(CANCER RESEARCH 36, 3554-3560, October 1976]

(14). All fish bearing the naturally occurring tumor havecutaneous or oral lesions as the 1st manifestation. Laboratory experiments have demonstrated that unaffected pikedevelop cutaneous lymphoma when kept in the same aquarium with lymphoma-bearing pike (6, 14). The tumors areeasily transplanted from pike to pike by i.c.,3 i.m., or i.p.inoculations of viable lymphoma cells (6, 14).

While it is clear from present evidence that the esocidlymphomas are transmissible, it has not yet been determined whether fish in the feral state contract the disease asa result of transfer of cells, transfer of an oncogenic agent,or perhaps transfer of both cells and a closely cell-associated agent. In an attempt to resolve this question, cytogenetic studies of the pike lymphoma were undertaken. Byanalogy with the transmissible venereal tumor of dogs (5, 8,15), transmission by cell grafting would be reflected byoccurrence of a single constant highly aneuploid karyotypeof the tumor cells, regardless of the geographic location ofthe host. On the other hand, chromosomal anomalies oflesser degree and of more variable nature would be moreconsistent with induction of the disease by some agent suchas an oncogenic virus. The use of sex chromosomes asmarkers was precluded by the fact that these are not identifiable in pike by currently available methods.

MATERIALS AND METHODS

Specimen Collection

SUMMARY

Chromosome analyses were performed on tumor cellsand normal hematopoietic cells of northern pike bearingmalignant lymphomas. The normal pike karyotype contains50 acrocentric chromosomes, among which the sex chromosomes are not identifiable by available techniques. Metaphases from 19 primary lymphomas showed a consistentpattern of chromosome anomalies consisting of 1 submetacentric marker, 1 minute marker, and 3 to 5 pairs of smallerthan-normal chromosomes, set within a mode of 50 and arange of 46 to 54. Occasional cells had 2 identical submetacentric markers. Cells from presumptively normal renal hematopoietic tissue in 41 lymphoma-bearing pike showed theeuploid karyotype, with the exception of 2 cases in whichsome cells with the neoplastic pattern were found. Thisprobably indicated occult neoplastic involvement of renalhematopoietic tissue, in spite of the absence of histologicalevidence of involvement. In many cases, some of the cellstaken directly from lymphomas bore the normal karyotype.It is not known whether these cells were reactive lymphoidcells or lymphoma cells that had not developed those anomalies typically associated with the tumors. The findings donot permit a conclusion regarding agent versus cellulartransmission. However, the pike lymphoma is an excellentexample of a neoplasm with a karyotypic― signature.―

INTRODUCTION

Normal and lymphoma-bearing pike were captured during the spring spawning run (water temperature about 7°)by means of a weir operated at the entrance of a spawningmarsh near Gaylord, Mich. They were anesthetized in MS222 (Sandoz Pharmaceuticals, Hanover, N. J.) to permitcareful examination and identification of Iymphoma-bearing specimens. Scales used for age determination weretaken from each fish, and the length and sex were recorded.Pike with active cutaneous lesions were photographed (Fig.1). Those fish selected for study were exsanguinated byseverance of the dorsal aorta. Peripheral blood smears andserum samples were collected from these bleedings.

Preparations for Cytogenetic Analysis

Tumor Cells by Direct Air-dried Technique. Cutaneouslymphoma lesions were scraped with a sterile scalpel andthe scrapings were suspended in Eagle's basal medium with

3 The abbreviation used is: ic. , intracutaneous.

Northern pike (Esox lucius) and muskellunge (Esox masquinongy) are 2 closely related fishes of the genus Esoxsubject to epizootics of malignant lymphoma (14). In NorthAmerica, the lymphomas in pike are geographically widespread, having been recognized from Alaska through Canada to New York State. The disease has also been found inabundance in pike in Ireland (9) and in the Baltic sea alongthe coasts of Sweden (6). The muskellunge is a speciesnative to North America and its distribution is limited toCanada and the United States. Lymphoma has been foundin muskellunge only in certain waters in Ontario, Canada(13).

