Hematopathology / MARGINAL ZONE LYMPHOMA INVOLVING BONE MARROW

Few studies have characterized or compared the pathologic features of bone marrow involvement by extranodal (EMZL), splenic (SMZL), and nodal marginal zone lymphoma (NMZL). We evaluated 45 bone marrow biopsy specimens from 39 patients with marginal zone lymphomas. As previously reported, bone marrow involvement was frequent (100%) in patients with SMZL. We also identified lymphoma involving bone marrow in 11 (44%) of 25 patients with EMZL and 1 of 2 patients with NMZL. The patterns of infiltration were mixed in all groups; however, the extent of involvement was greater in SMZL than in EMZL. In addition, germinal centers were present in bone marrow biopsy specimens involved by lymphoma in 4 patients with SMZL. Intrasinusoidal infiltration was common (10/12 [83%]) and prominent in patients with bone marrow involvement by SMZL, but was not invariably present. Intrasinusoidal infiltration of the bone marrow also was not specific for SMZL since similar infiltrates, although subtle, also were found in patients with other small B-cell lymphoproliferative disorders, including 6 (55%) of 11 patients whose bone marrow samples were infiltrated by EMZL. Marginal zone lymphomas are indolent B-cell lymphomas that include extranodal marginal zone lymphoma (EMZL), nodal marginal zone lymphoma (NMZL), and splenic marginal zone lymphoma (SMZL). The architectural and cytomorphologic features of these lymphomas support the hypothesis that they arise from B cells analogous to those of the marginal zone. EMZL, also known as mucosa-associated lymphoid tissue lymphoma (MALT lymphoma), involves mucosa-associated or related extranodal sites such as stomach, salivary glands, ocular adnexa, lung, skin, breast, and thyroid gland. EMZL tends to remain localized, occasionally progressing to a high-grade lymphoma. NMZL, also called monocytoid B-cell lymphoma, manifests with peripheral lymphadenopathy without involvement of extranodal sites. NMZL may have a shorter overall survival and failurefree survival than EMZL.1 SMZL also manifests as a lowgrade lymphoma, but is distinct from EMZL and NMZL because it manifests with marked splenomegaly and frequent bone marrow and peripheral blood involvement. The pathogenetic relationship among EMZL, NMZL, and SMZL is controversial and has been studied by morphologic, immunophenotypic, cytogenetic, and molecular studies. Morphologically, EMZL and NMZL are most similar with a proliferation of marginal zone cells including centrocyte-like cells, monocytoid B cells, occasional immunoblast-like cells, and other large cells.2-7 Reactive germinal centers with wellpreserved polyclonal mantle zones are common features of EMZL and NMZL. SMZL is characterized by a nodular expansion of the splenic white pulp by intermediate-sized lymphocytes that often surround germinal centers.8 A biphasic pattern may be present with centrocyte-like cells simulating a mantle zone enveloped by a zone of larger forms, but SMZL Hematopathology / ORIGINAL ARTICLE Am J Clin Pathol 2002;117:698-708 699 © American Society for Clinical Pathology is different from EMZL and NMZL in that both of these zones are composed of the same clonal B-cell population.9 Immunophenotyping of marginal zone lymphoma demonstrates B-cell lineage (CD19+, CD20+) with monotypic immunoglobulin light chain restriction, CD10–, CD5–, and CD43+/–. SMZL is often IgM+IgD+,8,9 whereas EMZL and NMZL are usually IgM+ and IgD–.10 Similar cytogenetic abnormalities in EMZL, NMZL, and SMZL include trisomy 3, trisomy 7, trisomy 12, and trisomy 18.11-16 Trisomy 3, the most frequent numeric chromosomal abnormality in EMZL, is likely a secondary chromosomal abnormality because it usually is seen in association with other chromosomal abnormalities and may be seen in other nonHodgkin lymphomas. On the other hand, the most common structural chromosomal aberration found in EMZL, t(11;18)(q21;q21),17-19 likely has a key role in lymphomagenesis since it often is seen as a sole abnormality. The fact that t(11;18) has not been found in NMZL or SMZL lends support to the hypothesis that EMZL is an entity different from NMZL and SMZL.14,20,21 Likewise, del7q31-32 is described only in SMZL and supports the distinct nature of this lymphoma.22 Marginal zone lymphomas also differ in the reported incidence of dissemination to bone marrow. SMZL almost always involves the bone marrow, with reported incidences ranging from 67% to 100% at diagnosis.13,23-27 Early reports of EMZL described rare dissemination to the bone marrow,13,28,29 but recently, EMZL has been reported to involve the bone marrow in up to 20% of cases at diagnosis.30 Only a few reports of bone marrow involvement by NMZL are present in the literature.1,31,32 Descriptions of the morphologic features of bone marrow involvement by any of the marginal zone lymphomas are limited, and a comparative study of bone marrow involvement has not been reported. However, 2 studies reported intrasinusoidal infiltration of bone marrow by SMZL.33,34 Since intrasinusoidal infiltration has not been reported in most other B-cell lymphoproliferative disorders, Franco et al33 suggested that intrasinusoidal infiltrates in bone marrow could be used to support the diagnosis of SMZL in patients who may not be able to undergo splenectomy. The purpose of this study was to determine and compare the pathologic features of bone marrow involvement in marginal zone lymphomas arising from different sites, concentrating on extent and patterns of bone marrow infiltration. By using immunohistochemical analysis to detect CD20+ cells in all