neoplastic sequelae of allogenic disease. i. theoretical considerations and experimental design

NEOPLASTIC SEQUELAE OF ALLOGENIC DISEASE. I. THEORETICAL CONSIDERATIONS AND

EXPERIMENTAL DESIGN*

Robert Schwartz, Janine Andr&-Schwartz, Martine Y. K. Armstrong and Lorraine Beldotti

Blood Research Laboratory, New England Medical Center Hospital, the Department of Medicine, Tufts University School

of Medicine and the Department of Bacteriology and Immunology, Harvard Medical School, Boston, Mass.

I t is the purpose of this paper to describe a newly defined sequel of the graft-versus-host reaction (GVHR) , the development of malignant lymphoma. This account is a progress report of our studies of this phenomenon; detailed descriptions of various phases of these experiments are in preparati0n.t

Theoretical Background

The aberrations of immunity so commonly encountered in malignant lymphoproliferative diseases' may represent an effect of the disease, the cause of the neoplasia, or a cellular process fundamental to both neoplasia and immunity. Whatever their significance might be, the elucidation of these disturbances has stimulated considerable interest in immunologic theories of neoplasia. One of these theories2 proposes that repetitive exposure of lymphoid cells to antigens could result in a proliferative response that culmi- nates in neoplasia. This concept is supported by the finding that repeated injections of antigens were associated with an increased incidence of reticulum cell sarcomas and plasma cell tumors in mice.3 I t is also sustained by the observation that some animals with Aleutian mink disease, a viral infection characterized by extreme plasmocytosis and hyperglobulinemia, develop a condition resembling multiple myeloma. The development of lymphoid neoplasms after protracted autoimmune disease in NZB may also be consistent with this theory.

Tyler' has proposed that neoplasms of lymphoid tissue could develop as the result of a mutational loss of a histocompatibility antigen by an immunologically competent cell. Normal cells with a full complement of histocompatibility antigens would then Serve as antigenic, and, hence, proliferative stimuli, for the abnormal, immunologically competent cell. This could result in a constant unrestrained growth of these cells. The loss of normal cell antigens from many types of neoplasmsg is consistent with this hypothesis.

4

'This investigation aided by grants from the U. S. Public Health Service numbers R01 AM 02037-07 and AM 07937-03 from the National Institute of Arthritis and Metabolic Diseases.

t A preliminary description of this work has been published (Schwartz, R. and Beldotti, L.: Malignant lymphomas following allogenic disease: transition from immunological to neoplastic disorders. Science, 1 4 9 1511, 1965).

804

Schwartz et al.: Neoplastic Sequelae 805

Tyler also proposed that the GVHR produced in inbred mice by the administration of parental lymphoid cells to their F1 hybrid might be a useful model for studying the pathogenesis of neoplasia. The transplanted parental cells (which may be designated as “A”) lack one or more of the histocompatibility antigens present in the F1 hybrid host (which may be designated as “AB”). The F1 recipient cannot react against parental cells,** but immunologically competent parental cells react against the hybrid.” The grafted cells are thus analogous to the hypothetical antigen-deleted, immunologically competent cell of lymphoid neoplasms.

Lymphoid Neoplasms in Allogenic Disease Against this theoretical background, and having in mind the clinical ex-

perience that certain autoimmune diseases are associated with a high incidence of lymphoma and leukemia,” we initiated the following experiments dealing with the relationships between immunologic and neoplastic disorders. The experimental model was a form of allogenic disease characterized by a prolonged course. The indolent nature of this GVHR must be stressed; acute or subacute forms of runting produced by injecting strongly histoincompatible cells into newborn or heavily x-irradiated recipients are too quickly lethal to allow time for the development of neoplasia. Serial histologic studies of the model to be described have demonstrated that, even if the mice appeared clinically normal, their lymphoid tissues were the sites of extensive, active, and continuing changes. Parenthetically, we believe that the absence of thymic atrophy, which is notable in this donor-recipient combination, may have a considerable bearing on later interpretations of the neoplastic sequelae.

