PREVALENCE OF NEOPLASIA IN 10 NEW ENGLAND POPULATIONS OF THE SOFT-SHELL CLAM

( M Y A ARENARIA)”

Robert S. Brown,t Richard E. Wolke,? Saul B. Sails,$ and Chris W. Browns

tMarine Pathology Laboratory Department of Animal Pathology

$Department of Oceanography and §Department of Chemistry

University of Rhode Island Kingston. Rhode Island 02881

INTRODUCTION

Neoplasia has been reported infrequently, but consistently, in mollusks since 1887, and the frequency of reports has increased dramatically during the last 10

Specifically, there have been 36 reports of neoplasia in 15 species of bivalve mollusks: four species of oysters, six species of clams, and five species of mussels. Farley4 has noted that five basic types of invasive neoplasms have been found in five genera and eight species of bivalve mollusks. The etiology of molluscan neoplasia, however, has remained enigmatic. In fact, reports suggest that multiple causative factors may be involved in molluscan carcinogenesis.

Reports of neoplastic growths in Sydney rock oysters, Crassostrea commer- c i a l i ~ , ~ . ~ suggested that pollution, either industrial (chemical and radioactive) or domestic, could be eliminated as an etiologic agent. Wolf noted that there was a paper mill 6 miles upstream from the affected site, but that the oysters between the mill and this site were not affected.5.e Similarly, the mud was eliminated as a source of carcinogens, because oysters grown off the bottom were also tumorous.

Sparks3 pointed out that the seasonal occurrence, progression of onset, and ap- parent contagious nature of the Ostrea lurida neoplasm in Yaquina Bay, Oregon was consistent with all known oyster epizootics of parasitic causation. On the other hand, no protistan of known pathogenicity was found in diseased oysters. Furthermore, F a r l e ~ ’ . ~ has demonstrated that nuclei of neoplastic molluscan cells contain intensely staining Feulgen-positive material (chromatin) and that protozoa do not. Therefore, it is highly unlikely that molluscan neoplasia is a result of infection by protozoan parasites.

Farley’ did not rule out pollutants in estuaries as etiologic agents of neoplasms, but most of the specimens collected were from the least polluted areas. He did note that the disease could conceivably be of viral origin.

found a high prevalence of atypical hyperplasia in Mya arenariu in four geographic locations but were unable to correlate the lesions with distinct causa- tive factors. On the other hand, Barry and Yevich,’” LaRoche,” and Yevich and B ~ ~ S Z C Z ~ ~ . ’ ~ have found a high incidence of gonadal tumors in clams contaminated by the oil spilled at Searsport, Maine in March 1971. These findings provided the im- petus for the studies reported herein.

Barry et

*Supported by American Petroleum Institute Contract 98-20-7372. This paper is contribu- tion 1730 from the Rhode Island Agricultural Experiment Station.

522

Brown et al.: Neoplasia in the Soft-Shell Clam 523

The purpose of our study was to determine the prevalence of neoplasia in popula- tions of the soft-shell clam, M. arenaria, and to determine if neoplasia was associated with varying types and degrees of environmental pollution.

MATERIALS A N D METHODS

Selection o$the Study Organism

M. arenaria was chosen because it is a sedentary long-lived filter feeder capable of accumulating substances from the environment and has been reported to be sus- ceptible to neoplastic disease.

Site Selection

Ten sites were sampled between Maine and Rhode Island. These sites had vary- ing types and degrees of pollution, including nominal (no visible sign or history of pollution in the area), heavy metal, industrial and/or domestic sewage, and petro- leum-derived hydrocarbons (TABLE l).

Organization of the Study

Ecologic studies of the 10 areas and hydrocarbon analyses of clams and associated sediments are in progress. We will report on preliminary findings of the histopathologic analyses of clam tissues to determine the frequency and type of neo- plasms and other lesions. In the final analysis, statistical correlations will be drawn among the ecologic, chemical, and histopathologic findings.