Field studies of both the pike and the muskellunge lymphoma strongly suggest horizontal transmission of the disease by contact or proximity during the act of spawning

I This work was supported in part by grants from the National Cancer

Institute of Canada, the National Sportsmen's Show, Environment Canada,the Anna Fuller Fund, and the Ontario Ministry of Natural Resources.

2 Research Scholar, National Cancer Institute of Canada.

Received April 19, 1976; accepted June 16, 1976.

3554 CANCERRESEARCHVOL. 36

Chromosomal Characteristics of Malignant Lymphoma in NorthernPike (Esox lucius) from the United States1

Jacqueline Whang-Peng, Ron A. Sonstegard,2 and Clyde Dawe

Cytogenetic Oncology, Medicine Branch, Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20014 (J. W-P., C. DI, and NationalCancer Institute of Canada, Department of Microbiology, University of Guelph, Guelph, Ontario, Canada (R. A. S.]

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Chromosomal Characteristics of Pike Lymphoma

Hanks' balancedsalts to which Colcemid had beenaddedata concentration of 0.05 @.tg/ml.The suspension was centrifuged for 5 mm at 1000rpm. The pellet was resuspendedin1% sodium citrate for 30 mm at pH 7.2 and temperature of20°.It was then contaminated with modified Carnoy's fixative(ethylalcohol:acetic acid, 3:1)and centrifuged for5 mmat 1000 rpm. The pellet was resuspended in fresh fixativealone and held at 4°overnight. The fixed suspension wasthen centrifuged for 5 mm at 1000rpm, and the pellet wasagain resuspended in fresh fixative. Two drops of cell suspension were placed on each freshly cleaned slide, and thechromosomes were spread by blowing on the slides. Somewere stained with standard Giemsa stain and others by thetrypsmn-Giemsa banding method.

Normal Hematopoietic Cells and Tumor Cells from Kidney, after Overnight Culture. Anterior kidney tissue wasaseptically excised and placed in a sterile glass tissue homogenizer, and Parker's Medium 199 supplemented with30% fetal calf serum, phytohemagglutinin M, streptomycin(50 j.tg/mI), and penicillin G (50 units/mI) were added. Thetissue was then gently homogenized and the cell suspension was transferred to a sterile centrifuge tube. The suspension was centrifuged at 1000 rpm for 10 mm and thepellet was then resuspended in the above-described medium to give a cell density of about 10@cells/mI. This suspension was incubated at 20°overnight and then treatedwith 0.05 @gof colchicine per ml for 1 hr. Air-dried spreadswere then prepared as described above. The trypsmn-Giemsabanding method of Seabright (12)was used.

Chromosome Analyses

In each sample, 30 to 50 metaphase figures were analyzed. They were scored as normal when they metthe critena proposed by McGregor (7). The normal karyotype of thenorthern pike contains 50 chromosomes, all acrocentric.

PathologicalAnatomy

All pike from which normal or lymphoma tissue was obtamed were necropsied and samples of the cutaneous lesion from those with lymphoma, kidney, liver, and spleenwere fixed in Bouin's fixative, dehydrated , embedded inparaffin, sectioned, and stained with hematoxylin andeosin. The presence and extent of spread of lymphomawere determined by light microscopic examination of thesetissues. Wright's stained blood smears were examined todetermine peripheral blood involvement. Routinely, tumor-bearing pike in field studies were divided into 4 categories according to the extent of spread of the lymphoma,as follows:

Stage I. Lymphoma is confined to skin and does notpenetrate beyond the compact layer of the dermis (Fig. 2).

Stage II. Lymphoma penetrates all layers of skin andinvades underlying muscle by direct extension (Fig. 3).

