BCL1, a Murine Model of Prolymphocytic Leukemia - NCBI - NIH

BCL1,

a

Murine

Model of Prolymphocytic

Leukemia

II. Morphology and Ultrastructure

MICHAEL J. MUIRHEAD, JAMES M. HOLBERT, Jr., JONATHAN W. UHR, and ELLEN S. VITETTA,

MD, MD,

MD,

From the Department of Microbiology, University of Texas Health Science Center at Dallas, Dallas, Texas, and Baptist Memorial Hospital, Memphis, Tennessee

PhD

The transplantable murine B-cell leukemia, BCL1, was examined by light and electron microscopy. The results show that the BCL1 cell closely resembles the prolymphocyte of human prolymphocytic leukemia (PLL). The spleen of animals transplanted with BCL1 is diffusely infiltrated by BCLI-prolymphocytes. Splenectomized animals given transplants of BCL1 develop a

lymphomalike disease with less leukemia and bone marrow involvement than the nonsplenectomized transplant recipients. Ultrastructural features of the BCL1 cell are identical in the splenectomized and nonsplenectomized animals. (Am J Pathol 1981, 105:306-315)

BCL, IS A transplantable murine B-cell leukemia

at random from the group of male BALB/c mice (Cumberland View Farms, Clinton, Tenn) studied in Part 1. Two of these mice were animals with intact spleens that had been given transplants of BCL,. Two other mice had been splenectomized and then given transplants of BCL,. The 5th mouse had been splenectomized but not transplanted and provided "control" tissues. The animals were sacrificed by cervical dislocation 14 weeks after tumor transplantation, when both the spleen-bearing and splenectomized transplant recipients were leukemic.'

with clinical and laboratory features of human prolymphocytic leukemia (PLL).' Among these are 1) "massive" enlargement of the spleen; 2) minimal lymph node enlargement; and 3) circulating lymphocytes of immature but not blastic morphology, usually exceeding 108/ml blood. The accompanying report' describes the growth kinetics and organ distribution of BCL, in spleen-bearing and splenectomized transplant recipients. The present paper is a morphologic appraisal of BCL,, which further substantiates the analogy between BCL, and human PLL. Although PLL is generally a more aggressive disease and has a more striking predilection for the spleen than chronic lymphocytic leukemia (CLL),2-4 the clinical features of PLL are not a diagnostic entity.2-5 Thus, the distinction between PLL and other B-cell leukemias rests in part on morphologic characteristics.25 Since this distinction has important prognostic and therapeutic implications,236' the purpose of this study was to establish that the BCL, cell is a prolymphocyte in the conventional morphologic sense.2'4'8

Blood Smears and Touch Preparations These were stained with Wright's.

Light Microscopy Bone marrow sections were obtained from the femur and tibia, which were decalcified before proSupported by NIH Grants Al-1 1851, AI-12789, CA-23115, and CA-28149. Accepted for publication August 13, 1981. Address reprint requests to Michael J. Muirhead, MD, Department of Microbiology, Southwestern Medical School, University of Texas Health Science Center, 5323 Harry Hines Blvd., Dallas, TX 75235.

Materials and Methods Leukemic Mice The procedures for tumor transplantation are given in the accompanying paper.' Five mice were selected

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cessing. Soft tissues were placed in Hollande's (726 mOsm) and FU-48 (432 mOsm) fixatives and embedded in paraffin. Sectioning was done on paraffin microtomes at the 4-, thickness setting. All sections for light microscopy were stained with hematoxylin and eosin (H&E). Transmission Electron Microscopy Tissues for electron microscopy (EM) were fixed in 0.1 M cacodylate-buffered 3Vo glutaraldehyde (final osmolarity 560 mOsm) followed by postfixation in cacodylate-buffered 2%o osmium tetroxide. They were then dehydrated and embedded in Spurr's medium. Semi-thin sections (1-2 ,u) were stained with toluidine blue and cut on a Porter-Blum microtome. These were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined in a Philips 300 electron microscope. Isolation of Peripheral Blood Leukocytes for Examination by EM Since routine centrifugation of peripheral blood from leukemic animals did not yield a clean buffy coat layer, the white blood cells were isolated on Ficoll-Isopaque as previously described.9 The entire Ficoll layer was removed to maximize yield and to obtain the most representative population of leukocytes. The cells were washed and then fixed in cacodylate-buffered 3'0o glutaraldehyde.