Collection and Maintenance of Clams

To date, 1829 clams have been collected from intertidal zones of the 10 sites. Clams were allowed to depurate any possible sediments accumulated during collec- tion for 2 days in flow-through seawater aquaria a t Narragansett, Rhode Island before being processed for histopathologic examination.

Histopathologic Analysis

Two histopathologic methods were employed to diagnose neoplasia in 1325 ani- mals examined to date. The first was light microscopic examination of all organ systems. Tissues were preserved in Dietrich’s solution, dehydrated in alcohol and xylene, embedded in paraffin, sectioned at 6 pm, and stained with hematoxylin and eosin, Giemsa, or Feulgen stains.

The second method, a modification of that employed by Farley,8 involves collec- tion of cardiac blood with a 23-gauge needle and I-ml syringe and examination of cir- culating blood cells by either phase-contrast optics for viable cells or bright-field optics for methanol-fixed Giemsa-stained cells.

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Brown et ul.: Neoplasia in the Soft-Shell Clam 525

Mortality Study

A study was conducted to determine if the neoplastic disease of M y a was ma- lignant. Eighty-two animals were collected from Bourne, Massachusetts, numbered individually, bled, and examined cytologically by the second method described above. A diagnosis of either neoplastic or nonneoplastic was made for each animal, based on the presence or absence of circulating anaplastic cells. The animals were maintained (as above) and monitored daily for mortality over a 3-month period. Seventy-one ani- mals from the same site were not bled and were similarly monitored to determine the effect of bleeding on mortality. Animals that died were examined histologically. The accuracy of diagnosis by the blood cytologic method was ascertained by comparing the blood cytology screening data to diagnosis by tissue examination.

RESULTS

Of the 1325 soft-shell clams examined histopathologically, 159 contained neo- plasms. The prevalence of neoplasia varied widely among sites and ranged from 0 to 64% (TABLE I). Sites 1-4 and 6-8 had either no neoplastic clams or a very low fre- quency of neoplasia. (It is difficult to judge the significance of the prevalence of neo- plasia at site I because of the small sample size.) However, the prevalence of neo- ,plasia was markedly greater at sites 5 , 9 , and 10.

Two types of neoplasms were found. In two of the nine neoplastic animals from site 9, and in all of the other animals, the neoplasms were composed of individual cells present in the connective tissues spaces, closely resembled lymphosarcomas found in higher vertebrates, and were probably of hematopoietic origin (FIGURES 1 & 2). These cells have been described previously.' Briefly, the neoplasms were com- posed of markedly anaplastic cells that contained large ovoid nuclei, which were often lobed or binucleate, with a single large, distinct nucleolus, clumped chromatin, and a thickened nuclear rim (FIGURES 3 & 4). The cytoplasm was scant and irregular in outline. The nuclear to cytoplasmic ratio of neoplastic cells (3.6) was double that of normal cells ( 1 3). The nuclei of neoplastic and normal cells were red after applica- tion of Feulgen stain, and Giemsa-stained preparations did not show the presence of microorganisms.

The second type of neoplasm was found in seven animals from site 9. Cells were found primarily within gonadal follicles, appeared to arise from the germinal epithe- lium, and therefore were apparently of gonadal origin (FIGURES 5 & 6).

The gonadal tumors were similar to those described by Barry and Yevich'" and Yevich and B a r s ~ c z . ' ~ . ' ~ At low power, the gonadal follicles often appeared as dis- tinct islands filled with uniform sheets of contiguous basophilic cells. Some of the cells in the center of these islands were necrotic. The appearances of foamy, vacuolated normal gonadal cells persisting within the neoplastic proliferation was reminiscent of the starry-sky effect seen in vertebrate lymphomas. Masses of the neoplastic cells occasionally formed a syncytium similar to that of foreign body giant cells.