Stage Ill. In addition to features of Stage II, there isinvolvement of kidney, spleen or liver.

Stage IV. In addition to features of Stage Ill, there isdemonstrable involvement of peripheral blood.

By gross and histological criteria, all of the lymphomabearing pike in this study proved to belong in Stages I and II.Exclusion of fish in Stages Ill and IV was probably effectedby the procedure of taking fish from the spawning run,which would exclude weaker fish in the terminal stage ofdisease.

RESULTS

Seventy-eight pike with lymphomas were entered into thestudy. Of these, 29 (36%) failed to yield useful preparationsfrom either tumor or kidney (normal hematopoietic) tissue.Satisfactory preparations were obtained from both the tumor and the kidney in 11 cases (14%), from the tumor alonein 8 cases (10%), and from the kidney alone in 30 cases(40%).

Data from the 11 pike in which both the tumor and thekidney were analyzed are presented in Table 1. All cellsobtained from kidney showed only the normal karyotype(Fig. 4N). In the tumors, chromosome number ranged from42 to 68, although most cells fell within a range of 47 to 52.The modal number for tumor cells was 50.

Many cells from all Stage I tumors contained 1 submetacentric chromosome with a heteropyknotic region at themiddle ofthe long arm. In looking fora mechanism of originof this submetacentric chromosome, we were handicappedby the lack of banding data for the pike karyotype. Therefore, length was the main criterion for arrangement of chromosomes in this work. Pair 12 showed a heteropyknoticregion, and since the long arm of the submetacentricmarker had a similar heteropyknotic region, we have presumed that this marker may be derived from a translocationbetween chromosomes 12 and 15. Besides the single abnormal submetacentric chromosome, tumor cells also had1 tiny minute marker chromosome. In addition, 3 to 5 chromosome pairs were smaller than those of normal karyotype(Fig. 4T).

In 2 of the 5 Stage II tumors, some of the cells contained 2identical submetacentric chromosomes (Fig. 5). Up to 15%of the aneuploid mitoses were in the tetraploid region. Inboth Stage I and II tumors, some cells had the normalkaryotype. Varying from case to case, 5 to 65% of the cellsfrom Stage I tumors were of normal karyotype. In Stage IItumors, however, no more than 3 to 4% of the cells had thenormal karyotype.

Table 2 lists data from the 8 pike in which only the tumorcells were satisfactory for analysis. Again, all the tumorswere found to have many cells with 1 submetracentricmarker, 1 minute marker, and 3 to 5 pairs of unusually smallacrocentrics. The chromosome number again ranged from42 to 54 with a mode of 50; up to 25% of the aneuploid cellswere in the tetraploid region, regardless of tumor stage.Two tumors in Stage I contained some cells of normalkaryotype, while no normal metaphases were seen in theremaining 3 examples in Stage I or in any of the 3 examplesin Stage II.

Table 3 presents data from the 30 lymphoma-bearing fishthat provided satisfactory preparations from the kidneyonly. The karyotype in all the cells examined from the kidneys of 28 fish was normal, regardless of the stage of the

OCTOBER1976 3555

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Chromosome studies of1 1 p1ke yielding successful spreads of both kidney andtumorKidneyTumor

chromosomedistributionN'

(%)Tetra

NFishSexStageN°(%)4246474849 50 51 52 535468ploid(%)8796MI10051010

5515058772FI1004010460408780MI1007140140658782MI10018

39 1810508888MI1001535351508833FI-Il100262612368776MII1001218

32 1866538777MII10015204910248789MII10063621

(6)'@1918 9

(3)(6)308828MII100325

(3)25373408892FII100186

58 660

Chromosome studies based onsuccessful spreads of tumorsonlyFishSexStageChromosome

distributionA'

