect large target areas and therefore, tumors which arise under strong endocrine influences might have multicellular origin. Of particular interest in females are thyroid and breast neoplasms.

VillA. Thyroid tumors 1. Thyroid adenoma. Many solitary nodules (adenomas) of the thyroid are assumed to arise under the influence of thyrotropin (formerly called thyroid-stimulating hormone). In fact, one previously postulated theory is that they are not true neoplasms, but represent hormone-influenced alterations in growth. This hypothesis, which predicts multicellular origin, may not be correct since single-enzyme phenotypes have been found in over 20 solitary follicular adenomas from as many GIc-6-P dehydrogenase heterozygotes (ref. 11 and Fialkow P. J., et al. unpublished observations). The finding that non-neoplastic thyroid cell proliferations as are found in hyperthyroidism with diffuse goiter (Graves' disease) retain their double-enzyme phenotypes (Fialkow, P. J., unpublished data), suggests that the single-enzyme phenotypes found in adenomas are unlikley to be chance overgrowth of one proliferating cell type by the other. Thus, it is likely that adenomas are clones and by inference, they may be true neoplasms. The presumed clonal origin of follicular thyroid adenomas does not preclude their development being dependent upon thyrotropin. For example, only rare cells in the thyroid may be susceptible to the tumor initiating effect of thyrotropin. Alternatively, the role of the hormone may be not to initiate tumorigenesis, but to promote proliferation of the clone in which the tumor-initiating event has already occurred. A similar mechanism obtains in experimental, radiation-induced thyroid tumors [135]. When thyroid hormone output is blocked (e.g. by thiouracil treatment), thyrotropin secretion is stimulated resulting in hyperplasia of the thyroid. Rats treated in this way and then exposed to radiation develop malignancy, but if tbyrotropin output is suppressed by administration of thyroid hormone, cancers do not develop. One way to interpret these findings is that radiation initiates the tumorigenic change and thyrotropin promotes cell division in the altered cells. Both are necessary for a cancer to develop.

309

2. Follicular carcinoma. The question of whether these cancers arise de novo in hitherto normal cells or from tumor progression in pre-existing adenomas is still debated. Single-enzyme phenotypes were found in the three follicular carcinomas studied (Fialkow, P. J., unpublished data). These findings do not distinguish between origin of the tumors from pre-existing adenomas or from single normal cells. However, the single-enzyme phenotypes exclude continuous recruitment of normal cells as a mechanism of carcinogenesis. The same is true for the one reported case of anaplastic thyroid carcinoma which also had a single-enzyme phenotype [11]. VIIIB. Breast carcinoma These malignancies are also strongly influenced by hormones. Double-enzyme phenotypes were reported in three breast carcinomas [136,137], but the considerable non-tumor cell admixture very frequently found in specimens of breast malignancy makes it difficult to interpret the Glc-6-P dehydrogenase results (e.g., see Section liD). We have observed several breast carcinomas with single-enzyme phenotypes (unpublished) suggesting that these cancers may also have clonal origin. IX. CARCINOMAS

IXA. Multiple "hit" and tumor-progression theories of carcinogenesis Several mechanisms have been postulated to explain the initiation and development of carcinomas. For example, these malignancies might form in a "field" comprising a large number of cells which undergo neoplastic alteration as the result of a potent carcinogen such as a virus or chemical. In this case the tumor would have multicellular origin. Another prevalent theory of carcinogenesis states that cancers require multiple mutation-like events or "hits". These multi-step mutation models were developed to explain the dependence of cancer mortality on age and they presume that malignancy develops only after a necessary series of "hits" occurs in a somatic cell. IXB. Embryonal tumors The simplest multistage mutation model of tumorigenesis involves only two steps and has been suggested for the embryonal tumors (retinoblastoma, neuroblastoma and nephroblastoma or Wilms' tumor) by Knudson, who finds support for this hypothesis in comparison of hereditary with non-hereditary cases [138]. Retinoblastoma occurs in hereditary (autosomal dominant) and non-hereditary forms. In the former disease, the first mutation is inherited through the germ cell and neoplasms are usually multiple and bilateral. On the other hand, in the non-hereditary retinoblastoma both mutations are somatic and must occur in the same cell. The probability for these mutations to occur is very low, and when they do, only a single retinoblastoma develops. If this hypothesis is correct, single-enzyme phenotypes should be found in hereditary and sporadic forms of retinoblastoma. On the other hand, if