Results The two nonsplenectomized leukemic animals had identical pathologic features. The same was true for the pair of splenectomized leukemic animals. Thus, no distinction is made between individuals belonging to the same pair.

Spleen Figures 1 and 2 show spleen from a BCL1-bearing animal examined by light and electron microscopy. There is diffuse involvement of both the red and the white pulp. Mitotic figures are rare. Figure 3 shows a touch preparation of the same spleen. Examined by light microscopy, the BCL1 cell appears as a mediumsized lymphocyte with a moderate amount of cytoplasm and a chromatin structure coarser than that of a lymphoblast but less aggregated than that of a small lymphocyte. Figures 4 and 5 are electron micrographs of individual BCL1 cells in the spleen. Their outstanding characteristics are: 1) prominent nucle-

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oli; 2) dispersed chromatin in the region of the nucleolus (central euchromatin); 3) aggregated chromatin adjacent to the nuclear rim (peripheral heterochromatin); and 4) large mitochondria with widely spaced cristae. Except for the mitochondria, these are salient characteristics of the prolymphocyte described by Galton et al.2 The mitochondria appear to be exaggerated versions of mitochondria in normal cells that have been activated to high adenosine tri-

phosphate (ATP) production.10 Blood-Animals With Spleens Both animals had white blood cell counts of approximately 200 x 106/ml. The blood smear (Figure 6) shows leukemia cells with occasionally prominent nucleoli, but in this preparation they do not have the typical appearance of prolymphocytes: the chromatin appears densely aggregated, and the cytoplasm is scant. Smudge cells are numerous. In contrast, these cells have the typical appearance of prolymphocytes when examined by EM (Figure 7): prominent nucleoli, peripheral heterochromatin, and a moderate amount of cytoplasm. Mitochondria are large and numerous, as in the spleen. Blood -Splenectomized Animals

These animals were less leukemic than the spleenbearing mice; their white blood cell counts were 30-50 x 106/ml. Figure 8 shows a representative blood smear. Smudge cells are frequent here, as they are on the smear from the spleen-bearing animal. In contrast to the latter, however, polymorphonuclear leukocytes are numerous in this preparation: the differential excluding smudge cells is 49% polymorphs, 43% lymphocytes. Figures 9 and 10 show that these lymphocytes examined by EM are indistinguishable from the leukemia cells of spleen-bearing animals. Catovsky has emphasized that examination of blood smears from patients with PLL may lead to an erroneous diagnosis of CLL.3 Similarly, BCL1 leukemia cells lack the typical appearance of prolymphocytes on blood smears, but EM reveals that they are morphologically identical to the cells in the spleen. The smudge cells on blood smear bear mention because they are so numerous. Since these presumably represent damaged BCL, cells, the large proportion of polymorphonuclear leukocytes on the smears of splenectomized leukemic animals (49%) may be misleading. The differential count of blood from these animals examined by EM is 2407o polymorphs, 76% (pro)lymphocytes. However, these cells were isolated

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Figure 1-Section of spleen from BALB/c mouse 14 weeks after BCL, transplantation. Arrow indicates BCL, cell. (H&E, x400) Figure 2-Electron micrograph of spleen shown in Figure 1. BCL, cells have the morphologic features of prolymphocytes. PL = BCL,-prolymphocyte. M = mitochondrion. (x 1600) Figure 3-Touch preparation of the same spleen, showing four BCL, cells. (Wright's stain, x 2000)

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Ficoll, which may have partially excluded the granulocytes. In any case, splenectomized leukemic animals clearly have not only lower total white blood cell counts but a smaller proportion of BCL1 cells in the white blood cell population a conclusion conon

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firmed by the immunofluorescence data reported in Part I of this study.

