How to cite this article: Naushad H, Choi WWL, Page CJ, Sanger WG, Weisenburger DD, Aoun P. Mantle cell lymphoma with flow cytometric evidence of clonal plasmacytic differentiation: A case report. Cytometry Part B 2009; 76B: 218–224.
Plasmacytic differentiation in mantle cell lymphoma (MCL) occurs rarely. However, no flow cytometric studies that demonstrate plasmacytic (PC) differentiation in MCL have been reported. Herein, we report a case of MCL with PC differentiation identified by flow cytometry.
Morphologic review was performed by hematoxylin and eosin (H&E) stained sections from paraffin-embedded lymph node, colon and bone marrow specimens, and Wright-Geimsa stained bone marrow aspirate smears and touch imprints. Immunohistochemical stains using antibodies against CD3, CD5, CD20, and cyclin-D1, and in-situ hybridization for kappa and lambda light chains were reviewed. Multicolor flow cytometry analysis was performed on the bone marrow aspirate with monoclonal antibodies to CD3, CD4, CD5, CD8, CD14, CD19, CD20, CD23, CD38, CD45, CD56, CD138, and kappa and lambda light chains. FISH analysis for t(11;14)(q13;q32) was performed on interphase cells.
The neoplastic cells had the cytologic features of MCL with nodal, bone marrow, and colonic involvement. In-situ hybridization for kappa and lambda light chains demonstrated clonal plasma cells in the lymph node and bone marrow biopsies. In addition, flow cytometric studies of the bone marrow aspirate showed three populations of neoplastic cells: a clonal B-cell population with typical MCL phenotype, a similar B-cell population in transition to plasma cells, and a clonal plasma cell population. The plasma cells retained CD5 expression and had the same light chain restriction as the clonal B-cells.
Mantle cell lymphoma (MCL) comprises ∼7% of all nonHodgkin lymphoma (NHL) in the western world (1). The median age of patients with MCL is about 60 years with a male predominance (2). Mantle cell lymphoma is an aggressive B-cell neoplasm with a median survival of only 3 years, and both the cytology and pattern of growth have been reported to be predictive of survival (3). Gene expression profiling studies have also identified a proliferation signature which is predictive of survival in MCL (4).
Mantle cell lymphoma is typically characterized by a nodular and/or diffuse proliferation of small to intermediate-sized lymphoid cells with irregular nuclear contours, a specific phenotype (CD5+, CD20+, cyclin D1+), and the characteristic t(11;14)(q13;q32). However, the cytology of mantle cell lymphoma is quite variable, including classical, lymphocytic, pleomorphic and blastoid variants (3, 5), and cyclin D1-negative cases have recently been reported (6). Plasma cells have traditionally been regarded as part of the non-neoplastic background in MCL since they were reported to be polyclonal (1). Recently, however, Young et al (7) reported two cases of MCL with clonal plasma cells that were shown to be derived from the same B-cell clone, thus demonstrating the occurrence of plasmacytic (PC) differentiation in MCL. Herein, we also report a case of MCL with PC differentiation. In our case, the flow cytometry findings were striking and showed a clear transition of the MCL cells to clonal plasma cells.
MATERIALS AND METHODS
Histology and Immunohistochemistry
Hematoxylin and eosin (H&E) stained sections from routinely fixed, paraffin-embedded lymph node, colon, and bone marrow specimens, and Wright-Giemsa stained bone marrow aspirate smears and touch imprints, were available for morphological examination. Immunohistochemical stains were performed using a Ventana ES automated immunostainer (Ventana Biotek, Tucson, AZ) with a streptavidin-biotin peroxidase detection system and antibodies against CD3 (PS1, Ventana Medical System, Tucson, AZ), CD5 (4C7, Novocastra, Newcastle upon Tyne, UK), CD20 (L26, DAKO, Carpentaria, CA) and cyclin D1 (SP4, NEOMARKERS, Fremont, CA). Antigen retrieval was performed as appropriate for the antibodies (7). In situ hybridization for immunoglobulin light chain mRNA was performed on a Ventana ES automated immunostainer using a streptavidin-biotin peroxidase detection system with Neat-Probe standard kits (Ventana).