Allogenic disease was induced in six-week-old, (C57BL/6 x DBA/P)F1 mice by four intraperitoneal injections of approximately 80 x lo6 C57BL/6 spleen cells. Two groups of BDFl recipients were used: (1) 65 mice that received 320 x lo6 C57BL/6 spleen cells and (2) 50 mice that received the same dose of spleen cells followed by five intraperitoneal injections of amethopterin, 3 mg/ kg. This treatment was undertaken because it reduces the mortality due to allogenic disease to approximately 30 percent. The mortality in the group of recipients not treated with amethopterin was 70 percent. These two groups of mice, together with six control groups, are listed in TABLE 1. In the system we employed, all deaths due to runt disease occurred during the first 150 days. The surviving mice were then maintained under standard laboratory conditions. The tissues of all groups of mice were examined grossly and micro- scopically a t the times indicated in TABLE 1. Approximately 70 percent of the untreated mice and 40 percent of the mice given methotrexate died of overt allogenic disease. One-half of the surviving mice developed transient, mildto severe, runting, usually during the first 50 to 60 days of the experiment. Thereafter, their appearance was normal except for those mice developing large tumor masses.

**It should be noted, however, that an unexpected and as yet poorly understood F1-versus-parent reaction can be elicited under certain experimental conditions.”

13

806 Annals New Y ork Academy of Sciences

0

+

TABLE 1 INCIDENCE OF MALIGNANT LYMPHOMAS IN EXPERIMENTAL

A N D CONTROL MICE

8 7/18

12 14/29

Strain

BDF

BDF

BDF

BDF

BDF

BDFl

C57BL/6

10

10

10

Parental Cells Administered

0/36

0/12

0/12

Spleen Spleen None

None Frozen-thawed spleen

Liver 0

0

*Number of mice with tumors/

12 0/12 10 0/24

Mean Age Malignant* hethopterin I (months) I Lymphomas

0

-k

0

In a subsequent experiment, a small group of BDFl recipients was given a single intraperitoneal injection of 9 x lo6 C57BL/6 spleen cells when ten days old. After 11 months, one of these mice has developed a lymphoid neoplasm.

The incidence of tumors for all groups of mice is shown in TABLE 1. Only those mice which received living parental spleen cells developed neoplasms. All tumors originated in lymphoid tissue. Except for one instance, the thymus was not affected and was either normal or slightly atrophied. Liver meta- stases were common and invasion of the kidney and lung was occasionally seen. Histologically, the tumor usually consisted of reticulum cells, histiocytes, lymphocytes and plasma cells. Multinucleated cells with a close resemblance to Reed Sternberg cells were occasionally seen. Eosinophils were abundant in some tumors, absent in others. The overall histologic picture in most mice was that of a granulomatous neoplastic profileration resembling Hodgkin’s disease in some areas and reticulum cell sarcoma in others.

Studies of Transplantable Tumors Arising during Allogenic Disease Several tumors developing in runted animals have been transplanted into

C57BL/6, DBA/2, and B D F L mice. Newborn, suckling and adult recipients were used. Intraperitoneal injections of tumor cell suspensions and sub- cutaneous implantations of tumor fragments have been employed. Thus far, the tumors have taken only in BDFl recipients. This indicates t ha t they are of host rather than of donor origin, a somewhat surprising discovery, if the theoretical considerations discussed previously are valid. However, many more experiments of this type are required before any definite conclusions can be drawn.

Two of these neoplasms have been carried in BDFl mice through approximately ten passages and possess certain interesting features:


Tumor 603. This neoplasm arose in a BDFl mouse nine months after it received 320 x lo6 spleen cells a t the age of six weeks. I t resembled Dunn’s Type B reticulum cell sarcoma,14 and involved the liver, spleen and lymph nodes. The thymus of this animal was normal. Cell suspensions of the neoplasm were injected into BDFl mice and about one-third of the recipients had a generalized lymphoma, including a thymoma, within four months. With succeeding passages, the tumor has become more virulent, and by its fifth passage it was 100 percent lethal within four to six weeks after injection into one month old BDFl mice. During its fourth passage, the tumor abruptly transformed from a granulomatous sarcoma, resembling Hodgkin’s disease, to a neoplasm consisting of sheets of primitive blast cells. A leukemic blood picture is common; extreme hypogammaglobulinemia or hypergammaglobu- linemia may occur. There is severe anemia and the antiglobulin test is negative. When thymoma cells are transplanted, the recipients develop a slowly growing, generalized neoplasm, including a massive thymoma. When lymph node or spleen cells are transplanted, the recipients rapidly develop a neoplasm characterized by generalized lymphadenopathy and splenomegaly without histologic evidence of a thymoma. Since the chromosomal abnormalities found in the “thymoma” variant and in the “lymph node” variant of this line of tumor differ (see below), we suspect that the original runted mouse was actually harboring two different, mutant, neoplastic cells.