bone marrow biopsy specimens, we evaluated the specificity of intrasinusoidal infiltration in SMZL. For comparison, we similarly evaluated random bone marrow biopsy specimens involved by other small B-cell lymphoproliferative disorders, including hairy cell leukemia, follicle center lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia (CLL). Materials and Methods We identified 39 patients with marginal zone lymphoma for whom 45 bone marrow biopsy specimens from January 1990 through December 1999 were examined at Northwestern Memorial Hospital, Chicago, IL. The diagnoses were as follows: EMZL, 25 patients; SMZL, 12 patients; and NMZL, 2 patients. Clinical data were reviewed to determine age, sex, site of primary involvement, and initial stage. Initial staging was assigned according to the Ann Arbor Staging System.35 Based on previously accepted criteria,3,6 the initial diagnosis in all cases of EMZL and NMZL was confirmed by review of H&E-stained sections of formalinor B5-fixed, paraffin-embedded tissue. By using the histomorphologic features described by Isaacson et al,9 the diagnosis of SMZL was confirmed in 6 cases by review of H&E-stained sections of formalinor B5-fixed, paraffin-embedded splenic tissue. The diagnosis in 6 additional cases of SMZL was based on cytologic characteristics of circulating lymphocytes and immunophenotyping by flow cytometry.36 These latter patients had splenomegaly with minimal or no lymphadenopathy but had not undergone splenectomy. Sections of 45 B5-fixed, decalcified, and paraffinembedded bone marrow biopsy specimens stained with H&E and 36 Wright-Giemsa–stained peripheral blood smears from the 39 patients were available for evaluation. Three patients with EMZL, 1 with NMZL, and 2 with SMZL each had 2 bone marrow biopsy specimens available for review from different times during their disease. Thirty-four bone marrow biopsies were done as part of the initial staging procedure for patients with the following diagnoses: EMZL, 23; NMZL, 2; and SMZL, 9. Extent of hematopoietic elements replaced by lymphoma and patterns of bone marrow infiltration were assessed. By using the avidin-biotin complex method, immunohistochemical studies for CD20 (prediluted; Ventana, Tucson, AZ) were performed on paraffin-embedded sections from all bone marrow biopsies. Pretreatment for CD20 was performed using Citra Antigen Retrieval Solution (Biogenex, San Ramon, CA). In some cases, monoclonal antibodies against the following antigens also were used: kappa light chain (prediluted; Ventana) and lambda light chain (prediluted; Ventana). Results of immunophenotypic characterization by flow cytometry were available for review from 1 or more samples from 13 patients with EMZL, 1 with NMZL, and 12 with SMZL. The samples included peripheral blood (6), bone marrow aspirates (14), and solid tissue (14). Flow cytometric analyses were performed by 3-color immunophenotyping using combinations of antibodies directly labeled with fluorescein isothiocyanate, phycoerythrin, or phycoerythrin-cyanin 5.1. The following monoclonal antibody combinations were Kent et al / MARGINAL ZONE LYMPHOMA INVOLVING BONE MARROW 700 Am J Clin Pathol 2002;117:698-708 © American Society for Clinical Pathology used for analysis of B cells: CD19/CD23/FMC7, CD19/CD5/CD20, CD19/CD79b/CD11c, CD45/CD19/CD10, CD19/CD25/CD103, and CD19/kappa/lambda. Antibodies to CD5, CD10, CD20, and CD23 were obtained from Beckman Coulter, Miami, FL. Antibodies to CD19, CD79b, FMC7, CD45, and CD103 were obtained from Immunotech, Marseille, France. Antibodies to CD11c and CD19/kappa/ lambda were obtained from DAKO, Carpinteria, CA. Antibody to CD25 was obtained from Becton Dickinson, San Jose, CA. T cells as a control population also were evaluated with an appropriate immunophenotyping panel. Fresh solid tissue was dissociated manually into a single-cell suspension and washed twice in phosphate-buffered saline containing 0.2% sodium azide. Peripheral blood or bone marrow specimens were prepared by lysis of 100 μL of sample using a modified protocol of the RBC lysis technique (Q prep, Coulter, Miami, FL), followed by addition of stabilizing reagent and 2 washes in phosphate-buffered saline containing 0.2% sodium azide. Analysis was performed on a Coulter XL/MCL flow cytometer (Coulter). For each specimen, 40,000 or more events were collected using a forwardand side-scattered light intensity region slightly greater than that of normal lymphocytes. Positive marker expression and assessment of intensity of marker expression were based on comparison with appropriate internal negative controls. Other Small B-Cell Lymphoproliferative Disorders Specimens from bone marrow biopsies performed at Northwestern Memorial Hospital between January 1996 and December 1998 that were infiltrated by other small B-cell lymphoproliferative disorders were randomly identified: 18 from 11 patients with hairy cell leukemia, 14 from 10 patients with follicle center lymphoma (Revised EuropeanAmerican classification of lymphoid neoplasms, grade I), 19 from 9 patients with mantle cell lymphoma, and 11 from 10 patients with CLL. All cases had initial diagnostic material available with immunophenotypic analysis by flow cytometry and were classified according to the Revised European-American classification of lymphoid neoplasms.10 The bone marrow biopsies were performed at different times during the disease. To evaluate the presence of intrasinusoidal infiltration, H&E-stained sections from these bone marrow biopsies and immunohistochemical studies for CD20 were similarly processed and examined as for the marginal zone lymphomas.