Flow Cytometry Immunophenotyping
Bone marrow aspirate was collected in sodium heparin and processed according to standard protocols. Bone marrow mononuclear cells were separated by density gradient centrifugation (Accu-Prep, Accurate Chemical, Westbury, NY), and the red blood cells lysed with 0.09% sodium chloride solution. The separated cells were then washed with phosphate buffered saline (PBS) and resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 1 mol/L of Hepes buffer, L-glutamine, penicillin, streptomycin, and 10% fetal calf serum. The separated cells were then placed in 5-mL-plastic tubes at a concentration of 0.5 × 106 cells per tube, and labeled using monoclonal antibodies to CD3, CD4, CD5, CD8, CD14, CD19, CD20, CD23, CD38, CD45, CD56, CD138, and kappa and lambda light chains (Table 1). Antibodies were used according to the manufacturer's recommendations. Cells were incubated with antibodies in 200 μL of cold PBS with 0.5% bovine serum albumin for 30 minutes at 4°C. After incubation, the cells were washed with PBS, and the cell pellets resuspended in 1% paraformaldehyde in PBS. Cytoplasmic staining was performed using FIX AND PERM Cell Permeabilization reagents (Caltag, Burlingame, CA). Multicolor flow cytometry analysis was performed using a Beckman Coulter FC500 flow cytometer (Beckman Coulter, Miami, FL) and Beckman Coulter Cytomics CXP software (Applied Cytometry Systems, Dinnington, England). For the lymphoma panel, 8,000 total events and 5,000 events in lymphocyte gate were collected. For the myeloma panel, 36,000 total events were collected.
Table 1. Antibodies Used for Immunophenotyping by Flow Cytometry
Flow cytometry studies of the lymph node were not performed.
Bone marrow and lymph node specimens were cultured for 24 h without stimulation using standard techniques. Fewer than five G-banded metaphases were available for the lymph node specimen for analysis. Twenty G-banded metaphases were examined from the bone marrow specimen. FISH analysis for detection of the t(11;14)(q13;q32) was performed on interphase cells from the 24-h-cell culture of the lymph node and bone marrow specimens using standard methods and the LSI IGH/CCND1 XT Dual Fusion Probe (Vysis, Downers Grove, IL). A total of 100 interphase cells from the lymph node sample and 200 interphase cells from bone marrow sample were examined. The abnormal reference range for this FISH assay is 3–100% and was established by the Human Genetics Laboratory at the University of Nebraska Medical Center in clinical studies. No additional FISH studies using probes for abnormalities seen in plasma cell dyscrasias were performed.
Molecular analysis of the bone marrow specimen for VDJ rearrangements was performed by DNA amplification using consensus primers to the heavy chain variable (framework II and framework III) and joining regions. Molecular studies were not performed on the inguinal lymph node.
A 70-year-old male presented with hematochezia, change in bowel habits and left-side abdominal pain. On physical examination, he had multiple enlarged lymph nodes including bilateral cervical, supraclavicular (1 cm), and axillary lymph nodes (2 cm). Laboratory testing showed hemoglobin of 12.7 g/dL, white blood cell count of 7.3 × 109/L, and a platelet count of 121 × 109/L. The serum lactate dehydrogenase level was 421 U/L (reference range, 313–618 U/L). Serum protein electrophoresis detected two monoclonal IgM-kappa proteins; quantification of the paraproteins was not performed. He did not have any symptoms related to his gammopathy. A computerized tomographic (CT) scan revealed enlargement of bilateral axillary, mediastinal, subcarinal, peri-esophageal, gastrophrenic, inguinal, and left obturator lymph nodes, and a large retroperitoneal mass measuring 11.3 × 9.3 cm. The CT scan also revealed thickening of the cecal wall and an enlarged spleen (18.1 cm). A bone survey showed no lytic lesions. An excisional biopsy of a right inguinal lymph node showed nodular MCL. A staging bone marrow aspirate and core biopsy showed extensive involvement by MCL with PC differentiation. He also had biopsy-proven involvement of the colon and terminal ileum by MCL. The patient was treated with rituximab, CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), and bortezomib (Ps-341) chemotherapy. The patient was stable 365 days after initial diagnosis, without complications.