Tumor 741. This neoplasm arose in a BDFl mouse eleven months after it received 9 x lo6 C57BL/6 spleen cells a t the age of ten days. The tumor involved mainly the liver- and consisted of nodules of primitive reticulum cells. These areas of tumor frequently formed hemorrhagic lakes surrounded by a collar of neoplastic cells, somewhat reminiscent of Dunn’s reticulum cell sarcoma, type A. The spleen was enlarged and showed intense extramedul- lary hematopoiesis. Neither tumor nor lymphoid follicles could be found in it. The thymus could not be identified, and serial sections of the mediastinum failed to disclose thymic tissue. The lymph nodes were atrophied and masses of histiocytes, often coalescing into giant cells, replaced the normal archi- tecture. There was severe anemia, reticulocytosis, and profound lymphocy- topenia. We interpreted these changes to indicate two distinct processes: (1) a primitive reticulum cell sarcoma and (2) active allogenic disease. These findings in the original tumor have been precisely reproduced through eight serial passages. Although tumor cells cannot be positively identified in histologic sections of the spleen, they are undoubtedly present since the tumor is readily passed by a suspension of spleen cells. Some of the recipients have a leukemic blood picture, with white blood cell counts as high as 80,000/mm.3 The abnormal cells in the blood and liver are vacuolated and resemble retic- uloplasmocytes. A massive, “monoclonal” gammopathy is sometimes present. Many of the recipients of this tumor develop a characteristic runted appearance within two to three weeks after its inoculation. Serial histologic exam- ination of BDFl recipients of the tumor has disclosed that the thymic and

14

808

lymph node atrophy are not the consequences of widespread malignant disease; atrophy of these organs begins a t least one week before any evidence of tumor growth can be discerned.

We are thus dealing with a neoplasm which arose in a runted animal and which faithfully reproduces the lesions of both runt disease and neoplasm, even after numerous serial passages through isogenic recipients. Our provi- sional interpretation of this phenomenon is that it represents a “functioning” lymphoid tumor with the capacity to induce allogenic disease without the antigenic stimulus required by normal lymphoid cells. Discriminant spleen cell assays, utilizing tumor cells of this line, have been carried out in eight day old BDFl and (C57BL/6 x A/Jax)F, mice. Reactions which could be expected if C57BL/6 cells were present in the tumor (i.e., splenomegaly in the (C57BL/6 x A/Jax)F1 recipients) have not been found. However, reactions suggestive of “anti-self” activity (i.e., splenomegaly in only the BDFl recipients) have occurred. While the results of these experiments require cautious interpretation, they are a t least consistent with the concept of an autoreactive lymphoid neoplasm inducing lesions of runt disease.

Annals New York Academy of Sciences

15

The Possibility of Oncogenic Viruses as Etiologic Agents The possible role of an oncogenic virus in the production of these tumors

must be seriously considered, since murine neoplasms, particularly lymphomas and leukemias, are so commonly associated with these agents. A tumor virus could be implicated by a t least two mechanisms: (1) release or activation of viruses consequent to the extensive and prolonged tissue damage occurring during allogenic disease; and (2) failure of immunologic anti-viral defense mechanisms during the runting process. In the first possibility, allogenic disease might be considered analogous to whole body x-irradiation, which results in the release or activation of latent tumor viruses harbored by the mouse. In the second mechanism, oncogenic viruses might achieve a selec- tive growth advantage in immunologically impaired, runted animals. In either eventuality, allogenic disease would serve as the background, or “trigger” mechanism for a virus-induced neoplasm.

For these reasons, intensive efforts to identify an oncogenic virus in these tumors have been undertaken. These include: (1) administration of cell free extracts and filtrates of neoplastic cells to newborn mice; (2) electron microscopic search of tumor material for virus particles; (3) growth of tumor cells in tissue culture; and (4) serological identification of tumor cell antigens known to be induced by oncogenic viruses. Thus far, none of these approaches has yielded convincing evidence implicating a virus as an etiologic agent in the lymphoma arising after allogenic disease. Nevertheless, because of the importance of this question, these experiments are being continued. I t may be of some interest that efforts to identify a virus in the lymphomas which arise in NZB mice afflicted with chronic autoimmune disease have also failed.6

16

17


Electron Microscopic Studies

Detailed electron microscopic studies have been carried out on the two transplantable tumors, 741 and 603, just described. Tissues were fixed in either buffered osmium tetroxide or 6.5 per cent glutaraldehyde followed by 2 per cent osmium tetroxide. After embedding in Epon 812 and ultrathin sectioning, the tissues were stained with either a one per cent solution of uranyl acetate or lead citrate;” some sections were double-stained. The samples were examined with an RCA EMU-3 microscope (50 kv; objective aperture 50 P I .

