Leukemic Phase of Mantle Cell Lymphoma, Blastoid Variant

Hematopathology / LEUKEMIC BLASTOID MANTLE CELL LYMPHOMA Leukemic Phase of Mantle Cell Lymphoma, Blastoid Variant Timothy P. Singleton, MD, Margaret M. Anderson, MD, Charles W. Ross, MD, and Bertram Schnitzer, MD Key Words: Malignant lymphoma; Mantle cell; Blastoid or blastic variant; Leukemic Six patients with mantle cell lymphoma, blastoid variant, involving the blood are described. The circulating blast-like cells suggested the possibility of acute leukemias, chronic lymphoproliferative disorders or peripheralized lymphomas. The WBC counts ranged from 3,700 to 249,000/pL (3.7-249.0 x 109/L) and the absolute lymphocyte counts from 1,000 to more than 200,000/pL (1.0 to >200.0 x 109/L). The peripheral blood smears showed a spectrum of cells, from small mature lymphocytes with irregular nuclei to mediumsized lymphocytes with blast-like chromatin. However, the morphologic features in a lymph node biopsy specimen and the immunophenotype confirmed a diagnosis of mantle cell lymphoma, blastoid variant. By flow cytometry the lymphoma cells expressed Bcell-associated antigens (CD19, CD20 and CD22), coexpressed CD5, lacked CD23, and expressed moderate intensity monoclonal surface immunoglobulin and CD20. Cytogenetic analysis showed the characteristic t(ll;14) in 2 of 4 analyzed specimens. Mantle cell lymphoma, blastoid variant, is part of the differential diagnosis for blast-like cells. Mantle cell lymphoma seems to be a distinct clinicopathologic entity.i2 The term mantle cell lymphoma has been proposed to replace mantle zone lymphoma, intermediately differentiated lymphocytic lymphoma, intermediate lymphocytic lymphoma, lymphocytic lymphoma of intermediate differentiation, and centrocytic lymphoma.12 In the Working Formulation, centrocytic lymphoma was included in the category of diffuse small cleaved cell lymphoma,3 but the latter term is generally not used today, to avoid confusion with follicular center cell lymphomas arising from germinal centers. Mantle cell lymphoma originates in the mantle zone, which is located adjacent to germinal centers.4 Mantle cell lymphoma consists of small B cells coexpressing CD5 and monoclonal surface immunoglobulin. It is associated with t(ll;14) involving bcl-\ and the immunoglobulin heavy chain genes, resulting in deregulated expression of cyclin D1/PRAD1.2-5-7 However, the grade has been disputed. Mantle cell lymphoma treated with traditional chemotherapy has the incurability of a low-grade lymphoma but the aggressive clinical course akin to intermediate grade.''2'8'9 A blastoid (blastic or lymphoblastoid) variant has been described based on morphologic changes in the lymph node and an even poorer prognosis.1,9"" We describe mantle cell lymphoma, blastoid variant, in leukemic phase, which, to our knowledge, has not been reported. There have been descriptions of the leukemic phase in classic mantle cell lymphoma, however, and some of these cases might have been the blastoid variant. 12~16 Since the cells have blast-like features morphologically, the differential diagnosis is broad and includes acute leukemias, chronic lymphoproliferative disorders, and other types of peripheralized lymphomas. Materials and Methods Patient Selection Between 1992 and 1995, 6 patients underwent lymph node biopsies that revealed the blastoid variant of mantle cell 495 Abstract