(%)424445464748

49 50 51525354Tetra ploidN(%)8799

8852885388758891877388048862M

M?MFFMMI

IIIIIIIIII40

340

34 532613

16 44 012 12 34 12

25 255 5 829 9 27 156 34 42 4

21 9 34 (6)b25 0 505

1225

22

6(9)0

50 55

925

05

126

255

9000000

onlyNo.

of fishStageof tu

morChromosome

distributionA―N

(%)48501(8774)

1 (8832)28I

IlorlI2

(8)b(22)98 70100

J. Whang-Peng et al.

Table 1

‘IN, cells with a normal karyotype; A, each cell contained 1 submetacentric, 1 minute marker; and 3 to 5 pairs of

small chromosomesexcept for values in parentheses.1@Numbers in parentheses, cells that had 2 submetacentric chromosomes each.

Table 2

@1SeeTable 1, Footnote a.

b See Table 1 , Footnote b.

lymphoma. The remaining 2 cases were at Stage I. In one ofthese, aneuploidy was present in 2% of the metaphases, inthe other, in 30%. All of the aneuploid cells in the latter casecontained 2 submetacentric marker chromosomes, whereasonly 1 submetacentric and the usual minute and small chromosomeswere seen in the other.

Pathological Observations

The histological features of the various stages of advancement of the muskellunge lymphoma have been described (11) and were found to be virtually identical for thepike lymphoma. In the present study, only the cutaneouslesions underlying muscle, kidney, spleen, and liver wereexamined for purposes of correlating pathological findingswith karyological findings. Two points are noteworthy concerning this correlation. First, the presence of normallymphoid cells within the lymphomatous lesions was nothistologically evident. The lymphoma lesions were ostensibly composed of diffuse, monomorphic masses of lym

Table 3Chromosome studies based on successful spreads from kidney

a See Table 1 , Footnote a.

b See Table 1 , Footnote b.

phoma cells of poorly differentiated type, corresponding tothe hemocytoblastic or immunoblastic level of differentiation. Second, as indicated by the staging of all cases asStages I or II, no involvement of kidney, spleen or liver wasrecognized by histological criteria. Yet, aneuploid cellsbearing marker chromosomes characteristic of the lymphoma were found in 2 cases (Table 3). This indicates thathistological recognition of small numbers of lymphoma

3556 CANCER RESEARCH VOL. 36

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Chromosomal Characteristics of Pike Lymphoma

The results of this study do not exclude either the viralconcept or the cell transplantation concept. The constantchromosomal anomalies observed are, in fact, consistentnot only with these 2 concepts, but also with the possibilitythat there may exist some genetic predisposition to particular chromosomal abnormalities which might occur in response to any of a variety of types of causal agents.

A point of particular interest was the occurrence of somenormal diploid cells in the tumor, at higher frequencies inStage I than in Stage II. This at first suggests that thecharacteristic chromosome anomalies are a relatively latedevelopment in the evolution of the tumors. It also mayfavor the concept of viral etiology over the cell transplantation concept, since a transplanted tumor would not beexpected to cycle back and forth between aneuploidy andeuploidy. However, it cannot be excluded that the normaldiploid cells observed may have been nonneoplastic cellsrepresenting the cellular immune response system. Suchnonneoplastic lymphoid cells have been observed in theresponse to polyoma virus-induced tumors (4).

Were it possible to identify the sex chromosomes in pike,this study would surely have had a less ambiguous result.Nonetheless, it has been shown that the pike lymphoma isone of the few neoplasms known to have a pathognomonickaryotype. When the mode of transmission of this diseasebecomes incontestably clear, the significance of such asignature karyotype will be better understood, and comparable examples perhaps yet to be discovered will be morereadily interpreted.

ACKNOWLEDGMENTS

The authors wish to thank Turid Knutsen and Elaine Lee for valuabletechnical assistance and EleanorTeall for preparation ofthe manuscript. Thephotomicrographs of histological preparations were kindly prepared byRalph L. lsenburg.