310 the second step is not mutational, hereditary retinoblastoma might have double-enzyme phenotypes whereas the sporadic form would have clonal origin and therefore, single-enzyme phenotypes. In unpublished studies we have found single-enzyme phenotypes in two instances of sporadic nephroblastoma and one case of neuroblastoma. One prediction of all somatic mutation theories, whether the number of steps or "hits" is one, two, or more, is that the resultant tumors should have clonal origin. This is especially true of multiple-"hit" models since the probability that the required number and sequence of mutations would accumulate in more than one cell in the same region is essentially nil. As shown in Table I, genetic marker studies suggest clonal origin for most tested cancers in which careful histologic studies were performed. However, at least one carcinoma of the colon and perhaps some carcinomas of the cervix may have multicellular origin. 1XC. Carcinoma o f the uterine cervix

Histopathological evidence for the sequential theory of carcinogenesis is strongest for these malignancies. It is generally held that tumor formation progresses from the benign cervical dysplasia to carcinoma in situ to invasive carcinoma, although not all dysplasias lead to in situ carcinoma and not all of the latter progress to invasive carcinoma. 1. Cervical dysplasia and carcinoma in situ. In contrast to most normal tissues from GdB/Gd a heterozygotes which almost invariably display double-enzyme phenotypes, single-enzyme phenotypes are not infrequently found in "normal" cervical epithelium. For example, in one study, single-enzyme phenotypes were found in 2 of 12 samples of cervical mucosa from three Glc-6-P dehydrogenase heterozygotes whereas all 44 samples of epithelium derived from other organs had the expected double-enzyme phenotypes [13]. That such "normal" cervical epithelium with a single-enzyme phenotype may result from pre-malignant changes was demonstrated in a study of invasive carcinoma of the cervix [139]. In that investigation "normal" cervical epithelial samples (2 x 2 mm) from ten Glc-6-P dehydrogenase heterozygotes were tested and one sample with a single-enzyme phenotype was uncovered. Microscopic examination showed squamous metaplasia. Subsequently, five GIc-6-P dehydrogenase heterozygotes with cervical dysplasia and one with carcinoma in situ were studied; single-enzyme phenotypes were found in seven separate samples of the carcinoma in situ and in eight dysplasia samples [140]. These observations strongly suggest that the non-invasive stages, dysplasia and carcinoma in situ, originate in single cells. 2. Invasive carcinoma. If the premalignant conditions are forerunners of invasive carcinoma, the latter should also have a single-enzyme phenotype. In one study of invasive carcinoma, five of eight neoplasms from as many patients exhibited a single-enzyme phenotype but tumors from the other three patients had doubleenzyme phenotypes. However, after careful histologic examination of tissues adjacent to those used for electrophoresis, it was suggested that admixture of normal cells was

3ll the most likely explanation for the double-enzyme phenotypes and clonal origin of invasive carcinoma of the cervix was favored [139]. Similarly, most of these malignancies that we studied had single-enzyme phenotypes (unpublished) suggesting clonal origin. A somewhat different conclusion was reached in another study involving seven GIc-6-P dehydrogenase heterozygotes with invasive carcinoma of the cervix [140]. Single-enzyme phenotypes were found in samples from five tumors, but all 10 samples from the remaining two tumors had two enzyme bands. It was sug, gested that invasive carcinoma of the cervix may arise from a single cell in some cases and from multiple cells in other cases. If continued study confirms multicellular origin of some invasive cancers of the cervix, two possibilities might be considered: the early stages (dysplasia, in situ carcinoma, etc.) of some cervical tumors may be circumvented so that the cancer is initiated in several cells, perhaps under the influence of a potent carcinogen; or the change from in situ to invasive carcinoma may involve recruitment of hitherto normal cells, perhaps through the release of infectious viruses or viral DNA.