Singleton et al / LEUKEMIC BLASTOID MANTLE CELL LYMPHOMA lymphoma, and the patients were found to have peripheral blood involvement. The patients were initially diagnosed at outside institutions and were referred to the University of Michigan Medical Center, Ann Arbor, for further care. Clinical data, including mode of presentation, therapy, and clinical outcome, were obtained by medical record review. Morphologic Features Immunohistochemistry Formalin-fixed paraffin-embedded tissues were deparaffinized in xylene and alcohol and then stained with the following antibodies: L26 (CD20, titer 1:500, DAKO, Carpinteria, CA), Leu22 (CD43, titer 1:100, Becton Dickinson, San Jose, CA), A6 (CD45RO, titer 1:50, Zymed, South San Francisco, CA), and anti-terminal deoxynucleotidyl transferase (TdT, rabbit polyclonal, titer 1:5, Supertechs, Bethesda, MD). For CD20 (L26), antigen retrieval was performed by microwaving tissue sections in 1.6 L of a 10-mmol/L concentration of citrate buffer (ph 6.0) inside a pressure cooker (Tender Cooker, Nordic Ware, Minneapolis, MN) in a 1,000-W microwave at full power for 15 minutes. TdT antigen was retrieved by microwaving tissue sections in a plastic slide holder (24-slide capacity) filled with a 10mmol/L concentration of citrate buffer (ph 6.0) in the 1,000W microwave oven at full power for 10 minutes. After either microwave protocol, the slides were cooled by rinsing in distilled water for 5 minutes. The immunohistochemistry was done with an automated stainer (Ventana 320, Ventana Medical Systems, Tucson, AZ) and the Ventana DAB Detection Kit. Briefly, the kit has the following reagents added in sequential steps (36 C): inhibitor for endogenous peroxidase, primary antibody for 32 minutes, biotinylated secondary antibody, avidin-biotin-complex with horseradish peroxidase, DAB Flow Cytometry Mononuclear cells were isolated from heparinized peripheral blood or bone marrow by Ficoll-Hypaque density gradient centrifugation. Two-color direct immunofluorescence staining was performed using the following panel of reagents: anti-k (TB28-2), anti-a, (1-155-2), CD2 (Leu 5b), CD3 (Leu 4), CD4 (Leu 3a), CD5 (Leu 1), CD7 (Leu 9), CD8 (Leu 2a), CD10 (anti-calla, W8E7), CDllc (Leu M5), CD14 (Leu M3), CD19 (Leu 12), CD20 (Leu 16), CD22 (Leu 14), and CD45 (HLe-1) purchased from Becton Dickinson. Anti-IgG, anti-iga, anti-igm, and anti-igd are polyclonal antibodies (Tago, Camarillo, CA). The stained cells were analyzed on a flow cytometer (FACScan, Becton Dickinson). Cytogenetics Analysis of peripheral blood (3 patients) and bone marrow (1 patient) was performed on cells harvested from short-term cultures by standard cytogenetic methods. Results Clinical Data The 6 patients, 5 men and 1 woman, ranged in age from 47 to 74 years at diagnosis (mean, 64 years) II able II. All patients were examined because of fatigue, weakness, anorexia, and/or palpable lymphadenopathy. The physical and radiologic examinations revealed generalized lymphadenopathy in all 6 patients. Five patients had splenomegaly, and 2 had hepatomegaly. The patients were referred to the University of Michigan with diagnoses of diffuse small cleaved cell lymphoma, chronic lymphocytic leukemia, and diffuse large cell lymphoma. However, these were reclassified based on the lymph node morphologic features and immunophenotype as mantle cell lymphoma, blastoid variant. Five patients had de novo blastoid variant, and the disease in patient 1 transformed Table II Clinical Features of Patients With Mantle Cell Lymphoma, Blastoid Variant, in Leukemic Phase Patient No. 1 2 3 4 5 6 496 Sex/Age (y) M/74 tvl/65 F/67 M/71 M/47 M/58 Sites of Involvement by Lymphoma Peripheral blood, lymph node, spleen, bone marrow Peripheral blood, lymph node, spleen, liver, bone marrow Lymph node, spleen, bone marrow Peripheral blood, lymph node, spleen, liver, bone marrow Lymph node, spleen, bone marrow Lymph node, bone marrow Clinical Outcome Died of disease, 4 y after diagnosis Alive with disease, 2 mo after diagnosis Alive with disease, 31 mo after diagnosis Alive with disease, 8 mo after diagnosis Alive with disease, 36 mo after diagnosis Alive with disease, 40 mo after diagnosis The peripheral blood and bone marrow aspirates were air dried and stained with Wright-Giemsa. Bone marrow and lymph node biopsy specimens were fixed in B5 or buffered formalin, processed by standard histologic methods, embedded in paraffin, and stained with H&E. (3,3'-diaminobenzidine tetrahydrochloride)/hydrogen peroxide, and copper enhancer.