Tumor 603. Two types of malignant cells were seen in this neoplasm: lymphocytic and reticular. In some tumors, one type of cell predominated, and in others there was a mixed picture. These cells underwent distinct changes as the tumor was serially transplanted.

The nucleus of the lymphocytic cells had a round or handmirror appearance, and that of the reticulum cells was sometimes folded or oval. In both cells, the nucleocytoplasmic ratio was high. Margination of the chromatin and an overall decrease in the density of the nucleoplasm was seen. In many samples, a granular, very dense material was seen in the interchromatinic zone. Circular, smooth vesicles or nuclear pores, sometimes surrounded by these dense granules, were occasionally seen. The nucleolus was hypertrophied and, in the earlier passages of the tumor, sometimes multiple. The nuclear membrane of some cells had a distinct invagination enclosing a moderately electron-dense homogeneous, oval, granule-like formation, 200-250 mp in diameter. No central nucleoid was seen in it. Dark, more opaque, and somewhat smaller, but similar formations were often seen directly against the nuclear membrane of cells having such invaginations. A cloudy-appearing electron light substance, or dark, fragmented electron dense material was seen in nuclear pores. These latter features were found only in poorly preserved, probably necrotic tissues, and not in well fixed samples.

The cytoplasm of both lymphocytic and reticular cells was packed with evenly distributed ribosomes, which were free, in rosettes, or on ergasto- plasmic lamellae. Many ribosomes were definitely larger than normal. The endoplasmic membranes were irregularly packed with ribosomes. In a few reticular cells, the ergastoplasm was quite abundant and some cells with this featurewere packed together in small clusters of three to five. Each cell of such a cluster had its own, well preserved plasma membrane, and the entire group of cells was bound together by a peripheral membrane or “barrier.”

The Golgi apparatus was well seen, even in the lymphocytic cells. It had packed membranes and very numerous vesicles. Many mitoses were observed, and one or two centrioles were commonly found.

The mitochondria were often normal in number and appearance. In poorly preserved areas, they were swollen, had altered cristae, and were moderately

810 Annals New York Academy of Sciences

electron dense. Even in well-fixed areas, conspicuous particles about 120 to 150

Numerous inclusions were seen in most of the neoplastic cells. They were most abundant in the reticular cells. These included: (1) minute, dense granules, evenly scattered throughout the cytoplasm, arranged in tight or loose clusters and either free or surrounded by a membrane; (2) larger, dense, oval or round isolated granules (“dense bodies”), (3) myelin figures; (4) vacuoles of variable sizes and shapes, usually appearing empty. Some vacuoles had small, dark, round formations a t their edges and within their lumens; others, usually flag or starshaped, contained a moderately electron dense, homogeneous material. In all cells, pinocytosis vacuoles were seen a t the periphery of the cytoplasm. (5) In many cells, particularly those in tumors beyond the foFrth serial passage, moderately electron dense, elongated formations, 3 to 4 A in length, were seen. (6) Some macrophages contained entire, lysed cells or huge fragments of cellular debris, together with large phagosomes and ferritin.

The plasma membranes of these cells were easily seen and regularly showed numerous shallow indentations. Intracellular debris was moderately abundant, as were collagen fibrils.

The lesions seen in these neoplastic cells are not specific, and have been described in other m a l i g n a n ~ i e s . ’ ~ ’ ~ ~ I t is known tha t evaluation of the degree of differentiation of a tumor varies considerably, not only within a given sample of the tumor, but also in its subsequent passages. Nevertheless, i t is our impression, which is supported by light microscopic evaluation of the tumor, that the original growth (i.e., the tumor derived from the runted mouse) resembled Hodgkin’s disease, while the later passages resembled an undifferentiated lymphoreticulosarcoma.