Histologic and Immunohistochemical Findings
The lymph node biopsy showed a typical cyclin D1-positive nodular MCL with weak positivity for CD5. Around the nodules, a few plasma cells were identified and showed a predominance of kappa by in situ hybridization stains for kappa and lambda light chains. The colon and terminal ileum biopsies showed diffuse MCL with scattered large lymphoid cells having more blastic chromatin, and an immunostain for cyclin-D1 was positive. However, significant PC differentiation was not seen in the colon biopsies.
The peripheral blood smear was unremarkable, and there was no rouleaux formation. The bone marrow was normocellular (30–40%) with a nodular and diffuse pattern of involvement by MCL. There were multiple para-trabecular and interstitial lymphoid aggregates and a diffuse increase in interstitial lymphoid cells and plasma cells. The lymphoid cells were small with irregular nuclear contours. Scattered plasmacytoid lymphoid cells and numerous mature plasma cells were also present (Figs. 1A and 1B). The lymphoid aggregates and interstitial lymphoid cells stained strongly positive for CD20 and cyclin D1 (Figs. 1C and 1D). The plasma cells were also cyclin D1-positive (Fig. 1D, inset). In situ hybridization stains for kappa and lambda light chains revealed a predominance of plasma cells and some lymphoid cells with kappa light chain expression (Figs. 1E and 1F).
Flow Cytometry Findings
Flow cytometry performed on the bone marrow aspirate revealed three clonal populations of cells (Fig. 2). A population of mature B-cells expressing CD5, CD19, CD20, CD38, bright CD45 and high-density monotypic surface kappa light chains was present at 27%. A second population of B-cells expressing CD5, CD19, high-density CD38, CD138, and monotypic surface and cytoplasmic kappa light chains was present at 11%. This population was negative for CD20 and had low-density expression of CD45, suggesting a transition toward PC differentiation. In addition, a third population of plasma cells expressing CD38, CD138, and monotypic cytoplasmic kappa light chains was present at 3 %. The plasma cell population was negative for CD20, CD45, and CD56, but retained the expression of CD5, CD19, and surface kappa, consistent with derivation from MCL.
Conventional cytogenetic studies performed on the lymph node yielded only five normal G-banded metaphase cells, a number insufficient to rule out the presence of an abnormal clone. FISH studies of the lymph node detected the IgH/CCND1 fusion in 95% of the interphase cells. Conventional cytogenetic studies of the bone marrow showed a normal karyotype (46XY) in 20 metaphases. FISH studies demonstrated a positive variant fusion signal for the t(11;14)(q31;q32) in 14.5% of the interphase cells, with the pattern of one green, two red and one fusion (yellow) signal (Fig. 3). Loss of IGH V gene segments within the IGH probe target, which is known to occur in physiologic V(D)J recombination, might produce a variant signal pattern (two red, one green, and one yellow fusion signal) in some samples rather than the usual dual fusion signal pattern of two yellow fusion signals, one on each of abnormal chromosomes 11 and 14, in addition to the single red and green signals expected from the normal chromosomes.
Plasmacytic differentiation is a feature of many B-cell lymphomas, particularly lymphoplasmacytic lymphoma and the marginal zone lymphomas, but it can also be observed in small lymphocytic lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma (8). Clonal PC differentiation in MCL is rare with only a few case reports in the literature (7, 9, 10). However, no cases of MCL with flow cytometric evidence of PC differentiation have been reported to date.