IXD. Carcinoma of the colon A double-enzyme phenotype was found in the one reported case of metastatic carcinoma of the colon studied with GIc-6-P dehydrogenase [141]. Because there was considerable admixture of non-neoplastic elements with tumor cells in the primary carcinoma, no firm conclusions about cellular origin could be reached. After the patient died, 28 metastatic nodules in the liver were analyzed. Of these, 7 had doubleenzyme phenotypes and histologic studies suggested that most of the Glc-6-P dehydrogenase was derived from stroma. On the other hand, the 21 nodules with predominantly type A or B enzyme were largely composed of tumor cells. The finding of some metastases with chiefly type B and others with type A enzyme indicates either that this carcinoma had a multicellular origin or that the patient had multiple primary tumors. Additional studies of cell markers in this disease would be of great interest. IXE. Anaplastic carcinoma of the nasopharynx This malignancy shows striking ethnic and/or geographic differences in prevalence [142]. For example, it occurs relatively frequently in certain ethnic groups of Chinese, but rarely in Western Europeans. 1. Glc-6-P dehydrogenase studies. The difficulties encountered in interpreting double-enzyme phenotypes in invasive solid malignancies are illustrated by study of anaplastic carcinoma of the nasopharynx [11]. The tumors are generally heavily infilrated with lymphocytes and also have residual normal epithelium and stroma. To study this disease, tumor biopsies are divided into several small samples, each measuring 3-8 mm in diameter. Individual samples are then divided into three equal portions; the two outer pieces are combined and tested for Glc-6-P dehydrogenase and the center piece is examined histologically and an estimate is made of the proportion of non-tumor cells.

312 Double-enzyme phenotypes were found in tumor biopsies from 26 of 33 Glc-6-P dehydrogenase heterozygotes with anaplastic carcinoma of the nasopharynx (ref. 11 and Fialkow, P. J. et al., unpublished observations). In these 26 biopsies there was considerable admixture of non-tumor cells with malignant epithelium and there was excellent correlation between the degree of admixture and the relative activity of the second enzyme type [11]. These observations together with the findings of single-enzyme phenotypes in the only seven tumors judged to be homogeneous with respect to neoplastic cells, strongly suggest clonal origin for this disease. 2. Epstein Barr virus. The only tumor aside from Burkitt lymphoma that shows a consistent association with EBV is anaplastic carcinoma of the nasopharynx. EBV genomes have regularly been found in tumor biopsies [99,100]. However, since the carcinomas are usually heavily infiltrated with lymphocytes and since EBV is specifically associated with B-lymphocytes, it was postulated that the EBV genomes detected in the tumor biopsies were present in the infiltrating lymphoid cells rather than in the carcinoma cells. That this hypothesis may not be correct was first suggested by in situ hybridization studies [143]. To resolve this question, preparations of malignant epithelial cells without lymphocytic infiltration were made by passaging the tumors in nude mice [144]. These animals have thymic aplasia and accept grafts of foreign tumors. Human carcinomas grown in nude mice maintain their original histological appearances and genetic markers, but the stroma and infiltrating cells are replaced by mouse stroma [144,145]. Three nude-mouse-passaged carcinomas were studied [144]. The proportion of mouse and human cells was estimated by GIc-6-P dehydrogenase enzyme analysis, the number of EBV genome equivalents per cell was determined by hybridization with EBV complementary RNA and the cells that carry EBV genomes were localized with tests for EBV-determined nuclear antigen (EBNA). The nude-mouse-passaged tumors retained their EBV genomes despite the virtual absence of lymphocytic infiltration and EBNA was detected in the large carcinoma cells [144]. Thus, it may be concluded that the EBV genomes found in biopsies from human nasopharyngeai carcinomas are localized to the cancer cells. The demonstrations that EBV is carried in provirus form by the carcinoma cells and that, like Burkitt lymphoma, this disease appears to be a malignant proliferation of a clone carrying EBV-genomes, suggest that the virus may be more than a mere passenger in anaplastic nasopharyngeal carcinoma. If this is the case, since EBV is ubiquitous, why does this malignancy show such striking geographic associations? One possibility is that there are different strains of EBV, one of which is associated with anaplastic nasopharyngeal carcinoma. Using reassociation kinetics, no differences were found between EBV DNA from this malignancy and from Burkitt lymphoma and infectious mononucleosis [117]. However, subtle differences would escape detection with this method. Additional explanations are that genetic susceptibility and/or environmental tumorigenic co-factors are responsible for the different virus-cell interactions. Genetic susceptibility has been documented in nasopharyngeal carcinomas [146], and there are data which suggest that certain HLA phenotypes may be associated with increased risk to this malignancy [147-149].