While it is often difficult to interpret certain features because of the poor fixation commonly encounted in neoplastic tissue, several findings seem especially noteworthy: (1) the constant increase in the number of ribosomes and their abnormally large size. It may be of interest tha t Weiss21 found similarly enlarged ribosomes in runted mice and rats. The significance of this is unknown. (2) the finding of dense particles on mitochondria. Nass and NassZ2 have described these formations and i t has been recently suggested tha t they might be “mitochondria1 ribosome^."^^ (3) the presence of nuclear pores inside the nuceloplasm. These were also found by Weiss21 in cells of runted animals; their significance is unknown. (4) the presence of elongated filaments in the c y t ~ p l a s m . ’ ~ ’ ~ ~ (5 ) the observation of lipoid inclusions.

Tumor 741. I n most samples, due to extensive necrosis, the tumor could not be distinguished from the liver tissue. In others, which had well fixed zones, the sheets of malignant cells invaded the spaces between otherwise normal appearing parenchymal cells. Mitotic figures were conspicuous. Three

were seen on some mitochondria.

18

19

13.21.25


types of malignant cells were seen: lymphocytic, reticular and a cell type intermediate between these two (“lymphoreticular” cells) (FIGURE 1).

The lymphocytic cells were of two types: lymphoblasts and more mature lymphocytes. The lymphoblasts were round and contained a circular nucleus. The nuclear chromatin was marginated and the nucleolus was abnormally large. The dark cytoplasm of these lymphoblasts was crowded with free ribosomes, which were often larger than normal. A centrosome was occasionally seen.

The more mature lymphocytes varied in size from small t o large; the latter type was numerous and difficult t o distinguish from a reticulum cell. What- ever their size, these cells presented the same nuclear features as those described in tumor 603, but no invagination of the nuclear membrane was seen. The cytoplasmic features were also quite comparable to what was seen in tumor 603; no elongated formations were seen.

FIGURE 1. Most of the tumor cells observed here are of the reticulum type. Large nucleoli are seen in their nuclei. Note, in their cytoplasm, the prominent Golgi apparatus, the numerous ribosomes and short lamellae of endoplasmic reticulum, the many dense inclusions, the conspicuous vacuoles and lysosomes (L) particularly in the cell of the lymphoreticular type (A). Upper left (C): a portion of the ribosome-packed cytoplasm and of the round nucleus of a “hemocytob1ast”-like cell are visible. Bottom left (B): a small portion of the cytoplasm of a parenchymatous cell of the liver is seen. Original magnification x 4,500, Print x 8,400.


The cells of the reticular type were also similar to those of tumor 603; their cytoplasm showed somewhat less numerous small inclusions but some contained lysed cellular fragments.

The “lymphoreticular” type cells exhibited a clearcut margination of the chromatin in their nuclei. Their cytoplasm showed numerous free ribosomes, some smooth or rough endoplasmic reticulum, normal size mitochrondia, a prominent Golgi apparatus, some dense bodies and tiny pinocytic vesicles. They had, in addition, large, oval formations, usually located a t one pole of the cell; in well-fixed zones of the tissues, they resembled large lysosomes with multivacuolated edges. In necrotic tissues, these formations were either filled by a homogeneous, moderately electron dense material, or appeared empty. Sometimes their contents were seen bulging from the surface of the cell as if being released into the extracellular environment. These cells had numerous microvilli. Vary rare sinusoids, with slightly enlarged lumina, were seen; the cells lining these sinusoids could not be distinguished from the tumor cells. No classical Kupffer cells were observed.

Even though some cellular changes seen in tumor 741 have already been there were, in addition, other intriguing features. First, the

round lymphoblasts, with their ribosome-packed cytoplasm, were reminiscent of the “hemocytoblast” observed during the homograft reaction. The lymphoreticular cells, on the other hand, contained numerous giant formations filled with a substance, the chemical nature of which is unknown. Judging by its electron density, it could be either globulin (which would be of interest since some of these mice have a monoclonal gammopathy) or lipid (since fat storing cells are commonly seen in the liver and since fatty degeneration is a common response of the liver to a variety of injuries). Similar cells have been seen in sarcoma I cells undergoing homograft reje~tion.’~-’~ The Kupffer cells of runted mice also show similar changes,28 although to a lesser degree. The liver sinusoids in runted mice are enlarged and their lumens are often filled with swollen endothelial and Kupffer cells.28 The liver sinusoids in mice with tumor 741 are obliterated by masses of neoplastic cells, most of them of the reticular type, which has some features of Kupffer cells. In view of the origin of tumor 741 in a runted mouse and since this transplantable neoplasm is associated with a syndrome resembling runt disease, these morphologic similarities could be more than coincidental. I t is conceivable that, both in runt disease and tumor 741, the lymphoreticular cells containing large lysosomes have a role in the destruction of the thymus.