The gonadal neoplastic cells were also markedly anaplastic but smaller and more irregular in outline than those present in the hematopoietic neoplasms. The nucleus was often eccentric, and the nucleolus frequently was difficult to distinguish from abundantly clumped chromatin. There was distinctly more clumped chromatin in gonadal neoplastic cells than in neoplastic cells of hematopoietic neoplasms.

Mitotic figures in neoplastic cells were present in more than 60% of the clams, and in some cases, as many as 10% of the cells (at 1OOx) were mitotically active. The

FIGURE 1. Hematopoietic neoplasm. Abundant anaplastic cells in connective tissue of male gonad. Hematoxylin and eosin. x 160.

FIGURE 2. High-power view of hematopoietic neoplasm. Note cellular detail. Hematoxylin and eosin. x375.

5 26

Brown el al. : Neoplasia in the Soft-Shell Clam 527

FIGURE 3. Electron micrograph of anaplastic cell from hematopoietic neoplasm. Note large, lobed nucleus with clumped chromatin, distinct nucleolus, and thickened rim. x 15,400.

5 28 Annals New York Academy of Sciences

FIGURE 4. Electron micrograph of normal blood cell. Cell is smaller than neoplastic cell (FIGURE 3), has a smaller nucleus, and indistinct nucleolus. Note cytoplasmic rnernbrane-bound electron-dense granules and pseudopods. x 15,400.

Brown et ul.: Neoplasia in the Soft-Shell Clam 5 29

F I G U R E 5. Normal gonadal follicles (bottom) and gonadal follicles filled with tumor cells (top). Note focal hyperplasia of digestive gland tubular epithelium (right, between follicles). He- matoxylin and eosin. x 37.

FIGURE 6. High-power view of gonadal neoplasm. Note proliferating tumor cells within follicle and vacuolated normal spermatogenic cells. Hematoxylin and eosin. x 375.

530 Annals New York Academy of Sciences

neoplastic cells were both invasive to adjacent types of tissues and were also found accumulated in discontinuous organ systems in nearly 100% of the animals. The latter finding may be analogous or homologous to metastasis of tumors in higher ani- mals, but it is difficult to compare the microscopic appearance of pathologic condi- tions in animals with such radically different anatomies. More than half of the ani- mals with neoplasms also had extensive necrosis of parenchymal tissue adjacent to accumulations of neoplastic tissue. No tumors were detectable by gross examination, but other lesions could be noted grossly. In particular, the majority of clams from site 5 had pale, watery tissue and paresis of the syphon and adductor muscles. Six percent of these moribund animals died within 2 days after collection. One hundred and fifty-three clams from site 5 were monitored by mortality over a 3-month period. Nineteen animals (12%) died during this period. Bleeding had no appreciable effect on mortality; this parameter was approximately the same for clams that had not been bled (9) and for those that had been bled (10). Twelve of the dead clams were autopsied and examined histopathologically. Advanced hematopoietic neoplastic disease was found in all 12 animals.

The diagnosis of molluscan neoplasia by microscopic examination of circulating blood cells was a rapid and accurate technique. The blood and tissue of 101 clams have been examined, and 94% of those diagnosed as neoplastic by phase-contrast mi- croscopy, in addition to 92% diagnosed as neoplastic by Giemsa-stained blood smears, were confirmed as neoplastic by histopathologic examination.

FIGURE 7 . Intestinal epithelium. Extracellular accumulations of orange to greyish brown bodies. Hematoxylin and eosin. x52.

Brown et al.: Neoplasia in the Soft-Shell Clam 53 I

FIGURE 8. Renal epithelium. Note pigmented bodies intracellularly. Hematoxylin and eosin. x 62.