Lymph Node Fourteen weeks after tumor transplantation the

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Figure 4-Electron micrograph of BCL, cell from the spleen. A large mitochondrion (M) is apposed to the nuclear membrane. (x 4000) Fig5-Electron micrograph of BCL, cell from the spleen. Note the pattern of central euchromatin and peripheral heterochromatin. (x 4000) Figure 6-Blood smear of a BALB/c mouse with an intact spleen 14 weeks after BCL, transplantation. There is a smudge cell adjacent to two leukemia cells. Chromatin appears densely aggregated and cytoplasm scant in this preparation. (Wright's, x 1500) Figure 7-Electron micrograph of leukemia cells from a BALB/c mouse with intact spleen 14 weeks after BCL, transplantation. The cells are morphologically identical to BCL, cells in the spleen. (x 2000) (All with a photographic reduction of 4%) ure

lymph nodes of animals with intact spleens were only slightly enlarged, even though as many as 30%7o of the lymph node cells were of tumor origin, as assessed by indirect immunofluorescence with the use of a tumorspecific antiidiotypic antibody. I In contrast, the lymph nodes of splenectomized transplant recipients were massively enlarged, and more than 50% of the cells were idiotype-positive.I The histopathologic charac-

ter of the lymph node in the spleen-bearing mice, however, is similar to that of the lymph node in splenec-

tomized transplant recipients (Figures 11 and 12). Like spleen, both peripheral and mesenteric lymph nodes are diffusely infiltrated by tumor cells. Figures 13 and 14 are electron micrographs of lymph node from a splenectomized leukemic animal and the splenectomized control animal, respectively. Lymph node

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Figure 8-Blood smear of a splenectomized BALB/c mouse 14 weeks after BCL, transplantation. Polymorphonuclear leukocytes (P) are numerous. L = BCL, leukemia cell. (Wright's Figure 9-Electron microstain, x 400) graph of leukemia cells from a splenectomized BALB/c mouse 14 weeks after BCL, transplantation. The lymphocytes are morphologically identical to BCL, cells in spleen-bearing mice. P = polymorphonuclear leukocyte. L = BCL, Figure 10-Elecleukemia cell. (x 2000) tron micrograph of a leukemia cell from a splenectomized BALB/c mouse 14 weeks after BCL, transplantation. (x 4000)

cells from the leukemic animal but not the control have the morphologic characteristics of prolymphocytes. Liver The histopathologic characteristics of liver are strik-

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ingly different in spleen-bearing and splenectomized leukemic animals (Figures 15 and 16). In the former, the hepatic sinusoids are dilated and filled with leukemia cells, whereas the parenchyma is relatively uninvolved. In contrast, sinusoidal dilatation is absent in the splenectomized animal, but a tumor nodule in

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I Figure 11-Section of lymph node from BALB/c mouse with intact spleen 14 weeks after BCL, transplantation, showing diffuse infiltration by BCL, cells (arrows). (H&E, x 400) Figure 12-Section of lymph node from a splenectomized BALB/c mouse 14 weeks after BCL, transplantation, showing diffuse infiltration by BCL, cells (arrows). (H&E, x 400) Figure 13-Electron micrograph of lymph node from a splenectomized BALB/c mouse 14 weeks after BCL, transplantation. PL = BCL,-prolymphocyte. (x 2000) Figure 14-Electron micrograph of lymph node from splenectomized BALB/c mouse that was not transplanted with BCL, cells. There are no prolymphocytes. (x 2000) (All with a photographic reduction of 4%)

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of lymphoma. Touch preparations of liver from b(oth spleen-bearing and splenectomized leukemic arnimals looked much the same as the touch preparati on of the spleen (Figure 3). Bione Marrow Figures 17 and 18 show bone marrow sections from

the splenectomized control and a splenectomized leukemic animal. Marrow from the latter contains increased numbers of medium-sized cells with palestaining nuclei and prominent nucleoli, compared with the control. Marrow from a leukemic spleenbearing animal (Figure 19) shows an obvious

dominance of the same cell type. Figures 20-22 preare electron micrographs of these same tissues. The con-

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Figure 15-Section of liver from a BALB/c mouse with an intact spleen 14 weeks after BCL, transplantation. The sinusoids are dilated and filled with BCL, cells (arrows), but the hepatic parenchyma is intact. H = hepatocyte. (H&E, x 400) Figure 16-Section of liver from splenectomized BALB/c mouse 14 weeks after BCL, transplantation. There is a tumor nodule within the hepatic parenchyma. The margins of the nodule are outlined by the arrows, and a rim of hepatic parenchyma is faintly visible in the upper right-hand corner. (H&E, x 250)

trol section shows some cells that resemble prolymphocytes. Marrow from the splenectomized leukemic animal contains many granulocytes; but 5-10% of the cells have the morphologic characteristics of BCL1, including the large mitochondria. The marrow of the spleen-bearing animal is heavily infiltrated with BCL1 cells, most of them containing large and bizarre mitochondria. The results indicate that transplanted BCL1 is a diffuse disease that involves every organ examined, although the extent of disease varies from organ to organ and between spleen-bearing and splenectomized transplant recipients. The latter have a lymphomalike disease with massively enlarged lymph nodes, tumor nodules in the liver, low-grade leukemia, and minimal bone marrow involvement. The morphologic features of the BCL, cell, however, are unchanged by splenectomy.