Careful search for virus particles in cells of tumors 741 and 603 was negative. However, the inherent difficulties of finding virus particles preclude a final opinion on the relationships between the tumor and such agent^.'^''^''^'^^

Chromosome Studies

The chromosomes of tumors 741 and 603 have been studied in each transplant generation. The findings in the first four generations are now reported.


A total of eleven animals was examined using tissue from one or more tumor sites. Direct chromosomal preparations were obtained using a modifica- tion of the technique described by Ford and H a m e r t ~ n . ~ ' The mice were injected intraperitoneally with colchicine and killed one to two hours later. Small pieces of the tissue to be examined were placed in one or two drops of saline on a glass slide and minced finely with scissors. A satisfactory cell suspension was obtained by squashing the particles with another glass slide. The material was then treated with hypotonic sodium citrate and fixed using an acetic acid-alcohol solution. Finally, four or five drops of the cell suspension were transferred to a slide wetted in 20 per cent ethyl alcohol and momentarily ignited. This ignition technique, based on the method of Kiossoglou, Mitus and D a m e ~ h e k , ~ ~ and used instead of Ford and Hamerton's Feulgen squash method, results in rapid drying and well-spread chromosomes. The prepara- tion was stained with Giemsa and permanently mounted.

Tumor 603. Cytogenetic observations were made on six animals in the first four transplant generations. In all but one animal, two or more tumor sites were sampled, and an average of 50 cells from each mouse was studied. Two distinct transplantation lines of this tumor appear to have been established, one with a modal number of 41 chromosomes and the other with a wide range of variation about a mode of 47 chromosomes, in contrast to the diploid number of 40 found in normal mice. The tumor associated with 41 chromosomes is the rapidly growing variant affecting, principally, lymph nodes and spleen. Although the thymus in these mice is histologically normal, most of the cells from this organ have been in the hyperdiploid range. The tumor associated with 47 chromosomes is the slowly growing variant characterized by a thymoma (TABLE 2 and FIGURES 2 & 3). I t is of interest to note that the animal examined during the first transplant generation showed only four hyperdiploid cells out of a total of 56. Although only six satisfactory mitoses were found in the tumor tissue, two of these had a chromosome count of 51, whereas two of the 50 spleen cells had 41 chromosomes. In the subsequent transplant generations, the great majority of cells examined were in the hyperdiploid range. Two marker chromosomes were seen in a small proportion of the cells in the first two transplant generations but were absent in the third and fourth passages. They consisted of a metacentric chromosome and a chromosome a t least 1.25 times longer than the next.

Tumor 741. This tumor was studied in five animals in the first four transplant generations. The liver tumor was sampled in each case and additional tumor sites were examined in two of the animals. Although mitotic forms were not plentiful and the quality of the metaphase plates was not always good, a minimum of 30 cells was analysed in each mouse. In contrast to the findings in tumor 603, chromosomal changes were not striking, the modal number remaining a t 40 (TABLE 3). There was some variability about the mode in that a small proportion of cells had 39, 41 or 42 chromosomes instead of 40. Hyperdiploid cells are not thought to occur under normal circumstances

Ani

mal

N

umbe

r

603-

1B (50)

603-

2B (61)

603-

3B (4)

603-

3B (5)

603-

4B (4)

Tra

nspl

ant

Gen

erat

ion

1 2 3 3

Tis

sue

1 N

umbe

r of

Cel

ls C

onta

inin

g th

e In

dica

ted

Chr

omos

ome

Cou

nts

Exa

min

ed f

or

Chr

omos

omes

Tum

or

Sple

en T

otal

:

Lym

ph n

ode

Sple

en T

otal

:

Tum

or

Thy

mus

T

otal

:

Tum

or

Thy

mus

L

ymph

nod

e T

otal

:

Thy

mus

Thy

mus

L

ymph

node

Tot

al:

Sple

en

-

< 39

1 1 1 1 2 1 1 2

44 2

-

-

rota

1 :e

lls

-

6 50

56

25

15

40

-

45 46

86

2

15

9

11

13

2

2 35

21

26

22

48

16

25

20

61

-

48 49

22

2

2

3 2 15

3

20

3

83

25

20

20

20

60

-

50 1 1

51 2 2

>51


FIGURE 2. Spleen cell with 41 chromosomes -from mouse bearing tumor 603 (fourth transplant generation) x 1200, original magnification.