313 IXF. Metastases

Mosaic systems can be used to determine if multiple tumors in the same subject arise independently of one another or through metastasis. For example, as discussed above, all tumors in a given GIc-6-P dehydrogenase heterozygote with Burkitt lymphoma have the same single-enzyme phenotype, A or B, and all tumors have the same cell-surface immunoglobulin. Thus, the multiple neoplasms ofBurkitt lymphoma result from metastastic spread of the malignant clone. Similar observations have been made in one case of lymphosarcoma [141] and in two patients with reticulum cell sarcoma [11,91]. Conversely, both B and A type uterine leiomyomas are frequently found in the same subject, indicating that each of the tumors has an independent origin [150,151]. If a carcinoma has multicellular origin, the GIc-6-P dehydrogenase system can be used to determine the number of cells from which metastases arise. Assuming that the afore-discussed patient with carcinoma of the colon had one malignancy with multicellular origin rather than two primary malignancies, the fact that 21 of 28 metastastic liver nodules had essentially single-enzyme phenotypes suggests that the metastases originated from one or a small number of primary tumor cells.

X. LEIOMYOMAS OF THE UTERUS The first application of the GIc-6-P dehydrogenase system for the study of tumorigenesis was made to these benign neoplasms of the uterine wall [10]. Leiomyomas which are the most common neoplasm in females are composed of muscle and fibrous tissue in varying proportions and are usually multiple. Thus far, tumors from 25 Glc-6-P dehedrogenase heterozygotes have been tested [150-153]. Samples of normal myometrium adjacent to the neoplasms exhibited double-enzyme phenotypes, but 184 of 185 non-necrotic leiomyomas had single-enzyme phenotypes. The one exception had considerable non-tumor cell admixture. Three or more leiomyomas were studied from each of 17 Glc-6-P dehydrogenase heterozygotes and in all but one case, some were of type B and others were of type A [154]. Thus, multiple tumors in the same patient arise independently of one another and not by metastasis. Explanations for the single-enzyme phenotypes other than clonal origin are not likely to be correct. For example, the patch size for normal adult myometrium is about 10000 cells [13] and assuming this size or even one larger by an order of magnitude, the chance that all 184 leiomyomas with single-enzyme phenotype arose from two adjacent cells which by chance had the same GIc-6-P dehydrogenase phenotype is less than 0.00l. The probability for origin from more than two cells is still lower. The ratio of B to A tumors is close to 1 : 1 (55 ~oB), indicating that selection on the basis of GIc-6-P dehydrogenase type alone does not account for the single-enzyme phenotypes. Furthermore, all but one of 17 patients from whom more than two tumors were studied had some type A, and some type B neoplasms. This

314 fact and the failure to detect mixed cell populations in small (presumably young) tumors or in the cores of larger ones [150] make the possibility that the single-enzyme phenotypes in leiomyomas are due to selective overgrowth during tumor development unlikely. Thus, the single-enzyme phenotypes in leiomyomas almost certainly reflect their single cell origin.