Hematopathology / ORIGINAL ARTICLE diagnosis, and the remaining 4 treated patients have progressive or recurrent disease at 8 to 40 months after diagnosis. One patient had gastric rupture due to extensive involvement by lymphoma and has not yet received chemotherapy. He was alive with extensive disease at 2 months after diagnosis. to the blastoid variant 4 years after the diagnosis of classic mantle cell lymphoma was made. The peripheral blood findings showed that the disease in 2 patients was in the leukemic phase of blastoid mantle cell lymphoma. One patient (patient 1) had classic mantle cell lymphoma with lymphocytosis that later transformed to the leukemic phase of the blastoid variant. The other 3 patients had documentation of the leukemic phase after therapy and subsequent transfer of their care to the University of Michigan; whether the leukemic phase was present previously is unknown. The hematologic data are from the time that leukemic phase of mantle cell lymphoma, blastoid variant, was documented ITable 21. The WBC counts ranged from 3,700 to 249,000/u.L (3.7-249.0 x 109/L). The absolute lymphocyte counts ranged from 1,000 to 244,000/uL (1.0-244 x 109/L). The hemoglobin concentration ranged from 8.3 to 12.7 g/dl (83-127 g/l), and the platelet counts ranged from 53 to 254 x 103/uL (53.0-254.0 x 109/L). Five of the 6 patients are alive. Five patients have been treated with systemic chemotherapy, 3 with 2 or more regimens. One died of lymphoma 4 years after Peripheral Blood Morphologic Features Bone Marrow Morphologic Features All 6 patients had bone marrow involvement at diagnosis and at the time of peripheral blood involvement. The ITable 21 Blood Cell Counts in Patients With Mantle Cell Lymphoma, Blastoid Variant, in Leukemic Phase Patient No. Previous Chemotherapy WBC Count, /ul (x 109/L) Absolute Lymphocyte Count, /ul (x 109/L) Hemoglobin Concentration, g/dl (g/l) Platelet Count, x KrVnL (x 10«/L) No No 249,000 (249.0) 23,100(23.1) 10,000(10.0) 11,400(11.4) 3,700 (3.7) 6,500 (6.5) >200,000 (>200.0) 14,000(14.1) 5,500 (5.5) 4,700 (4.7) 1,500(1.5) 1,000(1.0) (hematocrit, 33% [0.33]) 10.1 (101) 10.2(102) 8.3 (83) 12.7(127) 11.9(119) 83 (83) 152(152) 138(138) 176(176) 53 (53) 254 (254) B Image I I Peripheral blood smear in leukemic mantle cell lymphoma, blastoid variant. A, A spectrum of cells with slightly irregular nuclear contours, ranging from small mature lymphocytes with coarse chromatin to medium-sized cells with blast-like chromatin and nucleoli. B, Two more blast-like cells are shown (Wright-Giemsa, x1,000). 497 The circulating lymphoma cells showed a spectrum of morphologic features. The cells had slightly irregular nuclear contours and ranged from small lymphocytes with moderately coarse chromatin to medium-sized cells with finer blast-like chromatin llmage II. The blast-like cells had high nuclear/cytoplasmic ratios, relatively fine chromatin, and often 1 to 2 prominent nucleoli. The number of blast-like cells varied from infrequent to numerous. The smaller lymphocytes resembled the type that are seen in leukemic phase of classic mantle cell lymphoma.

