AFIP Wednesday Slide Conference - No. 26
April 12, 2000

Conference Moderator:
Dr. Alan Rebar, Diplomate, ACVP
Dean, School of Veterinary Medicine
Purdue University
West Lafayette, IN 47907-1240
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Case I - 93-1 (AFIP 2420938)
Signalment: 63-week-old, male Sprague-Dawley (Crl:CD®BR VAF/Plus®) rat
History: This animal was part of a 2-year carcinogenicity study. One year after the initiation of treatment, the animal was clinically normal but had a nucleated cell count of 143,300/mL. Four weeks later, the animal was sacrificed due to poor health. Clinical observations included weight loss, hunched posture, and pale mucous membranes.
Gross Pathology: Enlarged liver, spleen, lymph nodes, and kidneys. Liver was mottled; dark red and brownish-green. Spleen had multiple light foci; yellow and firm. Kidneys were mottled; tan and brownish-green. Appearance of lymph nodes was variable. Lumbar and renal lymph nodes were mottled; red and brownish-green.
Laboratory Results:


 Normal Range

 Week 52

 Week 57
(at sacrifice)
 RBC (/pL)    5,100,000  1,700,000
 Hemoglobin (g/dL)    11.7  5.0
 Hematocrit (%)    32.5  14.7
 MCV (fL)    64.0  86.4
 MCH (pg)    22.9  29.4
 MCHC (g/dL)    36.0  34.0
 Platelet count (/pL)    370,000  30,000
 Nucleated cells(/pL)    143,300  143,100
 Aspartate aminotransferase (lU/L)  <300  1688  not done
 Alanine aminotransferase (lU/L)  <100  109  not done
 Alkaline phosphatase (lU/L)  <100  373  not done
 Total bilirubin (mg/dL)  <0.2  0.8  not done

Other clinical chemistry results were unremarkable.
Contributor's Diagnosis and Comments:
Granulocytic leukemia (myelogenous or myeloid leukemia)
Examination of the blood film at low power confirms the markedly high nucleated cell count. The majority of nucleated cells have characteristics associated with the neutrophil cell line at different stages of maturation. Although atypical nuclear morphology (shape and chromatin pattern) and asynchronous maturation of nuclei and cytoplasm for many cells makes it difficult to do a standard differential cell count, our count resulted in the following relative numbers: 31% segmented neutrophils, 20% band neutrophils, 25% metamyelocytes, 9% myelocytes, 3% promyelocytes, 3% myeloblasts, 4% lymphocytes, and 5% metarubricytes and rubricytes. While the predominant cell counted was a segmented neutrophil, there are relatively few of these cells with typical morphology.
In contrast to typical morphology, the nuclei are not as tightly coiled or lobulated, the chromatin is not as dense and coarsely clumped, and the cytoplasm is more basophilic. The myeloblasts are distinguished by dark, basophilic cytoplasm and prominent nucleoli, but many have prominent azurophilic granules characteristic of primary granules that normally appear in promyelocytes. Many of the other immature cells are ring-forms, a characteristic of granulocyte nuclear morphology in rodents and frequently classified as metamyelocytes. It is possible that some of the cells with "ameboid-shaped" nuclei and blue-gray cytoplasm are monocytes or monocyte precursors, but special stains to distinguish granulocytic from monocytic cells were not done. As granulocytes and monocytes are derived from the same precursor, the possibility of myelomonocytic leukemia exists in this case.
Red blood cell morphology is characteristic of a regenerative anemia, and features marked anisocytosis and polychromasia, increased Howell-Jolly bodies, and several nucleated red blood cells. The high mean corpuscular volume is also consistent with prominent reticulocytosis. In contrast to the nonregenerative anemia typically associated with leukemia, rats with granulocytic leukemia frequently have regenerative characteristics (Moloney, 1974). The appearance of nucleated red blood cells in peripheral blood has been proposed as an early indicator of chemically-induced granulocytic leukemic, but there are many other causes of metarubricytosis (Takayama, 1993).
There are few platelets per oil immersion field, confirming the thrombocytopenia indicated by the electronic counter. Thrombocytopenia is a frequent feature of leukemia. Histopathologic examination identified infiltrating neoplastic cells consistent with the granulocytic lineage in many tissues, including liver, spleen, lung, lymph nodes, kidneys, and eyes.
Among inbred rats, spontaneous granulocytic leukemia occurs at very low incidence; less than 0.5% in literature references (Moloney, 1974; McMartin et al., 1992) and 0.3% in our laboratory the past 5 years. Ionizing irradiation and chemical agents (e.g., methylcholanthracene, dimethylbenz(a)anthracene, fluorenylenebisacetamide, ethyl nitrosourea, and butyl nitrosourea) may increase the incidence of granulocytic leukemia (Moloney, 1974; Takayama, 1993; Gal et al., 1973; Gal et al., 1990). It is readily transplantable, but attempts to transmit the disease by cell-free ultrafiltrate are infrequently successful even though C type viral particles have been observed ultrastructurally (Chen et al., 1972; Gal et al., 1990). As this animal belonged to a low-dose group in the carcinogenicity study and other animals were not similarly affected, its leukemia was considered spontaneous.
AFIP Diagnosis: Morphologic Diagnosis: Blood film: Chronic granulocytic leukemia, with regenerative anemia and thrombocytopenia, Sprague-Dawley rat, rodent.

