Hematologic Abnormalities Preceding Myeloid Leukemia in Three Cats

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1 Vet. Pathol. 16: (1979) Hematologic Abnormalities Preceding Myeloid Leukemia in Three Cats B. R. MADEWELL, N. C. JAIN and R. E. WELLER Section of Clinical Oncology, Departments of Veterinary Surgery and Clinical Pathology, University of California, Davis, Calif. Abstract. Cytopenia were recognized in three cats infected with feline leukemia virus. In one cat, marrow blast cells were increased in number, and a diagnosis of aleukemic leukemia was made. The disease progressed slowly for 3?4 months before terminating in acute myelomonocytic leukemia, recognized as a blast crisis in blood. In the other two cats, neutropenia and altered granulopoiesis in bone marrow preceded development of myeloid leukemia. Preleukemia and smoldering leukemia are terms used to describe a group of hematologic abnormalities that precede acute myeloid leukemia in man. The term preleukemia refers to patients with acquired chronic cytopenias who develop myeloproliferative disorders (acute myeloid leukemia or myelomonocytic leukemia) months or years later [5]. These syndromes also are called hemopoietic dysplasias [27]. Laboratory abnormalities have dominated the clinical picture of preleukemia; anemia, neutropenia and thrombocytopenia occurring singly or in various combinations have been the most common findings [29]. In none of these disorders is there an excess of leukemic blast cells in either the bone marrow or blood at the onset, and the conclusion that the condition was preleukemic can be made only in retrospect when the picture has progressed to an acute myeloid leukemia [2, 6, 11. In contrast to the preleukemic group, the smoldering or aleukemic leukemias show more blast cells in marrow from the onset, while the blood shows cytopenias without blast cells [6, 211. For a period from several months to 2 or more years, however, the disease does not progress rapidly, but runs a prolonged benign course before terminating typically in acute myeloid leukemia [6, 281. Recognition of early subclinical stages of leukemia and those clinical states conducive to the development of leukemia are important to define the onset of leukemia. The diagnosis of leukemia requires recognition of an increased number of mature and immature cells in blood and alteration of hematopoietic tissues [2]. Case Histories and Results Cat 1 An 8-year-old male Siamese cat (4.7 kilograms) had anorexia and lethargy. 51

2 Myeloid Leukemia in Cats 51 1 It had mild dehydration, pale mucous membranes, a severe non-responsive anemia and leukopenia (table I). The erythrocyte indices revealed slight macrocytosis; there was marked anisocytosis, but reticulocytes were seen rarely. Many of the platelets were large and pleomorphic. Autoagglutination of blood was noted, and a direct antiglobulin (Coomb s) test was positive. The lupus erythematosus cell phenomenon was not seen in vitro. The indirect immunofluorescence antibody test for feline leukemia virus was positive in blood leukocytes and platelets. Membrane immunofluorescence to feline oncornavirus cell membrane antigen was positive at a 1:5 serum dilution, but negative at 1 :25. Serum antibodies to feline infectious peritonitis were not detected by indirect immunofluorescence. Bone marrow smears were cellular, and 9% of the cells were poorly differentiated, some showing differentiation toward progranulocytes and others toward monocytes (fig. 1). Mature neutrophils and erythroid precursors rarely were seen, while megakaryocytes were easily found on scanning. A diagnosis of aleukemic (myeloid) leukemia was made on the basis of blood and bone marrow findings. Treatment was supportive and included cross-matched fresh whole blood transfusions and glucocorticosteroids. Five weeks later the cat had improved clinically. A complete blood count again showed leukopenia (white blood cell count, 2999/pl) and anemia (red blood cell count, 3.3 IX 16/pl). Erythrocytes were macrocytic (mean corpuscular volume, 63.4/pm3), but few reticulocytes were seen. The platelet count was elevated to 389,O/pl. The Coomb s test was negative. Fifteen weeks after first examination the cat was lethargic. It had moderate peripheral lymphadenopathy and pale mucous membranes. Hemogram findings showed frank leukemia (leukocyte count, 178,4OO/p1; fig. 2) and severe anemia (table I). Erythrocytes were slightly macrocytic but no reticulocytes were seen. Percutaneous needle aspiration of a prescapular lymph node revealed a predominance of poorly differentiated mononuclear cells similar to those seen in blood. Bone marrow aspiration smears were cellular. Ninety-five percent of the cells were myeloblasts; the remaining 5% included monoblasts, monocytes, progranulocytes and myelocytes. Red cell precursors or megakaryocytes were not seen. Occasional mature neutrophils, lymphocytes and eosinophils were seen. The iron stain was negative. Cytochemical studies on blood leukocytes and bone marrow were done to confirm the diagnosis of myelomonocytic leukemia. Many of the abnormal mononuclear cells stained strongly for monocytic markers (lipase, non-specific esterase) and for granulocytic markers (peroxidase, alkaline phosphatase) 17, 3, After diagnosis, a trial course of anticancer drug treatment was given. One week after intravenous administration of 9 milligrams of doxorubicin HC1, the total leukocyte count had dropped to 14,6O/pl (non-segmented neutrophils, 146/pl; segmented neutrophils, 73/p1; lymphocytes, 166/pk tumor cells, l2,118/pl). Despite the initial prompt reduction of circulating tumor cells, there was severe nonresponsive anemia and rapid resurgence of tumor cells in blood. The cat was killed 23 days after onset of treatment. Necropsy findings confirmed the clinical diagnosis of a myeloproliferative disorder. The spleen, liver, bone marrow and lymph nodes were extensively infiltrated with a

3 Dav <I 7 3.4' rare rare rare I %( 34) 1,36 39%( 1,326) I %( 34) 48%( 1,632) 3%( 12) 8%(272) 48, Pleomorphic; many large Cat 3 ~ ~ in I N Day I I rare 5 2 % e 4,7' %- 72%(29,34) L 5, 13%(5,291) 3 P 6%(2,442) a 2%(814) E % 1,221 z 2%( 814) 17447) 4%( 1,628) 236, Many large Factor RBC (X IO"/mm~") Hemoglobin, g/dl PCV. % MCV, fl MCHC, % MCH, pg Plasma protein, g/dl Plasma fibrinogen, mg/dl Reticulocytes, % Nucleated RBC (/I wbc) WBC' Blast cells Progranulocytes Myelocytes Metam yelocytes Neutrophils Segmented Non-segmented Lymphocytes Monocytes Eosinophils Basophils Unclassified cells Thrombocytes Platelet morphology Day I I I I rare 1.9 1,26 52%(988) 2%(38) 42%(798) 2%( 3 8) 2%(38) Present Pleomorphic; some very large Table I. Hematologic findings in three cats Cat I Cat 2 - Day 13 Day Day <I 178,4 1,784 I%( 1,784) 4%(7,I36) 95%( 169,48) 139, - ' Absolute values are expressed as cells/pl of blood. Leukocyte counts corrected for nucleated red blood cells. ~~ ,4 1,156 34%( I, 156) 49%( 1.666) ) 172, Occasional; very large --~- I I few 3 2,7' 52%( 1,44) 6%( 162) 4%( 18) 2%( 54) 27 1 %(27) 35%(945) I8. Occasional; very large

4 Myeloid Leukemia in Cats Fig. 1: Poorly differentiated cells in marrow. Cat 1, initial examination. Some contain prominent nucleoli; large cells with indented nucleus seems to be monocytoid. Fig. 2 Three myeloblasts in blood. Cat 1, 4 months after first examination. Fig. 3: Myeloblasts with large nucleoli in marrow. Cat 2, initial examination. Fig. 4 Myeloblast in cat 2 after 1 year. neoplastic cell population appearing as undifferentiated blast cells. The cells had a narrow rim of purple cytoplasm and a large oval nucleus with slight chromatin clumping and prominent nucleolus. Mitotic figures were found readily. Cat 2 A 2-year-old male domestic short-haired cat (3.4 kilograms) was examined because of lethargy and weight loss. Physical abnormalities included fever (4" C ), moderately enlarged peripheral lymph nodes, and mild hepatosplenomegaly. Abnormal findings included leukopenia and neutropenia and high plasma fibrinogen (table I). Many platelets were large.