XI. PARTHOGENIC ORIGIN OF OVARIAN TERATOMAS Cystic teratomas of the ovary are benign neoplasms lined by skin epithelium with its associated underlying structures such as sebaceous glands and hair shafts. In addition to these ectodermal elements, structures such as cartilage and bone from other germ layers are usually found. On this basis, the neoplasm was presumed to be derived from "parthogenic" development of a totipotential ovum [155]. If the teratoma arises from a germ cell after it has undergone the first meiotic cell division, a tumor from a heterozygote might express only one allele as a consequence of segregation [153]. For example, in a patient heterozygous for A and B at an hypothetical X-linked or autosomal locus, one daughter cell would be A and the other, B after the first meiotic cell division (if no crossover occurred between the locus and the centromere). Thus, the normal tissues would type as A/B and the teratoma, as AorB. Thirty-nine cystic teratomas of the ovary from 33 females were studied for markers at three autosomal loci, phosphoglucomutase-I, 3, and 6-phosphogluconate dehydrogenase. Homozygous phenotypes (i.e., single gene expressions) were detected in 6 of 12 tumors from patients heterozygous at the phosphoglucomutase-1 locus, in 7 of 11 tumors from phosphoglucomutase-3 heterozygotes and in 4 of 5 tumors from females heterozygous at the 6-phosphogluconate dehydrogenase locus [153,156]. Thus, it is very likely that the cystic ovarian teratoma arises from a germ cell after the first meiotic cell division. These conclusions are strongly supported by studies with chromosome markers in five patients with teratomas [157]. The normal tissues were heterozygous for 17 chromosomal polymorphisms detected with banding techniques. In contrast, the teratomas were uniformly homozygous. Presumably, no heterozygous chromosomal phenotypes were found because, in contrast to the isoenzyme markers, the morphologic markers are so close to the centromere that crossing over is unlikely to occur between them and the centromere. The isoenzyme marker loci are farther away from the centromere. Since each tumor phenotype is uniform and is non-mosaic, it arises from a single cell. In summary, the isoenzyme and chromosomal markers indicate that cystic ovarian teratomas have a parthogenic origin from a single germ cell after the first meiotic cell division. In contrast, extragonadal teratomas do not manifest single gene expression indicating their origin from somatic cells or from germ cells that fail to undergo meiosis and procede directly to mitosis [158].

315 XII. ATHEROSCLEROSIS Atherosclerosis, the leading cause of death in the United States, is a progressive disease of medium- and large-sized arteries leading to erosion of vessel walls and focal obstructions of blood flow. The characteristic lesion, the atherosclerotic plaque, is initially a focal thickening of the inner layer (intima) of the artery wall and is composed primarily of collagen and smooth muscle cells. Later, cell degeneration and lipid deposition occur in the center of the plaque, and, in advanced stages, it loses its fibrous cap and becomes ulcerated. The causes of the collagen deposition, smooth muscle cell proliferation and fatty degeneration are unknown. Virchow suggested in 1856 [159] that injury of the intima plays a central role and recently this possibility has received much attention (e.g., refs. 160 and 161). An alternative to injury-repair hypotheses is that plaque formation results from proliferation of smooth muscle cells transformed by a somatic mutation [162]. The somatic mutation hypothesis predicts clonal origin of plaques and to test it, atherosclerotic plaques removed at autopsy from Glc-6-P dehydrogenase heterozygotes were studied [162]. The plaques showed single- or predominately singleenzyme phenotypes [162,163] and the findings were interpreted to suggest that plaques arise from a clone of mutant transformed cells [162]. Alternate possibilities to explain the single-enzyme phenotypes are discussed in Section liE and include origin of the plaque from a patch of cells with like GIc-6-P dehydrogenase phenotype and evolution of single enzyme phenotypes through selection or repetitive sampling as may occur if plaque formation develops after repeated cycles of cell proliferation. If the plaques are clonal, the possibility that not all clonal growths result from mutations or other tumorigenic-like events must also be considered. For example, it has been postulated that clonal senescence of smooth muscle cells in the artery could release progenitors of atypical smooth muscle cells in the intima from a hypothetical feedback inhibition thereby resulting in cell proliferation and plaque formation [164]. Thus, there are explanations for the single-enzyme phenotypes other than clonal origin and explanations for clonal proliferation other than neoplasia. These possibilities are now being investigated. In any event, the suggestion that plaques are tumor-like growths is stimulating and deserving of further study.