Herein, we report a case of MCL with clear evidence by flow cytometry of a transition of MCL cells to clonal plasma cells. Our case had the typical morphology of MCL in the lymph node and also showed para-trabecular and interstitial lymphoid aggregates, as well as diffuses interstitial infiltration, in the bone marrow. An increase in plasmacytoid lymphoid cells and plasma cells was also observed in the bone marrow aspirate smears. The differential diagnosis in the bone marrow included MCL with PC differentiation, lymphoplasmacytic lymphoma, or a second unrelated plasma cell dyscrasia. The patient was also evaluated for monoclonal serum proteins and a skeletal survey was performed. He did not have lytic bone lesions, but two monoclonal IgM-kappa proteins were found in the serum. The presence of monoclonal immunoproteins in the serum has been reported in 18% of patients with MCL (11). In addition to plasma cells, the bone marrow also showed a population of plasmacytoid lymphocytes suggesting a transition from the MCL. Upon further evaluation, the plasma cells were found to be clonal and shared the same light chain restriction as the MCL, supporting the notion that it was a true PC differentiation rather than an unrelated plasma cell dyscrasia. Finally, the flow cytometry studies in this case demonstrated a clear transition of the MCL cells to plasma cells.
Normal B-cell differentiation to plasma cells occurs either through the germinal center (GC) or via non-GC pathways (12–14). In the two cases reported by Young et al (7), the clonal plasma cells were found in and around reactive GCs, suggesting that the PC differentiation occurred in MCL cells that invaded or were recruited into GCs. The plasma cells in those cases were negative for CD5 by paraffin immunostaining. In our case, we also studied the lymph node biopsy by paraffin immunostaining and found evidence of PC differentiation with kappa light chain-restricted plasma cells. However, in our case, flow cytometry immunophenotyping of the bone marrow demonstrated that the plasma cells retained CD5 expression. One explanation for the detection of CD5 on the plasma cells in our case is the use of a more sensitive flow cytometric technique. The lack of GCs in the lymph nodes and bone marrow of our case suggests that the PC differentiation in MCL may also occur via a non-GC pathway.
The presence of t(11;14) as detected by FISH is insufficient to differentiate between MCL with plasmacytic differentiation and MCL with concurrent plasma cell myeloma. To support further the clonal relationship between the MCL and the plasma cells, molecular analysis for immunoglobulin heavy chain gene rearrangements was attempted on the bone marrow specimen. These studies did not demonstrate a clonal rearrangement with primers for framework II and III. However, the VDJ breakpoint is not amplifiable by the currently used primers in up to 30% of B-cell lymphomas.
Flow cytometry is useful for the characterization of B-cell NHL with PC differentiation and the plasma cell dyscrasias (15–18). A recent study by Seegmiller et al (8) used flow cytometry to compare the phenotype of plasma cells in various B-cell NHLs and plasma cell myeloma. That study reported that the neoplastic plasma cells in B-cell NHL are more likely to express CD19, CD45, and surface immunoglobulin, and less likely to express CD56, as compared to plasma cell myeloma (8). In our case, the plasma cells were CD5+, CD19+, CD45−, and CD56−, and showed surface light chain expression in addition to cytoplasmic light chain restriction, which is concordant with the findings reported in the literature (8, 15–18). In addition, flow cytometry was able to demonstrate the presence of a lymphocyte population with phenotypic features that overlapped both the MCL cells and the clonal plasma cells. This population most likely corresponds to the plasmacytoid lymphocytes observed morphologically, and provides the additional evidence that the MCL cells and plasma cells are clonally related.
Only a few cases of MCL with PC differentiation have been reported in the literature, and the clinical significance of this morphology is unknown. However, distinguishing MCL with PC differentiation from MCL with a concurrent but unrelated plasma cell dyscrasia may have treatment implications. Furthermore, other B-cell lymphomas with PC differentiation have been reported to show post-treatment relapses with only PC histology (19, 20). The distinctive phenotype of the plasma cells in this case, with coexpression of CD5 and CD19, renders flow cytometry immunophenotyping of great value in evaluating possible relapses that show only PC histology.
In conclusion, our case clearly demonstrates clonal PC differentiation in a case of MCL by flow cytometry, and underscores the fact that plasma cell differentiation can be seen in MCL. However, in such cases, a thorough morphologic, immunophenotypic, and FISH evaluation is necessary to exclude the possibility of an unrelated plasma cell dyscrasia. In such cases, the use of flow cytometry to differentiate B-cell NHL with PC differentiation from plasma cell myeloma may be particularly helpful.