5 5 14 Madewell, Jain and Weller Other abnormalities included severe proteinuria and moderately increased serum activity of glutamic oxalacetic and glutamic pyruvic transaminases. The indirect immunofluorescent antibody test for feline leukemia virus was positive on blood leukocytes. Smears of a peripheral lymph node aspirate contained predominantly small lymphocytes with occasional prolymphocytes and lymphoblasts. Bone marrow smears were moderately cellular. Granulocyte maturation was complete, but immature granulocytes exceeded mature neutrophils. Some granulocyte precursors were large and abnormal (fig. 3). Rubricytic maturation was complete, and the myeloid: erythroid ratio was 1.2:l. Fifteen percent of the cells were mature lymphocytes; megakaryocytes were present. The cat was treated with polyionic solutions. One month later the hemogram was essentially unchanged. Over the ensuing 12 months, the cat remained generally unthrifty and was treated intermittently for cellulitis and abscesses. Moderate to severe neutropenia (2 to 4 neutrophils/pl) persisted. The cat was reexamined 11 months after the initial entry because of weakness and pallor of mucous membranes. Lymph nodes were not enlarged. Hematologic examination showed severe anemia, leukopenia and thrombocytopenia (table I). The anemia was slightly macrocytic, but few reticulocytes were seen. Myeloblasts and other granulocyte precursors were present, but there were no mature neutrophils (fig. 4). Occasional large platelets were seen. The feline leukemia virus fluorescent antibody test was positive on blood leukocytes. Serum antibodies to the feline infectious peritonitis virus were not detected by indirect immunofluorescence. Severe proteinuria persisted, and liver transaminase values remained moderately high. Bone marrow smears were diluted with blood. The predominant cells were myeloblasts and progranulocytes; some had cytoplasmic and nuclear vacuolization. Rubricytic maturation was complete, but precursor cells were scarce. The myeloid: erythroid ratio was 15:l. Fifteen percent of the cells were lymphocytes, and megakaryocytes were present. The iron stain was negative. On the basis of blood and bone marrow findings, a diagnosis of undifferentiated or blast cell myelogenous leukemia was made. The cat was killed because of cachexia; permission was not obtained for necropsy. Cat 3 A 2-year-old male domestic short-haired cat (4.5 kilograms) was referred to the University of California Veterinary Medical Teaching Hospital because of a positive indirect immunofluorescent antibody test for feline leukemia virus. Ophthalmoscopic examination showed unilateral (left) chorioretinitis in the area centralis and temporal quadrants. Abnormal hemogram findings included leukopenia and neutropenia with a left shift to metamyelocytes and nucleated red blood cells (table I). Many platelets were large and pleomorphic. The cat s immune response to infection was negligible as measured by membrane immunofluorescence to feline oncornavirus cell membrane antigen and by the 51Cr release method for complementdependent cytolytic antibody [ 1 1 ].