Singleton et al / LEUKEMIC BLASTOID MANTLE CELL LYMPHOMA patterns of marrow involvement were focal (nodular), interstitial, or diffuse. Although occasional paratrabecular aggregates were noted, this pattern did not predominate. The aggregates often were poorly circumscribed. In the core biopsy specimens, the lymphoid cells were small and occasionally blast-like. In the marrow aspirates, the lymphoma cells were similar to those described in the peripheral blood, with few to numerous blast-like cells. Lymph Node Morphologic Features Immunophenotype Immunohistochemical stains were performed on the paraffin-embedded lymph nodes. In all cases, the neoplastic cells coexpressed CD20 (L26) and CD43 (Leu22) but lacked CD45RO (A6) or TdT. Image 21 Lymph node morphology in mantle cell lymphoma, blastoid variant. There are medium-sized lymphoid cells with slightly irregular nuclear contours, moderately fine chromatin, and a high mitotic rate. There are scattered histiocytes but no prolymphocytes, paraimmunoblasts, or large lymphoid cells (H&E, x400). 498 Cytogenetics Cytogenetic studies were performed on the peripheral blood from 4 patients and on a bone marrow aspirate from 1. Two patients had t(ll;14), and 2 had normal cytogenetics. One of the patients with normal cytogenetics had suboptimal cell growth. Discussion Mantle cell lymphoma is thought to arise in the mantle zone adjacent to the germinal center.4 Clinically, it is important to recognize because it generally has a more aggressive course than low-grade lymphomas but does not seem to be cured with traditional chemotherapy. 128 ' 9 A blastoid (lymphoblastoid or blastic) variant with blast-like morphologic features, high mitotic rate, and poorer prognosis than the classic type has been described.i,9_11 We describe the blastoid variant with peripheral blood involvement, which has not been previously reported. When confronted with these circulating blast-like cells, the differential diagnosis is broad and includes acute leukemia, prolymphocyte leukemia, peripheralized large cell lymphoma, chronic lymphocytic leukemia, or peripheralized follicular center cell lymphoma. There have been descriptions of the leukemic phase in classic mantle cell lymphoma, however, and some of these cases might have been the blastoid variant.'2-16 For example, 1 report describes a mantle cell lymphoma with a high mitotic rate, peripheral blood lymphocytosis, an aggressive clinical course, and difficulty distinguishing the disease from acute leukemia.16 And in a series of 12 patients, 2 had an aggressive disease with an S phase fraction that was in the range of high-grade lymphomas.13 Also, cases published previously as leukemic nodular poorly differentiated lymphocytic lymphoma with cytologic features akin to lymphoblasts might represent leukemic blastoid mantle cell lymphoma.17 In leukemic mantle cell lymphoma, blastoid variant, there is a spectrum of circulating lymphoma cells with slightly irregular nuclear contours, ranging from small lymphocytes with moderately coarse chromatin to mediumsized blast-like cells with finer blast-like chromatin. The Five patients had a blastoid variant of mantle cell lymphoma at diagnosis. One originally had a classic mantle cell lymphoma that later transformed to a blastoid variant. The lymph nodes with mantle cell lymphoma, blastoid variant, were diffusely replaced by lymphoma in 4 cases. Two cases had a vaguely nodular pattern. A high mitotic rate was observed in all, and scattered histiocytes were present Image 21. The lymphoma cells were small to medium-sized with slightly irregular nuclear contours, moderately fine blast-like chromatin, and small or inconspicuous nucleoli. No pseudofollicular proliferation centers, prolymphocytes, paraimmunoblasts or large lymphoid cells were present. Immunophenotyping was performed on the peripheral blood or bone marrow specimen by flow cytometry. The cases expressed CD 19 (6/6), CD20 (6/6, moderate intensity in all), CD5 (6/6), and FMC7 (4/4) but lacked CD 10 (0/6) and CD23 (0/4). All cases expressed monoclonal surface immunoglobulin of moderate intensity (6/6). Three cases expressed A. light chain, and 3 expressed K. Moderate intensity IgM with weak to moderate intensity IgD was present in 4 of 6 cases, and moderate intensity IgM without IgD was present in 2 of 6.