FIGURE 3. Tumor cell with 47 chromosomes from mouse bearing tumor 603 (third transplant generation) x 1200, original magnification.


although hypodiploid cells are not uncommon.33 I t seems likely that the majority of the liver cells studied were indeed neoplastic, since few, if any, mitotic forms can be demonstrated in normal liver tissue, using the present technique. A long marker chromosomes was occasionally recorded in each of the four transplant generations.

Primary structural changes were frequently seen in both types of tumors and consisted of gaps, breaks and loss of chromatin, involving one or both chromatids.

Gross chromosomal abnormalities have been described in a variety of mouse tumors, be they primary or transplanted, spontaneous or induced by viral and other oncogenic agent^.^^.^^ The nature of the cytogenetic changes that occur in tumors has been extensively studied in the ascites tumors of rats and I t was shown that most ascites tumors were made up of mixed cell populations that varied in their chromosome content about a mode usually different in number from the normal diploid complement. The finding that this new modal set was perpetuated on transplantation led to the stem-line concept. The implication of this hypothesis is that with the induction of neoplasia a number of different cell types emerge, one or more of which may have growth advantages over the others. This successful, or stem-line, type con- tinues through successive generations; less successful variants that arise are eliminated.

The findings in the present study are consistent with the stem-line hypothesis. In tumor 603, two distinct clones have been established with modal numbers of 41 and 47 respectively, whereas in tumor 741, the mode remains diploid a t 40.

TABLE 3 TUMOR NUMBER 741 TRANSPLANTED FROM A R U N T E D

741-2B (17) 741-3B (49)

741-4B (8) 4

BDFi HOST

Number of Cells Con- Tissue Examined for Chromosomes

taining the Indicated Total Chromosome Counts Cells

Tumor Liver Lymph node

Total:

39 39

3

3

Liver I 1 3 Liver

Liver

Total:

3 4

1 1 2


speculated that chromosomal aberrations might be responsible for the induction of tumors. I t is still not clear whether such defects precede the onset of malignancy or whether they merely represent epiphen~mena.~’ It was decided to study the chromosomes a t different stages of the graft-versus-host reaction, as well as the chromosomes of the GVHR- associated neoplasms to determine whether or not chromosomal changes occur during the immunologic response or only after the malignancy has appeared. A small number of runted animals has so far been examined, and structural abnormalities, consisting principally of gaps and breaks, have been noted. However, until more animals have been studied, it is not possible to comment on the significance of these findings.

39 Over sixty years ago, Boveri

Discussion

Lymphomas and leukemia may arise in a variety of settings which appear to have the disorganization of lymphoid tissue in common. These circumstances include autoimmune disease in men and m i ~ e , ~ . ~ Aleutian mink disease, agammaglobulinemia and allogenic disease. I t is possible that these conditions are related only superficially; each of them could have a unique defect in lymphoid cells. Furthermore, it is also possible that whatever the lymphoid abnormality might be, its role in the pathogenesis of neoplasia is permissive rather than primary. The presence of an oncogenic virus, released from damaged lymphoid cells or free to multiply in an immunologically de- fective individual, is always difficult to disprove. Powerful arguments con- tending that negative results are worthless in the search for oncogenic viruses have been presented by Gross.42 Nevertheless, the significance of such agents in the etiology of a t least certain types of lymphomas seems, a t present, a matter of perspective. For example, viruses could represent the final common pathway to neoplasia which is entered only after completion of a distinctive and necessary event, which could be triggered by x-irradiation or a chemical carcinogen. This point of view is not meant to minimize the importance of virusesin the pathogenesis of malignancy; it is only intended to emphasize the significance of events preceding what might be an inescapable, final episode.