Conference Note: Conference participants agreed with the contributor's diagnosis. Discussion centered on the abundant information that could be gleaned from this simple peripheral blood smear. Marked leukocytosis (predominantly neutrophils with atypical morphology as described by the contributor) and thrombocytopenia, as well as polychromasia (regenerative anemia) are evident microscopically.
Leukemia is classified as acute or chronic based on the degree of differentiation of the involved cell line. If the average cell type is in the early stages of leukocyte development, the leukemia is deemed "acute", and when neoplastic cells are more differentiated, the leukemia is referred to as "chronic". Conference participants favored adding the designation of chronic to the morphologic diagnosis.
Contributor: Hazleton Wisconsin, Inc., P0 Box 7545, Madison, WI 53707-7545.
1. Chen L, Handler EE, Handler ES, Weiss L: An electron microscopic study of the bone marrow of the rat in an experimental myelogenous leukemia. Blood 39:99-112, 1972
2. Firth CH, Ward JM, Chandra M: The morphology, immunohistochemistry, and incidence of hematopoietic neoplasms in mice and rats. Toxicol Pathol 21(2):206-218, 1993
3. Gal F, Somfai S, Szentirmay Z: Transplantable myeloid rat leukaemia induced by 7,12-dimethylbenz(a)anthracene. Acta Haemat 49:281-290, 1973
4. Gal F, Sugar J, Csuka 0: Granulocytic leukemia, rat. In:, Monographs on Pathology of Laboratory Animals. Hemopoietic System, eds. Jones TC, Ward JM, Mohr U, Hunt R D, pp. 39-45. Springer-Verlag, New York, pp 39-45, 1990
5. McMartin DN, Sahota P 5, Gunson DE, Hsu HH, Spaet RH: Neoplasms and related proliferative lesions in control Sprague -Dawley rats from carcinogenicity studies. Historical data and diagnostic considerations. Tox Pathol 20:212-225, 1992
6. Moloney WC: Primary granulocytic leukemia in the rat. Cancer Research 34:3049-3057, 1974.
7. Takayama S: Erythroblasts as an index of initial stage of 2,7-FM, ENU, and BNU-induced rat leukemia. Acta Pathologica Japonica 43:222-229, 1993
Case II - A74093(AFIP 2681381)
Signalment: 35-week-old, male, Tg.AC mouse
History: This vehicle-treated (negative-control) mouse was found dead in week 27 of an alternative to carcinogenicity testing study. The study was part of the International Life Sciences Institute (ILSI), Health and Environmental Sciences Institute (HESI) initiative.
Gross Pathology: The liver and spleen were enlarged.