XllI. CONCLUDING REMARKS Studies of tumors from patients with mosaicism provide important clues to the nature of the cells from which the neoplasms arise and to their modes of origin and progression. Malay hematopoietic neoplasms involve stem cells despite the fact that the predominant manifestations are over-abundances of differentiated cells (e.g., chronic myelocytic leukemia, multiple myeloma, etc.). Ovarian teratomas have a parthogenic origin from germ cells after the first meiotic cell division. Demonstration of clonal origin of disorders such as myelofibrosis, paroxysmal nocturnal hemo-

316 globinuria and idiopathic chronic cold agglutinin disease strengthens the hypotheses that they are neoplasms. Most tumors appear to have clonal origin and this is compatible with somatic mutation theories of carcinogenesis. However, some benign hereditary and viral "tumors", one reported case of carcinoma of the colon and perhaps a few invasive carcinomas of the cervix may be exceptions. The clonal origin of chronic leukemias (myelocytic and lymphocytic) excludes the hypothesis that these neoplasms result from continuous recruitment of normal cells. It is not known yet whether untreated acute lymphoblastic leukemia is clonal, but under very exceptional circumstances hitherto normal cells can be recruited to form leukemia recurrences. Burkitt lymphoma, the malignancy in man for which there is much circumstantial evidence of a viral cause, has clonal origin. Thus, either the viral-induced oncogenic change is a rare event such as an unusual chromosomal rearrangement, or the virus is but one of several factors that must be present in a cell to allow its malignant outgrowth or the virus alters many cells initially, but only a rare clone is able to escape surveillance mechanisms. The contrast between clonal origin of this putative viral malignancy and multicellular origin of the benign viral "tumor", venereal wart, suggests that virus-infected cells give rise to a malignant clone only if one or more additional events occur. Of particular interest is the demonstration that early relapses after remissions of Burkitt lymphoma represent re-emergences of the original malignant cell lines, whereas some late recurrences are the result of outgrowth of malignant cell lines which differ from the originally detected clones. One possibility is that these late recurrences result from new inductions of disease. It is important to know how frequently this occurs in other lymphomas. The fact that even the late recurrences which may result from new induction of disease are clonal indicates that continuous recruitment of normal cells to the malignancy does not occur in Burkitt lymphoma. Thyroid adenomas presumably arise under the influence of thyrotropin, yet these growths have clonal origin. Thus, they may be true neoplasms rather than endocrine-influenced growth disturbances, but this does not preclude their development being dependent upon thyrotropin. It is possible that only a rare cell is susceptible to thyrotropin or that the role of the hormone is to promote proliferation of a cell in which an oncogenic change has already been initiated. Unfortunately, genetic marker approaches to the study of human tumorigenesis are limited in most instances to neoplasms that synthesize immunoglobulin or arise in GIc-6-P dehydrogenase heterozygotes. When other suitable X-linked markers are discovered, many more tumors can be investigated. Of particular interest would be neoplasms for which at least one pathogenetic factor is known. Examples are the lympho-reticular tumors that arise in patients with genetic or exogenously-induced immunodeficiency, radiation-associated malignancies, bladder carcinomas in patients exposed to aniline dyes, vaginal carcinomas in young women whose mothers ingested estrogens, and tumors associated with inherited gene mutations (e.g., multiple polyposis of the colon, xeroderma pigmentosa, multiple endocrine adenomatosis,

317 retinoblastoma), etc. Hopefully, such studies will lead to better u n d e r s t a n d i n g of the factors that initiate a n d p r o m o t e h u m a n tumorigenesis and, ultimately, to more effective preventive a n d therapeutic measures.

ACKNOWLEDGEMENTS I a m grateful to the m a n y colleagues w i t h o u t whose help the studies done in my l a b o r a t o r y could n o t have been accomplished. These studies were supported by G r a n t N u m b e r s G M 15253 a n d C A 16448 from the N a t i o n a l Institutes of Health, D H E W , a n d by designated research funds of the Veterans A d m i n i s t r a t i o n .

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