REFERENCES

1. Banfield, w. G., Woke, P. A., Mackay, C. M., and Cooper, H. L. MosquitoTransmission of a Reticulum Cell Sarcoma of Hamsters. Science, 148:1239-1240, 1965.

2. Brindley, D. C., and Banfield, W. G. A Contagious Tumor of the Hamster.J. NatI. Cancer Inst., 26: 949-957, 1961.

3. Brown, E. R., Hazdra, J. J., keith, L., Greenspan, I., kwapisski, J. B. G.,and Blamer, P. Frequency of Fish Tumors Found in a Polluted Watershedas Compared to Nonpolluted Canadian Waters. Cancer Res., 33: 189-198,1973.

4. Dawe, C. J., Whang-Peng, J., Morgan, W. D., Hearon, E. C., and knutsen, T. Epithelial Origin of Polyoma Salivary Tumors in Mice: EvidenceBased on Chromosome-Marked Cells. Science, 171: 394—397,1971.

5. Karlson, A. G., and Mann, F. C. The Transmissible Veneral Tumor ofDogs. Observations on Forty Generations of Experimental Transfers.Ann.N.Y.Acad.Sci.,64:1197-1213,1952.

6. Ljungberg, 0. Epizootiological and Experimental Studies of Skin Tumours in Northern Pike (Esox lucius L.) in the Baltic Sea. Progr. Exptl.Tumor Res., 20: 156-165, 1976.

7. McGregor, J. F. The Chromosomes of the Maskinonge (Esox masquinongy). Can. J. Genet. Cytol., 12: 224-229, 1970.

8. Miles, C. P., Moldavannu, G., Miller, D., and Moore, A. ChromosomeAnalysis of Canine Lymphosarcoma: Two Cases Involving Probably Centric Fusion. Am. J. Vet. Res., 31: 784-790, 1970.

9. Mulcahy, M. F. Lymphosarcoma in the Pike, Esox lucius L. (Pisces;Esocidae) in Ireland. Proc. Royal Irish Acad., 63: 103-129, 1963.

10. Mulcahy, M. F., and O'Leary, A. Cell-Free Transmission of Lymphosarcoma in the Northern Pike, Esox lucius L. (Pisces; Esocidae). Experimentia, 26: 891, 1970.

cells among a large population of normal hematopoieticcells is not always possible. Karyotypic analysis appearstobe a more sensitive method to recognize a small componentof lymphoma cells.

DISCUSSION

Normal diploid cells of the pike contain 50 acrocentricchromosomes. Because of the small size of many of thesechromosomes, difficulty was experienced in obtaining goodspreads for analysis. The technical problem was evengreater for application of banding methods such as trypsintreatment, fluorescence, and alkaloid treatment. No consistently satisfactory technique for cytogenetic analysis ofpike cells has yet been devised.

The unique and nearly constant pattern of aneuploidy inthe lymphoma cells, consisting of 1 submetacentric and 1minute marker, plus 3 to 5 pairs of smaller than normalchromosomes, was an especially interesting finding. Suchidentical aneuploidy in tumor cells from different individualfish requires interpretation in the light of the 2 contendingtheories regarding the nature and mechanism of transmission of the disease. According to 1 theory, the diseasemight be transferred by grafts of lymphoma cells occurringthrough mating activities. Supporting this theory are experimental observations showing (a) that the tumor can betransferred by rubbing viable tumor cells into abraded skinsurfaces (6, 14) and (b) that the lymphoma can be contracted by normal pike kept in the same aquarium withlymphoma-bearing pike (6, 14). The venereally transmissible canine tumor is an example of a neoplasm that apparently can be transferred by cellular graft. A constant karyotype has been found in the canine tumor, whether transplanted experimentally, or whether incurred through coitusor biting (5, 8, 15). Another example of a cellularly transmissible tumor is a reticulum cell sarcoma of the Syrian hamster, shown to be transferred by feeding the fresh tumortissue (2) or by intermediation of a mosquito as vector (1).