Contributor's Diagnoses and Comments: Lung: Erythremic myelosis
Erythremic myelosis is a tumor of the erythroid cell line, exclusively (Valli, 1993). Erythroid leukemia involves neoplastic proliferation of both erythroid and neutrophil precursors and may progress to acute myelogenous leukemia (M2) (Jain NC, 1993). In the present case, bone marrow cytology was diagnostic for erythremic myelosis. While the cytomorphology of the neoplastic cells in other tissues was not adequate to definitively diagnose this condition, the presence of metarubricytes and leukemic cells in multiple organs, along with effacement of splenic and hepatic parenchyma, was highly suggestive of this neoplasm. The basophilic material in the pulmonary vasculature associated with these cells was identified as DNA (Feulgin positive) and considered a terminal event along with intravascular coagulation. Erythremic myelosis was reported as the cause of death in this mouse.
Erythremic myelosis was present in 6% (9/150) of the Tg.AC control mice. All of the animals had splenomegaly and 44% had hepatomegaly, due to infiltrates of neoplastic cells. Trempus, et al., (1998) reported <5% incidence of erythroid leukemias (erythremic myelosis), along with splenomegaly and hepatomegaly, in untreated Tg.AC mice. In the present study, other causes of splenomegaly were nonspecific lymphoid hyperplasia and extramedullary hematopoiesis.
Other common lesions in the present study included odontogenic tumors (24% incidence, 25% of which were fatal), gastric papillomas (28% incidence), gastric hyperplasia and/or hyperkeratosis (nonglandular mucosa), and diffuse retinal atrophy (100% incidence).
The Tg.AC transgenic mouse has a v-Ha-ras oncogene linked to the embryonic zeta-globin promoter. The mouse was developed in order to study the development of the embryonic hematopoietic system and is currently being investigated for its potential usefulness as an alternative to long-term carcinogenicity testing. This transgenic mouse has a propensity to develop skin papillomas, even from non-genotoxic agents. It has been speculated that the transcription factors required for expression of the embryonic zeta-globin promoter are active in the adult mouse, resulting in expression of the inserted v-Ha-ras oncogene and aberrant proliferation of erythropoietic cells (Trempus, et al., 1998).
AFIP Diagnosis: Lung: Intravascular leukocytic neoplasm, Tg.AC mouse, rodent.

Conference Note: Conference participants agreed that the proliferation of round cells within the pulmonary vasculature represents a neoplastic process. They further agreed that additional materials, particularly bone marrow, are necessary to make a more definitive diagnosis. Most would also have liked a peripheral blood smear and specimens of other parenchymatous organs to examine microscopically. Spontaneous neoplasia of the erythroid cell line occurs only rarely in mice and is unreported in rats. Most reports in mice involve the experimental inoculation of Friend leukemia virus or Rauscher virus. Irradiation and trimethylbenzene(a)anthracene exposure have caused the disease in rats.

Contributor: Schering Plough Research Institute, PO Box 32, 144 Route 94, Lafayette, NJ 07848.
1. Firth CH, Ward JM, Chandra M: The morphology, immunohistochemistry, and incidence of hematopoietic neoplasms in mice and rats. Toxicol Pathol 21(2):206-218, 1993
2. Jain NC: Essentials of Veterinary Hematology. p. 315. Lea & Febiger, Philadelphia, PA, 1993
3. Trempus CS, Ward S, Farris G, Malarkey D, Faircloth RS, Cannon Re, Mahler JF: Association of v-Ha-ras Transgene Expression with development of erythroleukemia in Tg.AC transgenic mice. Am J Pathol 153:247-254, 1998
4. Valli VEO: The hematopoietic system. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 3, pp. 122-124. Academic Press, Inc., San Diego, CA, 1993
Case III - 1804565 (AFIP2595297)
Signalment: One-year-old, mixed breed, castrated male feline.
History: This cat was presented for vomiting and was in renal failure. The cat had eaten several leaves from an Easter lily.
Gross Pathology: Gross lesions were not described.
Contributor's Diagnosis and Comments: Severe nephrotoxic acute tubular nephrosis (necrosis) with massive proteinuria; etiology, Easter lily toxicosis.
The kidney had a severe tubular nephrosis (necrosis) with preservation of tubular basement membranes, which primarily affected proximal tubules. Tubular lumina contained flocculent eosinophilic debris. Surviving tubules contained proteinaceous exudate, which was most prominent in the renal medullary zones. There was patchy, mild interstitial congestion and hemorrhage, but glomeruli were histologically normal in appearance. The localization to proximal tubules with preservation of tubular basement membranes is supportive of a nephrotoxin rather than ischemic origin.
In a single abstract, the nephrotoxicity of ingested Easter lily (Lilium longiflorum) was described.2 Renal failure develops between 48 and 96 hours post-exposure. The causative nephrotoxic principle has not yet been determined. Three cats have been conclusively diagnosed with Easter lily toxicosis since the sentinel case in April, 1992. The diagnosis is based on the history of consumption of the plant and the supportive histopathology. A variety of nephrotoxins have been described in Jubb, Kennedy, and Palmer.1 Decontamination procedures when instituted within 6 hours of plant ingestion are prophylactic against the onset of renal failure.2
AFIP Diagnosis: Kidney: Necrosis, tubular (nephrosis), diffuse, with granular casts and tubular proteinosis, mixed breed, feline.