What, then, is the significance of the development of lymphomas in allogenic disease? The GVHR has been proposed as a laboratory model of the hypothetical lymphoid cell abnormality underlying au to imm~niza t ion~~ or, to use Burnet’s exceedingly useful term, the “forbidden clone.”44 While it is technically correct that the GVHR is actually set into motion by isoimmuni- zation, this disorder nevertheless illuminates the consequences of the presence, within a suitable host, of “abnormal,” “forbidden,” or “mutant” immunocytes. Tyler,’ and Walford and Hildemann,45 as well as ourselves,” have proposed that the central feature linking this experimental model to autoim- munity and neoplasia is histoincompatibility between an immunologically competent cell and “host” or “patient” tissues (FIGURE 4). This histoincom-

2

4 41

818 Annals N e w Yolk Academy of Sciences

INITIATING AGENT

NORMAL. CELL

PROLIFERATION

FIGURE 4. A possible mechanism of neoplasia. An immunologically competent cell loses a histocompatibility antigen (black space). Normal cells possessing this antigen act as constant proliferative stimuli t o the deleted cell. Such a continued proliferation could result in a lymphoma. patibility could come about by mutational loss of a tissue antigen from a lymphoid cell. I t might also be the result of the direct implantation of mater- nal immunocytes into the fetus. Such a possibility is supported by the report by Zeulzer et al.46 of a male infant who died of a syndrome having features of both runt disease and leukemia and in whom XX chromosomes were found. Bloom's syndrome,47 a rare disorder of infants characterized by low birth weight, retarded growth, dermatitis, chromosomal aberrations, and an increased incidence of leukemia, might also develop on this basis.

In testing the hypothesis outline in FIGURE 4, Walford and Hildemann45 used coisogenic C3H mice that differed only at a locus, H-1, which controlled a relatively weak histocompatibility antigen. Newborn C3H (HIB) mice were injected with adult C3H (H'") spleen cells. No overt runting occurred, but, after one year, a high incidence of lymphomas was found in the recipients. The control mice of their experiments also developed lymphomas, bu t these did not reach peak incidence until the age of two years. The experiments of Walford and Hildemann, which clearly complement ours, demonstrate that overt allogenic disease is not necessary for the development of neoplasia. While further work is needed in this area, this finding seriously weakens the argument that the lymphomas are due to a secondary effect of runting, such


as infection or impaired immunity. The central feature of their experiment and ours is the presence, over a suitably long time, of histoincompatible lymphoid cells. As TABLE 1 indicates, the administration of immunologically in- competent histoincompatible cells (e.g., parental liver cells) does not give rise to tumors. Thus, the three essential requisites of the “leukemogenic graft” are: (1) histoincompatibility, (2) immunologic competence, and (3) a prolonged residence within the host. The recipient of such a graft must: (1) possess antigens foreign to the graft and (2) be immunologically tolerant of the transplanted cells. The element of overt runt disease appears unnecessary, but it is not presently known if any of the more subtle effects of the GVHR are required. Davies4’ and his associates found a higher incidence of leukemia in heavily x-irradiated recipients of allogenic bone marrow cells than in comparable recipients of isogenic bone marrow cells. Although somewhat compli- cated by the leukemogenic effects of x-irradiation, their data are consistent with our own observations, as well as those of Walford and Hildemann.

Still other studies have indicated a decrease in the incidence of lymphomas and leukemias in heavily x-irradiated recipients of homologous bone marrow. Here again, the effects of x-irradiation are clearly of major importance.

The presently available data do not indicate how neoplasia develops following the transplantation of ostensibly normal histocompatible tissue to a tolerant host. They only suggest that the initial hypothesis may have some validity. The lack of growth of the tumors in the parental strain which served as the spleen cell donor may seriously weaken the idea that the grafted cells are the source of the neoplastic cells. Nevertheless, as pointed out before, many more studies of this type need to be done. For example, preliminary experiments with the combination BALB/C + (BALB/C x A/Jax)FI indicate that some recipients develop plasmocytomas and monoclonal gammopathies, typical of the parental line. Other experiments, utilizing mice having the Ts marker chromosome, should be particularly useful in this respect.

The exploitation of animal models permitting studies of the effects of protracted histocompatibility differences is just beginning. The transplantable lymphoma 741, described above, is a vivid example of the complexities which can result from an encounter between lymphoid cells having different genetic backgrounds. The practical importance of these findings to those attempting the transplantation of allogenic bone marrow or lymphoid tissue remains to be determined. Nevertheless, it might be appropriate to consider them along with other possible hazards of tissue transplantation.

49

A ckno wledgrnent

Miss Lynn Mirtl and Mrs. Lynne K. Kilham gave invaluable technical assistance.


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