There was no difficulty in distinguishing normal from neoplastic cells in the blood. In addition to being morphologically distinct, the behavior of the neoplastic cells also differed markedly from that of normal cells. Extravasated neoplastic cells did not ag- gregate or adhere firmly to the glass slide, as did normal cells. The neoplastic cells maintained a spherical shape, had distinct anaplastic characteristics, especially the prominent nucleolus, and were nonmotile, whereas normal cells flattened to the glass surface, formed pseudopods, and moved in ameboid fashion.I5

Other prominent nonneoplastic lesions also found were hyperplasia of epithelial tissues and accumulations of orange-brown bodies. Epithelial hyperplasia was noted in clams from both polluted and nonpolluted areas. Hyperplasia was much greater in the digestive glands of animals from polluted areas. Also striking was the high preva- lence of orange-brown bodies in clams from polluted sites.

Pigmented bodies that varied in size from 2 to 20 pm were present in many organs but were markedly abundant in the intestines, digestive gland, and kidney. The bodies were irregular in shape, opaque, and greyish brown to orange-brown in he- matoxylin and eosin stained-preparations. The degree of pigmentation varied from body to body and within individual bodies. Smaller, darker bodies were often seen to lie within larger lighter-colored structures. In most instances, the bodies were sur- rounded by a round halo. The structures were found both intra- and extracellularly. Accumulations of pigment were present between intestinal epithelial cells and close to the basement membrane (FIGURE 7). Large accumulations often caused cell dis- tortion.

Similar accumulations were found free in connective tissue and other organs throughout the animal. Flattened crescent-shaped nuclei in close proximity to the pigmented bodies suggested their presence within phagocytes. Smaller, multiple ac- cumulations of the pigment were present with renal tubular epithelial cell cytoplasm (FIGURE 8).

5 32 Annals New York Academy of Sciences

The material resembled a ceroid of lipofuchsin pigment, and the structures did not appear to be ameboid in nature, although they were similar to bodies previously photographed in the intestinal epithelium of oysters."

It is not known if these renal pigments and orange-brown bodies were identical substances, even though they often had a similar appearance. They were resistant to such lipid solvents as ethanol and xylene (during histologic processing) and carbon tetrachloride (exposure of an unstained histologic section on a microscopic slide to 50 ml of absolute carbon tetrachloride for 30 min).

DISCUSSION

It is now widely accepted that mollusks are susceptible to proliferative disorders that histologically resemble neoplasms in higher animals. Furthermore, these diseases can occur in epizootics and are found in such widely geographically separated areas as the E a ~ t ' . ~ . ~ ~ . ~ ~ - ~ ~ . ~ ~ and Westl8 Coasts of the United States and Au~tra l ia .~~" Our findings of neoplasia, in some instances a t very high frequencies, from five of 10 geographically separate sites examined are in agreement with pre- vious reports of hematopoietic neoplasms in Mya by Farley' and Yevich and B a r s z c ~ ~ * * ' ~ and with the finding of gonadal and hematopoietic neoplasms in Mya from Searsport, Maine.10*12*13

It is interesting to note that in Mya, only hematopoietic neoplasms have been found in southern New England and predominantly gonadal neoplasms have been found in northern New England. Perhaps the relative differences in tidal flow and temperature affect the pathogenesis of the disease. There may also be differences in genetic susceptibility to different types of neoplasms in geographically isolated clam populations. A genetic basis for susceptibility to neoplasia is well known in higher ani- mals and may be true in oysters. Frierman and Andrews17 and Frierman" found two inbred strains that were very susceptible to hematopoietic neoplastic disease. The hard-shell clam, often found associated with Mya, is susceptible to gonadal neo- plasms in southern New England but is apparently not susceptible to hematopoietic neoplasms. 19.20

Farley,4*R Frierman and Andrews," and Frierman" have presented good indirect evidence that molluscan neoplasms are malignant. Additional corroborating evidence of this very important point, required to define proliferative disorders in mollusks as true neoplasms, was presented in the mortality study in this paper. Twelve clams that were diagnosed as having neoplastic disease by cytologic examination of circu- lating blood eventually died, and the diagnosis of neoplasia was confirmed by his- topathologic examination of the tissue. In addition, the neoplasms found in the field survey exhibited most of the traits associated with malignancy by vertebrate stan- dards. These features included marked anaplasia, high degrees of mitotic activity, in- vasiveness, metastasis, and morbidity.