Conference Note: The diffuse distribution of the necrosis and the maintenance of basement membranes lead all conference participants to favor a toxic insult over an ischemic one. Conference participants discussed hemoglobinuric nephrosis, onion/garlic toxicity, aminoglycoside antibiotic toxicity and a wide variety of other possible causes. History of ingestion of the plant or the presence of identifiable plant material in the gastrointestinal tract is needed for specific diagnosis.
Contributor: Animal Health Diagnostic Lab, P0 Box 30076, Lansing, MI 48909-7576.
1. Maxie MG: The Urinary System, In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N. 4th ed, vol 2, pp. 487-490. Academic Press Inc, San Diego, Ca, 1993
2. Hall JO: Nephrotoxicity of Easter lily (Lilium longiflorum) when ingested by the cat (abstract 49) J Vet Int Med 6(2):121, 1992
Case IV - HN1156 (AFIP 2602966)
Signalment: Dog, mongrel, male, 13-year-old
History: Seven months earlier there was swelling of the right hind limb and then a large mass developed. Radiography revealed evidence of pulmonary and internal iliac lymph node metastases. Euthanasia was performed at the owner's request.
Gross Pathology: The large mass in the right hind limb was 13.5 x 10.5 x 12.5 cm in size, yellowish-white, with mild hemorrhagic streaks and necrosis. The lungs showed multiple, spherical white nodular masses, up to 2.5 cm in diameter, diffusely disseminated throughout the pulmonary parenchyma. Nodules are also found around the left elbow joint and internal iliac lymph node.
Laboratory Results: RBC = 28,000 cells/ul, WBC= 42,000 cells/ul, hemoglobin was 10.2%, and hematocrit was 30,6%.
Contributor's Diagnosis and Comments: The neoplasm in the lung was diagnosed as metastatic hemangiopericytoma, originating from the skin of the right hind limb. The same morphology and pattern of cells were also found in the masses around left elbow joint and in internal iliac lymph node. It was possible that dissemination of this neoplasm from the primary subcutaneous mass occurred hematogenously and via lymph vessels.
The histologic features of the pulmonary masses were consistent with canine hema-ngiopericytoma. The masses were composed of the prolife-ration of spindle and polygonal cells with whorled arrangement. In dogs, the use of the term "malignant" in diagnosis seems to be avoided because the frequency of metastases to other organs is extremely low despite the frequency of the neoplasm in the subcutis of this species. Canine hemangiopericytoma is usually a distinct neoplasm, arises from pericytes, is slow growing, and is limited to the dermis and subcutis in most cases, but the neoplasm occasionally infiltrates between muscle bundles, tendons, and nerves, especially when located on the distal extremities. Canine hemangiopericytoma usually appears in dogs older than 6 years, and most neoplasms occurs in animals between 8 and 14 years of age. Metastasis is rare and has been previously reported in just 2 cases. When metastasis of canine hemangiopericytoma occurs, it spreads primarily to regional lymph nodes, lung, and liver.
AFIP Diagnosis: Lung: Spindle cell sarcoma, high grade, metastatic.