6 Myeloid Leukemia in Cats 5 I5 Fig. 5: Myeloblasts with prominent nucleoli and undulated nuclear outline in blood. Cat 3, 5 months after initial examination, Wright-Leishman stain. Fig. 6 Three myeloblasts, one promyelocyte with distinct cytoplasmic granules, and a densely stained rubricyte in marrow. Cat 3, 5 months after initial examination. WrightLeishman stain. Fig. 7: Myeloblasts in lymph node aspirate. Cat 3, 5 months after initial examination. Wright-Leishman stain. Bone marrow smears were cellular. Erythroid and myeloid maturation sequences were complete and the mye1oid:erythroid ratio was 1.2:1. The predominant cells were myeloblasts and progranulocytes. Megakaryocytes were increased in numbers. Ten percent of the cells were mature lymphocytes, and occasional normal plasma cells were seen. The iron stain was negative. Over the ensuing 5 months the cat was given symptomatic treatment for several conditions including gingivitis, stomatitis, bite wounds and pyrexia. The cat was reexamined because of weakness 5 months after the initial presentation. Abnormal physical findings included pyrexia (4"C ), stomatitis, hepatosplenomegaly and peripheral lymphadenopathy. Hematologic findings included severe non-responsive anemia and granulocytic leukemia (table I). Myeloblasts and progranulocytes contained prominent nucleoli and often had an undulated nuclear outline (fig. 5). The platelet count was normal, but many platelets were large. Bone marrow smears were cellular. The cells were predominantly immature granulocytes (fig. 6). Myeloblasts and progranulocytes were most numerous. Granulocytic maturation was complete, but there were many more immature cells than mature neutrophils. Rubricytic maturation appeared complete, and the mye1oid:erythroid ratio was 2.5: 1. One percent of the cells were mature lymphocytes. Megakaryocytes were increased in numbers and some were immature. The iron stain was positive. Percutaneous needle aspirates of a peripheral lymph node and spleen showed a predominance of myelo-

7 516 Madewell, Jain and Weller blasts and progranulocytes (fig. 7). The indirect immunofluorescent antibody test for feline leukemia virus remained positive on blood leukocytes. The cat was killed. At necropsy, the liver and spleen were enlarged, and the lungs were mottled red and moist. There was generalized lymphadenopathy. The bone marrow was very red and soft. The lung, kidney, liver, spleen, bone marrow and lymph nodes were examined histologically. All tissues contained a pleomorphic cellular population compatible with our clinical diagnosis of a myeloproliferative disorder. Morphology varied. Some cells had a moderately large nucleus and diffuse chromatin pattern and others had a small, round, dark nucleus. Megakaryocytes were distributed through the tissues. Other changes included a focal lipid pneumonia and membranous glomerulonephritis. Discussion The myeloproliferative disorders, described in man in as those syndromes characterized by abnormal proliferation of hematopoietic cells, have been described in the cat [8, 31. The term disorder is particularly appropriate to this group of hematologic abnormalities as each disorder is a spectrum of disordered function rather than a simple morphologic entity. The myeloproliferative disorders appropriately are viewed as a continuum of pathophysiologic disorders. In animals, leukemia virus infections have provided considerable data on abnormalities in hematopoiesis. In cats, both degenerative and proliferative diseases of bone marrow have been recognized clinically and induced experimentally with leukemia virus. Feline leukemia virus is commonly associated with myeloproliferative disorders and type-c viral particles have been identified in bone marrow cells in various myeloproliferative diseases [ 14, 151. Myeloproliferative diseases, including myeloid leukemia and reticuloendotheliosis also have been seen in several cats experimentally infected with feline leukemia virus [ 18, 19,261, It is generally accepted that feline leukemia virus is a cause for myeloproliferative disorders, but a specific strain of virus has not been isolated that will consistently induce these disorders. Thus, the pathogenesis of myeloid leukemia has not been studied as extensively as the more readily induced lymphoid neoplasms [24]. Many degenerative diseases of bone marrow and blood cytopenias have been recognized in cats infected with feline leukemia virus, but documentation of progression of these diseases to myeloid leukemias has been limited. The nonresponsive anemias have been studied in considerable detail [16, 17, 23, 251. A syndrome of panleukopenia also has been described in cats in association with feline leukemia virus infection. The syndrome is reported to mimic panleukopenia (parvovirus) infection, but is considered distinct from that disease [ 13, 141. Lymphopenia reflects lymphoid depletion [I], while neutropenia presumably reflects disturbance in granulopoiesis induced by viral infection. In clinical studies, leukopenia has been seen in feline leukemia virus infected cats that have concurrent bacterial infections [3]. Neutropenia also has been seen in cats after experimental infection with feline leukemia virus [16, 261. In one study, bone

8 Myeloid Leukemia in Cats 5 17 marrow samples were collected from cats rendered pancytopenic by feline leukemia virus. Cytologic examinations showed decreased hematopoiesis of granulocyte, erythrocyte and megakaryocyte cell lines; the maturation sequence of the marrow cells present was considered normal [26]. In another study involving experimentally infected cats, total leukocyte counts varied, but moderate neutropenia was most common [ 171. Cytologic examination of bone marrow smears showed relative increase in the percentage of myeloid cells. This increase was caused by erythroid hypoplasia ~71. Our three cats had feline leukemia virus-induced disturbances in hematopoiesis that preceded myeloid leukemia. For cat 1, a diagnosis of myeloid leukemia could not be made on the basis of our initial blood examination. Hemogram findings reflected anemia, macrocytosis, neutropenia and lymphopenia. Bone marrow examination, however, allowed a diagnosis of myeloid (myelomonocytic) leukemia. The increased numbers of blast cells in marrow accompanying blood cytopenia is termed smoldering or aleukemic leukemia. Three and a half months later, the disease terminated in an acute myelomonocytic leukemia. The term myelomonocytic indicates that all myeloid (marrow) cells are involved, and mono emphasizes the monocytoid morphologic features [22]. In cat 2, the initial hematologic abnormalities included neutropenia and lymphopenia, and ultimately terminated as pancytopenia. A diagnosis of granulocytic leukemia subsequently was made on the basis of finding increased numbers of immature myeloid cells in blood and a predominance of myeloblasts and progranulocytes in marrow. In cat 3, initial blood examination showed neutropenia and nucleated red blood cells. Bone marrow cytology showed a predominance of myeloblasts and progranulocytes, but erythroid and myeloid maturation was complete. Five months later, however, granulocytic leukemia was diagnosed on the basis of blood and bone marrow cytology. In cats 2 and 3 abnormal proliferation of myeloid precursors, leading to anemia, and leukopenia (and thrombocytopenia in cat 2) preceded the development of myeloid leukemia. The cytopenias seen initially in these cats retrospectively can be termed preleukemic states or hemopoietic dysplasias since the derangements in hematopoiesis preceded classical myeloproliferative disorders. It is emphasized, however, that preleukemic syndromes are ill-defined, since the risk for developing classic myeloproliferative disorders after cytopenia is not absolute, and the distinction between hemopoietic dysplasia and myeloid leukemia is not always distinct [6, 2, 271. The incidental necropsy findings of membranous glomerulonephritis was presumed related to feline leukemia virus infection [9]. Simultaneous alteration of bone marrow cell lines characterize many of the myeloproliferative disorders in cats and man [4, 31. In our three cats there were disturbances in granulopoiesis, erythropoiesis and megakaryopoiesis. The term stem cell disorder has been proposed [2] as a definition for concurrent hematopoietic abnormalities. In vitro studies of bone marrow cells from people with preleukemia have provided evidence supporting a concept of acute myelogenous leukemia as a disturbance of cellular maturation [lo, 121. Similar in vitro studies are necessary to define the pathogenesis of feline myeloproliferative disorders.