A 2nd theory would consider the pike lymphoma to beinduced by some oncogenic agent, such as a virus, thatregularly produced identical chromosomal rearrangementsand identical marker chromosomes. Although not proved tobe of viral etiology, human chronic myelogenous leukemiaexhibiting the Ph1 chromosome is an example of a neoplasm with a pathognomonic chromosomal anomaly. Inblastic crisis, the Ph' chromosome may be reduplicated,resulting in 2 Ph' chromosomes or occasionally even 3/cell.This phenomenon could be compared with the occurrenceof 2 submetacentrics, or even of 3 (6), in some of the pikelymphomas. Evidence in support of a viral etiology of thepikelymphoma has been reportedby Mulcahyand O'Leary(10), Sonstegard (14), and Brown et al. (3), all of whomfound it possible to induce lymphoma in the pike by injection of cell-free filtrates. On the other hand, Ljungberg (6)was unable to induce the lymphoma with cell-free filtratesof lymphoma of the pike from the Baltic Sea. Until a virus isisolated from the pike lymphoma and is shown to be able toinduce the disease, a viral etiology must remain presumptive.

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11. Mulcahy, M. F., Winqvist, G., and Dawe, C. J. The Neoplastic Cell Type inLymphoreticular Neoplasms of the Northern Pike, Esox lucius L. CancerRoe., 30: 2712-2717, 1970.

12. Seabright, M. A Rapid Banding Technique for Human Chromosomes.Lancet,2: 971-972, 1971.

13. Sonstegard. R. Lymphosarcoma in Muskellunge (Esox masquinongy).In: W. E. Ribelin and G. Migaki (eds.), Pathology of Fishes, pp. 902-924.

Madison, Wis.: University of Wisconsin Press, 1975.14. Sonstegard, R. A. Studies ofthe Etiology and Epizootiology of Lympho

sarcoma in Esox (Esox lucius L. and Esox masquinongy). Progr. Exptl.Tumor Res., 20: 141-155, 1976.

15. Weber, W. T. , Nowell, P., and Hare, W. Chromosome Studies of aTransplanted and a Primary Canine Venereal Sarcoma. J. NatI. CancerInst., 35: 537-547, 1965.

Fig. 1. Gross appearance of a primary cutaneous lymphoma on the caudal peduncle of a feral northern pike. Scale pockets are distended with tumor andthe central part of the lesion is ulcerated.

Fig. 2. Histological section of a Stage I lymphoma. Lymphoma cells (see Fig. 1) penetrate partway through the compact layer of the dermis, but not to themuscle (see Fig. 3). H & E, x 65.

Fig. 3. Histological section of a Stage II lymphoma. Lymphoma cells have penetrated all levels of the dermis and are infiltrating trunk muscles. H & E. x 65.Fig. 4. N. normal pike karyotype from kidney, which contains 50 acrocentric chromosomes; no identifiable sex chromosome was seen. T, karyotype of a

Stage I tumor cell with 50 chromosomes, containing 1 submetacentric chromosome (Sub) with a heteropyknotic region at the middle of the long arm. Thismarker presumably is derived from translocation between chromosomes 12 and 15. This cell also had 1 tiny minute marker (Mi), and 3 to 5 smaller pairs ofchromosomes.

Fig. 5. Two karyotypes (A and B) from Stage II tumor. They had 53 and 52 chromosomes, respectively. Both had 2 identical submetacentric markers.

3558 CANCERRESEARCHVOL. 36

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3559

Chromosomal Characteristics of Pike Lymphoma

OCTOBER1976

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3560 CANCERRESEARCHVOL.36

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1976;36:3554-3560. Cancer Res   Jacqueline Whang-Peng, Ron A. Sonstegard and Clyde Dawe 

) from the United StatesEsox luciusNorthern Pike (Chromosomal Characteristics of Malignant Lymphoma in

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