amount of cytoplasm, prominent nucleolus, central

Discussion

euchromatin, and peripheral heterochromatin. This finding reinforces the argument, clearly supported by pathophysiologic and immunologic data,' that BCL1 is a murine analog of human PLL. In contrast, a previous study13 concluded that BCL1 is a "well-differentiated lymphocytic lymphoma." This group of investigators qualified their morphologic diagnosis by noting that BCL, has a striking predilection for the spleen and terminates with a "leukemic phase." The nuclear morphology of the BCL, cell in their preparations appears the same as that of a small lymphocyte. Our own experience suggests that the appearance of the BCL, cell when it is spread on glass or stained with H&E in routine sections is misleading, because the chromatin pattern is poorly delineated. The chromatin structure of BCL, is neither that of a blast cell nor that of a small lymphocyte, but intermediate between the two. In morphologic terms, BCL, is an "immature" lymphocyte. The immunologic pheno-

The BCL, cell has the morphologic characteristics that define the "prolymphocyte"2'4 8: a moderate

and thereby provides confirmation of the morphologic interpretation.

1.1-1

type of BCL1 is that of an "intermediate" B cell'2-17

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Figure 17-Section of bone marrow from a splenectomized BALB/c mouse that was not given transplants of BCL, cells. MK = megakaryocyte. (H&E, x 400) Figure 18 -Section of bone marrow from a splenec-

tomized BALB/c mouse 14 weeks after BCL, transplantation. Some cells have the morphologic features of BCL, (arrows). (H&E, x 400) Figure 19-Section of bone marrow from a BALB/c mouse with an intact spleen 14 weeks after BCL, transplantation. There are many cells with the morphologic features of BCL, (arrow). (H&E, x 400)

Quantitative data based on the identification of BCL1 cells by idiotype analysis' is generally consistent with the morphologic data presented in this paper. However, there are some discrepancies that bear mention. Indirect immunofluorescence with an anti-

idiotypic antibody indicated that the bone marrow of leukemic spleen-bearing animals contained 5-20%o BCL1 cells.1 In contrast, the light-microscopic and EM sections of bone marrow from spleen-bearing animals show very heavy tumor cell infiltration-

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Figure 20-Electron micrograph of bone marrow from a splenectomized BALB/c mouse that was not given transplants of BCL, cells. ( x 2000) Figure 21Electron micrograph of bone marrow from splenectomized BALB/c mouse 14 weeks after BCL, transplantation. Polymorphonuclear leukocytes predominate, but one cell has the morphologic features of the BCL,prolymphocyte (PL). M = mitochondrion. (x 2000) Figure 22-Electron micrograph of bone marrow f rom a BALB/c mouse with an intact spleen 14 weeks after BCL, transplantation. All cells appearing here have the morphologic features of BCL,-prolymphocytes (PL). M = mitochondrion. (x 2000)

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about 5007o of the cells in these sections are morphologically identical to BCL1 cells in spleen and blood, where idiotype-positive cells predominate. There is a quantitatively smaller discrepancy between the morphologic and immunofluorescence data obtained from bone marrow of splenectomized leukemic mice.

EM indicates that 5-lOWo of the cells have the morphologic characteristics of BCL1, whereas immunofluorescence showed virtual absence of idiotypepositive cells in the marrow of these animals.' Since the immunofluorescence and the pathologic examinations were not performed on tissues from the

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same individual mice, it is impossible to confirm that all the animals had comparable tumor burdens. It is possible that not all BCL1 cells carry the idiotypic determinants or that morphologic analysis cannot distinguish between BCL, and certain cells of host origin. The use of an independent BCL1 marker such as karyotype'8 should provide a more definitive means of determining whether all BCL1 cells bear the BCL,-immunoglobulin idiotype. Nevertheless, morphologic examination as well as immunofluorescence appear to be useful for the study of BCL, kinetics and organ distribution, including the response to therapy, which is now being implemented with tumor-specific antiidiotype antibody coupled to one of the peptides of the plant toxin ricin. 19 It is to be hoped that the therapeutic outcome will make the analogy between this murine leukemia and human disease especially relevant and exciting.