9 518 Madewell. Jain and Weller Acknowledgements This study supported in part by a grant from the Max C. Fleischmann Foundation, Reno, Nev. We thank Ms. Rachel Smith, Clinical Pathology, Veterinary Medical Teaching Hospital, Davis, Calif., for technical assistance. References 1 ANDERSON, L.J.; JARRETT, W.F.H.; JARRETT..; LAIRD, H.M.: Feline leukemia virus infection of kittens: mortality associated with atrophy of the thymus and lymphoid depletion. J Natl Cancer Inst , BERNARD, J.: Preleukemic states. Blood Cells 2:5-7, COTTER, S.M.; HARDY, W.D.; ESSEX, M.: Association of feline leukemia virus with lymphosarcoma and other disorders in the cat. J Am Vet Med Assoc 166:449%454, DAMESHEK, W.: Some speculations on the myeloproliferative syndromes. Blood , DREYFUS, B.: Preleukemia states. Blood Cells 2:33-55, FISHER, W.B.; ARMENTROUT, S.A.; WEISMAN, R.; GRAHAM, R.C.: Pre1eukemia -a myelodysplastic syndrome often terminating in acute leukemia. Arch Intern Med , GARDNER, M.B.; ESRA, G.; CAIN, M.J.; ROSSMAN, S.; JOHNSON, C.: Myelomonocytic leukemia in an Orangutan. Vet Pathol 15:667-67, GILMORE, C.E.; HOLZWORTH, J.: Naturally occurring feline leukemia: clinical, pathologic, and differential diagnostic features, J Am Vet Med Assoc , GLICK, A.D.: HORN, R.G.; HOLSCHER. M.: Characterization of feline glomerulonephritis associated with viral-induced hematopoietic neoplasms. Am J Pathol , GOLDE, D.W.; CLINE, M.J.: Human preleukemia. N Engl J Med 288: , GRANT, C.K.; WORLEY, W.B.; DEBOER. D.J.: Detection of complement-dependent lytic antibodies in sera from feline leukemia virus-infected cats by the chromium-5 1 release assay. J Natl Cancer Inst , GREENBERG, P.L.; NICHOLS, W.C.; SCHRIER, S.L.: Granulopoiesis in acute myeloid leukemia and preleukemia. N Engl J Med 284: , HARDY, W.D.; HESS, P.W.; MACEWEN, E.G.; MCCLELLAND, A.J.; ZUCKERMAN, E.E.: ESSEX, M.; COTTER, S.M.; JARRETT..: Biology of feline leukemia virus in the natural environment. Cancer Res X , HARDY, W.D.; MCCLELLAND, A.J.: Feline oncornaviruses in Handbook of Laboratory Animal Science, ed. Melby and Altman; CRC Press, Cleveland, HERZ, A.; THEILEN. G.H.; SCHALM, O.W.; MUNN, E.J.: C-type virus in bone marrow cells of cats with myeloproliferative disorders. J Natl Cancer Inst 44: , HOOVER, E.A.; KOCIBA, G.J.: Bone lesions in cats with anemia induced by feline leukemia virus. J Natl Cancer Inst , HOOVER, E.A.; KOCIBA, G.J.; HARDY, W.D.; YOHN, D.S.: Erythroid hypoplasia in cats inoculated with feline leukemia virus. J Natl Cancer Inst JARRETT,.: Personal commun., JARRETT, W.F.H.; ANDERSON, L.J.; JARRETT,.; LAIRD, H.M.; STEWART, M.F.: Myeloid leukaemia in a cat produced experimentally by feline leukaemia virus. Res Vet Sci 12: , KILLMANN, S.: Preleukemia: does it exist? Blood Cells , KNOSPE, W.H.; GREGORY, S.A.: Smoldering acute leukemia. Arch Intern Med , LINMAN, J.W.; BAGBY, G.C.: The preleukemic syndrome (hemopoietic dysplasia). Cancer , 1978

10 Myeloid Leukemia in Cats MACKEY, L.: Feline leukaemia virus and its clinical effects in cats. Vet Rec , MACKEY, L.; JARRETT, W.F.H.: Pathogenesis of lymphoid neoplasia in cats and its relationship to immunologic cell pathways. I. Morphologic aspects. J Natl Cancer Inst , MACKEY, L.; JARRETT, W.; JARRETT,.; LAIRD, H.: Anemia associated with feline leukemia virus infection in cats. J Natl Cancer Inst , PEDERSEN, N.C.; THEILEN, G.H.; KEANE, M.A.; FAIRBANKS, L.; MASON, T.; ORSER, B.; CHEN, C.; ALLISON, C.: Studies of naturally transmitted feline leukemia virus infection. Am J Vet Res , RICCI, P.; BACCARANI, M.; ZACCARIA, A,; SANTUCCI, M.A.; TURA, S.: Clinical contribution to the knowledge of hemopoietic dysplasias; long-term follow-up of 13 patients with refractory anemia. Acta Haematol (Basel) 6: 1-2, RHEINGOLD, J.J.; KAUFMAN, R.; ADELSON, E.; LEAR, A,: Smoldering acute leukemia. N Engl J Med 268: , SAARNI, M.I.; LINMAN, J.W.: Preleukemia-the hematologic syndrome preceding acute leukemia. Am J Med , SCHALM, O.W.; JAIN, N.C.; CARROLL, E.J.: Veterinary Hematology, pg. 23, 3rd ed.; Lea & Febiger, Philadelphia, YAM, L.T.; LI, C.Y.; CROSBY, W.H.: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol , 1971 Request reprints from Bruce R. Madewell, Department of Veterinary Surgery, University of California, Davis, CA (USA).