Conference Note: As noted by Yost and Jones in their 1958 publication on canine hemangiopericytoma, "Although there is disagreement as to exact origin and function of the principle cell type, the structure of the tumor and its behavior in the host leave little room for disagreement concerning its existence as a distinct entity in the dog." Little has changed over the last 42 years concerning this common tumor. The cell of origin remains a subject of controversy. Canine hemangiopericytoma (CHP) has little resemblance to human hemangiopericytoma. Many believe that cells of peripheral nerve sheaths rather than pericytes give rise to these tumors. Thus, other names are used such as neurofibroma, neurofibrosarcoma, schwannoma and peripheral nerve sheath tumor of the skin and subcutis of dogs. Whatever their origin, these neoplasms are prone to local recurrence and can be locally aggressive in some cases. Metastasis is rare.
As with the cell of origin, diagnosis of this case proved to be controversial. Classic CHP (or whichever name is preferred) is histologically distinctive. It is a dermal and/or subcutaneous neoplasm characterized by collagenous whorls that are sometimes centered on blood vessels, cells arranged in a storiform-like pattern of short interlacing bundles, and collagenous, sparsely cellular areas interspersed with more highly cellular areas that contain little collagen. Cells are predominantly spindle-shaped. Most nuclei are plump, ovoid, have stippled and marginated chromatin, and one or two fairly prominent nucleoli. Mitotic rate has been reported to range from 0 to 12 per 10 high power fields.
In the present case, the neoplastic cells are arranged in bundles that vary from short to intermediate to long, and whorls that are sometimes centered on blood vessels. A small, relatively uniform amount of collagen is present between the cells. Nuclei are elongate to irregularly ovoid, vesicular, and occasionally contain one or two prominent nucleoli. There are scattered foci of necrosis. Mitotic rate ranges up to 10 per high power field.
Thus, this neoplasm has features of CHP, but it differs from the classic tumor in some respects. It should be remembered that whorling and storiform-like patterns can be seen in other tumors such as malignant peripheral nerve sheath tumor and fibrohistiocytic tumors such as dermatofibrosarcoma protuberans.
Only a few unstained sections were available from the lung. The contributor graciously provided a paraffin block from the mass in the right hind limb. The histologic findings for the limb tumor were very similar to the metastatic tumor except for the presence of abundant myxomatous matrix that formed accumulations between the spindle cells in some areas. The mitotic rate was similarly high. By immunohistochemistry, the pulmonary masses were lightly positive for S-100 protein and negative for desmin and smooth muscle actin. The limb tumor was also lightly positive for S-100 protein and negative for desmin, smooth muscle actin, muscle actin (HHF35) and glial fibrillary acidic protein.
In a recent study, 23/45 canine hemangiopericytomas were positive for muscle actin (HHF35) and 16/45 were positive for S-100 protein. Nine of the 16 S-100 positive tumors were also positive for muscle actin. This suggests that either CHP has a variable immunophenotype or different types of tumors were considered to be CHP. In any case, immunohistochemistry does not resolve the diagnosis in this case.
If one's basic interpretation is that this tumor is CHP, then the differences from the classic tumor can be attributed to the fact that this one is malignant. Alternatively, the differences can be considered evidence that the tumor is not CHP. We are confident of the diagnosis of high grade spindle cell sarcoma.
Contributor: Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060, Japan
1. Graves GM, Bjorling DE, Mahaffey E: Canine hemangiopericytoma: 23 cases (1967-1984). J Am Vet Med Assoc 192:99-102, 1988
2. Mills JHL, Nielsen SW: Canine hemangiopericytomas - A Survey of 200 Tumours. J Sm Ani Pract 8:599-604, 1967
3. Perez J, Bautists MJ, Rollon E, de Lara FC, Carrasco L, Martin de las Mulas J: Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog. Vet Pathol 33:391-397, 1996
4. Pulley LT, Stannard AA: Tumors of the Skin and Soft Tissues. In: Tumors in Do-mestic Animals, ed. Moulton JE, pp. 48-51. University of California Press, Berkeley, CA. 1990
5. Richardson RC, Render JA, Rudd RG, Shupe RE, Carlto WW: Metastatic canine hemangiopericytoma. J Amer Vet Med Assoc 182:705-706, 1983
6. Yost DH, Jones TC: Hemangiopericytoma in the dog. Am J Vet Res 19:159-163, 1958
J Scot Estep, DVM
Captain, United States Army
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202)782-2615; DSN: 662-2615
* The American Veterinary Medical Association and the American College of Veterinary Pathologists are co-sponsors of the Registry of Veterinary Pathology. The C.L. Davis Foundation also provides substantial support for the Registry.
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