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References 1. Muirhead MJ, Isakson PC, Krolick KA, Uhr JW, Vitetta ES: BCL,, a murine model of prolymphocytic leukemia: I. Effect of splenectomy on growth kinetics and organ distribution. Am J Pathol, 1981, 105:000000 2. Galton DAG, Goldman JM, Wiltshaw E, Catovsky D, Henry K, Goldenberg GJ: Prolymphocytic leukemia. Br J Haematol 1974, 27:7-23 3. Catovsky D: Hairy-cell leukaemia and prolymphocytic leukaemia. Clin Haematol 1977, 6:245-268 4. Costello C, Catovsky D, O'Brien M, Galton DAG: Prolymphocytic leukaemia: An ultrastructural study of 22 cases. Br J Haematol 1980, 44:389-394 5. Katayama I, Aiba M, Pechet L, Sullivan JL, Roberts P, Humphreys RE: B-lineage prolymphocytic leukemia as a distinct clinicopathologic entity. Am J Pathol 1980, 99:399-412 6. Konig E, Meusers P, Brittinger G: Efficacy of doxorubicin in prolymphocytic leukaemia. Br J Haematol 1979, 42:487-488 7. Sibbald R, Catovsky D: Complete remission in prolymphocytic leukaemia with the combination chemotherapy-CHOP. Br J Haematol 1979, 42:488-490 8. Bearman RM, Pangalis GA, Rappaport H: Prolym-

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phocytic leukemia: Clinical, histopathological, and cytochemical observations. Cancer 1978, 42:2360-2372 Ligler FS, Vitetta ES, Uhr JW: Cell surface immunoglobulin: XXII. Reappearance of surface IgM and IgD on murine splenocytes after removal by capping. J Immunol 1977, 119:1545-1546 Novikoff AB, Holtzman E: Cells and Organelles. New York, Holt, Rinehart and Winston, 1976, p 125 Slavin S, Strober S: Spontaneous murine B-cell leukaemia. Nature 1978, 272:624-626 Knapp MR, Jones PP, Black SJ, Vitetta ES, Slavin S, Strober S: Characterization of a spontaneous murine B cell leukemia (BCL,): I. Cell surface expression of IgM, IgD, Ia, and FcR. J Immunol 1979, 123:992-999 Warnke RA, Slavin S, Coffman RL, Butcher EC, Knapp MR, Strober S, Weissman IL: The pathology and homing of a transplantable murine B cell leukemia (BCL,). J Immunol 1979, 123:1181-1188 Krolick KA, Isakson PC, Uhr JW, Vitetta ES: Murine B cell leukemia (BCL,): Organ distribution and kinetics of growth as determined by fluorescence analysis with an anti-idiotypic antibody. J Immunol 1979, 123:19281935 Krolick KA, Isakson PC, Uhr JW, Vitetta ES: BCL,, a murine model for chronic lymphocytic leukemia: Use of the surface immunoglobulin idiotype for the detection and treatment of tumor. Immunol Rev 1979, 48: 81-106 Isakson PC, Uhr JW, Krolick KA, Finkelman F, Vitetta ES: Acquisition of cell surface IgD after in vitro culture of neoplastic B cells from the murine tumor BCL,. J Exp Med 1980, 151:749-754 Isakson PC, Krolick KA, Uhr JW, Vitetta ES: The effect of anti-immunoglobulin antibodies on the in vitro proliferation and differentiation of normal and neoplastic murine B cells. J Immunol 1980, 125:886-892 Strober S, Gronowicz ES, Knapp MR, Slavin S, Vitetta ES, Warnke RA, Kotzin B, Schroder J: Immunobiology of a spontaneous murine B cell leukemia (BCL,). Immunol Rev 1979, 48:169-195 Krolick KA, Villemez C, Isakson P, Uhr JW, Vitetta ES: Selective killing of normal or neoplastic B cells by antibodies coupled to the A chain of ricin. Proc Natl Acad Sci USA 1980, 77:5419-5423

Acknowledgments We wish to express our gratitude to Dr. James A. Pitcock, Mrs. Peggy S. Brown, and Mrs. Kay Nabers for their tremendous technical assistance and Ms. Daisi Marcoulides for excellent secretarial assistance.