H e m a t o p a t h o l o g y / ORIGINAL ARTICLE nuclear clefts. The follicular lymphomas often express CD10 but lack CD5 and are associated with a t(14;18). The peripheral blood smear in the mixed cell type of chronic lymphocytic leukemia15 has less pleomorphism and rounder nuclei and may have intermingled prolymphocytes. However, some cases of chronic lymphocytic leukemia may have irregular nuclear contours, and these may be difficult to distinguish morphologically from mantle cell lymphoma. Immunophenotyping by flow cytometry is helpful in this differential diagnosis. Chronic lymphocytic leukemia expresses CD23 and weak intensity surface immunoglobulin and CD20, while mantle cell lymphoma lacks CD23 and expresses moderate to strong intensity surface immunoglobulin and CD20. Staining with FMC7 is seen more often with mantle cell lymphoma. However, a lymph node biopsy may be necessary for precise classification. The lymph node in chronic lymphocytic leukemia contains paraimmunoblasts and pseudofollicular proliferation centers, which are absent in mantle cell lymphoma. We described 6 patients with the leukemic phase of mantle cell lymphoma, blastoid variant. Morphologic examination, including a lymph node biopsy, and immunophenotype are generally required for a definitive diagnosis. Cytogenetic studies may be confirmatory. When confronted with a peripheral blood smear containing blast-like cells, the possibility of mantle cell lymphoma should be considered. From the Department of Pathology, University of Michigan Medical Center, Ann Arbor. Address reprint requests to Dr Singleton: Department of Pathology, University of Michigan Medical School, Medical Science Building IM5242, 1301 Catherine Rd, Ann Arbor, MI 48109-0602. References 1. Harris NL, Jaffe ES, Stein H, et al. A revised EuropeanAmerican classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood. 1994;84:1361-1392. 2. Banks PM, Chan J, Cleary ML, et al. Mantle cell lymphoma: a proposal for unification of morphologic, immunologic, and molecular data. Am] Surg Pathol. 1992;16:637-640. 3. National Cancer Institute-sponsored study of classifications of non-hodgkin's lymphomas: summary and description of a working formulation for clinical usage: the Non-Hodgkin's Lymphoma Pathologic Classification Project. Cancer. 1982;49:2112-2135. 4- Weisenburger DD, Chan W C. Lymphomas of follicles: mantle cell and follicle center cell lymphomas. Am J Clin Pathol. 1993;99:409-420. 5. Ott MM, O t t G, Kuse R, et al. T h e anaplastic variant of centrocytic lymphoma is marked by frequent rearrangements of the bcl-l gene and high proliferation indices. Histopathology. 1994;24:329-334. 499 smaller lymphocytes resemble those seen in leukemic mantle cell lymphoma of the classic type. In the lymph node, mantle cell lymphoma may have a mantle zone, vaguely nodular, or diffuse architectural pattern. Large noncleaved cells or large transformed immunoblast-like cells are rarely, if ever, seen. Pseudofollicular proliferation centers, paraimmunoblasts, and prolymphocytes are absent. Compared with the classic type of mantle cell lymphoma, the blastoid variant has more pleomorphism, slightly larger nuclei, finer blast-like chromatin, and a higher mitotic rate in lymph node biopsy specimens. The blastoid variant and classic type are reported to have an identical immunophenotype, except for proliferation markers, and our study supports this finding. 5 ~ 710 The neoplastic cells express B-cell-associated antigens (CD 19, CD20, CD22), coexpress CD5, and lack CD23. There is moderate to strong intensity monoclonal surface immunoglobulin and CD20. Also, mantle cell lymphoma is associated with t(ll;14). This translocation has been reported to involve the juxtaposition of the bcl-l gene at 11 q 13 with the immunoglobulin heavy chain gene at 14q32 and results in deregulation of cyclin D1/PRAD1. 5-7 We found the t( 11; 14) in 2 of 4 patients. When confronted with a peripheral blood smear containing blast-like cells, the differential diagnosis is broad and includes acute leukemias, chronic lymphoproliferative disorders, and other types of peripheralized lymphoma. Although cases with numerous blast-like cells suggest acute leukemias, the latter has blasts with finer chromatin and fewer admixed mature lymphocytes with coarse chromatin. In leukemic blastoid mantle cell lymphoma, the chromatin pattern of most blast-like cells is generally not as finely granular as that seen in L2 lymphoblasts, and the small circulating lymphocytes of blastoid mantle cell lymphoma typically have coarse chromatin with a sharp distinction between the parachromatin and euchromatin as opposed to the more homogeneous smudgy chromatin of LI lymphoblasts, in well-prepared smears. Ancillary studies, however, may be necessary to make a definitive diagnosis. Acute lymphoblastic leukemia (French-American-British types LI and L2) lacks surface immunoglobulin and expresses TdT; blastoid mantle cell lymphoma has monoclonal surface immunoglobulin and lacks TdT. Acute myeloid leukemia expresses myeloid antigens. Large cell lymphomas rarely coexpress CD5, they may have cytoplasmic vacuoles, and they have larger cells, often with coarser chromatin. However, the chromatin pattern of large cell lymphoma may be blast-like. Prolymphocytic leukemia is distinguished by minimal lymphadenopathy and the morphologic features of the prolymphocyte with its prominent central nucleolus, coarser chromatin, and a round nuclear contour. The small cleaved cell of follicular center cell lymphoma is smaller with coarser chromatin and

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