It is interesting to note that the blood of neoplastic animals, monitored in the 3-month mortality study, appeared opalescent due to a massive number of circulat- ing neoplastic cells. This finding suggested that the animals had advanced neoplastic disease; yet, only 12 clams died. This relatively low mortality rate was perhaps due to the cool fall and winter water temperatures and/or to the fact that neoplasia in Mya is a chronic disease.

The finding of orange-brown bodies in M y a has not been previously reported. Morphologically similar particulates have been reported in Crassostrea virginica exposed to waste motor oilz1 and in Mercenaria mercenaria from polluted waters.22 We have also found particulates in tissues of M. mercenaria and the shipwoim, Teredo navalis. The significance and further identification of these bodies are under investigation.

Brown et uf.: Neoplasia in the Soft-Shell Clam 533

The fact that most molluscan neoplasms are not detectable grossly has meant that costly and time-consuming histopathologic examinations have been required to diagnose neoplastic disease. We suggest that the cytologic examination of molluscan blood be incorporated as a routine procedure for the diagnosis of molluscan neo- plasia. The technique is accurate, simple, quick, and inexpensive, and bleeding does not result in death of the animal. It also offers the advantage of working with a living animal, thus providing possibilities for experimental cell culture, electron mi- croscopy, pathogenesis, and search for biologic causative agents.

As stated under INTRODUCTION, the etiology of molluscan neoplasia is not well known. The results presented in this paper are still too preliminary to help clarify this confusing situation. There is a surprising dichotomy of results from petroleum- derived hydrocarbon (PDH)-polluted sites. Clams from Providence and Portland had no neoplasms, whereas other PDH-polluted sites, namely, Bourne, Searsport, and Quonset, had a high prevalence of neoplasms. These results suggest that the type and degree of hydrocarbon pollution are possibly related to the frequency of neo- plasms and other lesions in Mya, but they are by no means theonly causative factors.

The finding of neoplasia in nominally polluted areas, such as Sandy Point, Wick- ford, and Quonochontaug, suggests that the etiology of neoplasia may not necessarily be exposure of clams to high concentrations of pollutants. It is interesting to note that in a recent report that described three epizootics of neoplastic disease in mollusks, Farleyl did not find any evidence for environmental relationships nor was an etiology readily apparent. The possibility that neoplastic disease in M y a has a genetic basis or is a result of an infectious agent cannot be excluded by our results.

SUMMARY

Neoplasia was a prevalent disease of the soft-shell clam and was found in widely geographically distinct sites in New England. Two types of neoplasms were recognized. Most were of hematopoietic origin, except in clams from Maine, which also had gonadal neoplasms. Both types were apparently malignant neoplasms, based on their characteristic anaplastic appearance, invasiveness, metastasis, mitotic activity, associated tissue necrosis, and mortality. Diagnosis of neoplasia in the living mollusk was achieved rapidly and accurately by cytologic examination of circulating blood. The etiology of the neoplasms was not identified.

In general, nonneoplastic lesions, such as epithelial hyperplasia and accumula- tions of orange-brown bodies, were more common in clams from polluted areas.

ACKNOWLEDGMENTS

The authors thank Richard Appeldoorn for collecting clams, Victoria Murray and Dennis Mesko for preparing tissues for histopathologic analysis, and Martha Tanner for typing the manuscript. The senior author appreciates the advice of the histopathologists at the National Marine Fisheries Service Laboratory at Oxford, Maryland. This author specifically thanks C. Austin Farley for his assistance in initiating this project and for many inspiring conversations.

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5 34 Annals New York Academy of Sciences

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