Japanese Journal of Clinical Oncology 33:232-237 (2003)
© 2003 Foundation for Promotion of Cancer Research
A Case of Primary Plasma Cell Leukemia with Hairy-cell Morphology and Lambda-type Bence–Jones Protein. Immunohistochemical and Molecular Analysis
1 Division of Pathology and Laboratory Medicine, Iwata City Hospital, Iwata, Shizuoka, Departments of 2 Hematology and 3 Pathology, Seirei Hamamatsu Hospital, Hamamatsu, Shizuoka, 4 Hematology Division, National Cancer Center Hospital, Tokyo and 5 First Department of Pathology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCLINICAL SUMMARYPATHOLOGICAL FINDINGSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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A case of primary plasma cell leukemia with hairy-cell morphology and lambda-type Bence–Jones protein is reported. Most of the atypical cells in the peripheral blood of this case were small lymphoid cells or lymphoplasmacytoid lymphocytes with numerous cytoplasmic hairy projections. Plasmablastic cells and ‘tadpole’-like cells were also present in the bone marrow. Immunohistochemically, these atypical cells expressed the cytoplasmic lambda light chain and surface CD38 proteins but were negative for B-cell markers such as CD19, CD20 and CD79a. VLA-5 (CD49e), which is supposed to be expressed in mature populations of plasma cells, was negative. A sequence analysis of the variable region gene in the light-chain (VL) and heavy-chain (VH) loci of immunoglobulin demonstrated significant somatic hypermutation and intraclonal nucleic acid sequence variations. To our knowledge, the intraclonal diversity of these loci has been previously reported in some cases of monoclonal gammopathy of undetermined significance (MGUS), but never in a case of multiple myeloma. The immunohistochemical and molecular characteristics of this case allowed us to delineate the origin of the leukemic cells with hairy cell-morphology as germinal center B-cells, which would be at a more immature stage than the presumable origin of multiple myeloma.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCLINICAL SUMMARYPATHOLOGICAL FINDINGSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Plasma cell leukemia (PCL) is a rare form of plasma cell dyscrasia and is classified into two clinical types. The primary type occurs in individuals without being preceded by multiple myeloma, whereas the secondary type is a rare complication of the late-stage multiple myeloma (1,2). In certain PCL cases, the leukemic cells are more immature than usual and a definitive diagnosis is difficult to obtain without electron microscopic (3) or immunohistochemical studies. Here, we report a case of plasma cell leukemia containing the lambda-type Bence–Jones protein. The tumor cells in the peripheral blood of this case were small lymphoid or lymphoplasmacytoid cells with numerous cytoplasmic hairy projections. These cells did not show any of the characteristics of mature plasma cells (4) and their plasma cell nature was difficult to identify using conventional microscopic examinations. Immunohistochemical staining with monoclonal antibodies is useful for establishing a diagnosis of plasma cell leukemia or multiple myeloma. In addition to identifying the plasma cell morphology, Kawano et al. (5) claimed that immunohistochemical staining using monoclonal antibodies, such as CD38, VLA-5 and MPC-1, could also reveal the stage of maturation. Furthermore, a molecular analysis of the immunoglobulin variable region now provides invaluable information on the status of tumors with a B-cell lineage, thus confirming the proper categorization of B-cell tumors at the molecular level (6,7). To characterize the nature of the cell origin in this unique case of plasma cell leukemia, we analyzed the immunoglobulin light chain (VL) and heavy chain (VH) gene loci of the leukemic cells.
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CLINICAL SUMMARY |
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TOPABSTRACTINTRODUCTIONCLINICAL SUMMARYPATHOLOGICAL FINDINGSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Anemia, thrombocytopenia and a considerable number of atypical lymphoid cells with cytoplasmic hairy projections were discovered in a 56-year-old man. A physical examination disclosed cervical lymphadenopathy (the size of a red bean) and splenomegaly. The hematological data were as follows: Hb, 12.2g/dl; RBC, 392x104/µl; WBC, 25 250/µl (differentials: stab cell, 0%; segmented leukocyte, 16%; eosinocyte, 0%; basophil, 0%; monocyte, 2%; lymphocyte, 15%; atypical cells, 67%). The total protein level was 7.1 g/dl and the
-globulin level was 12.2%. Immunoelectorophoresis showed a reduction in normal immunogloblins (IgG, 657 mg/dl; IgA, 0 mg/dl; IgM, 0 mg/dl) and urinary lambda-type Bence–Jones protein was also detected. The serum LDH value was 497 IU/l. Atypical cells occupied 78% of the bone marrow nuclear cells. X-ray examinations showed no evidence of osteolytic bone lesions. Gallium and bone scintigraphy showed a mild accumulation in the paranasal sinus, but the lymph nodes, liver, spleen and bone were clear. The patient was diagnosed as having primary plasma cell leukemia and DMVM therapy (doxorubicin, methotrexate, vinblastine and methylpredonine) was initiated. Complete remission was obtained and the patient has been disease-free for 7 months since his first chemotherapy treatment. |
PATHOLOGICAL FINDINGS |
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TOPABSTRACTINTRODUCTIONCLINICAL SUMMARYPATHOLOGICAL FINDINGSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Histological Findings
Most of the atypical cells in the peripheral blood were small lymphoid or plasmacytoid cells with cytoplasmic hairy projections, resembling those of hairy-cell leukemia (Fig. 1). Immunohistochemistry revealed that these cells were strongly positive for the surface CD38 protein (Fig. 2) and the cytoplasmic lambda light chain protein. In addition to these cells, plasmablastic cells and ‘tadpole’-like cells were also found, representing about 10% of the nuclear cells in the bone marrow (Fig. 3). These hairy-cell leukemia-like cells lacked tartrate-resistant acid phosphatase reactivity and a bone marrow clot section did not exhibit the characteristic loosely packed arrangement of cells with a perinuclear halo. More than 90% of these leukemic cells expressed the surface CD38 and cytoplasmic lambda light chain (Fig. 4A and B), but these cells were negative for lymphocytic markers, such as CD19 (0.2%) and CD20 (0.9%), and also CD45 (0.0%), CD5 (0.0%), CD10 (0.1%), CD11c (0.1%), CD22 (1.8%), CD23 (0.5%), CD25 (0.0%), CD79a (0.9%), FMC-7 (0.9%) and HLA-DR (13.9%). Bone marrow clot sections were negative for cytoplasmic IgG, IgM and IgA. These cells were also negative for VLA-5 (8) using immunohistostaining and 4.4% of these cells were VLA-5 (CD49e)-positive using flow cytometry. On the other hand, these cells were positive for several adhesion molecules, such as CD46 (99.7%), CD49d (VLA-4, 90.8%), CD54 (99.8%), CD55 (89.2%) and CD59 (98.7%), using flow cytometric analysis.
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Molecular Pathological Findings
Total RNA was extracted from a frozen peripheral blood clot by using Isogen (Nippon Gene, Fukuyama, Japan) following the instructions supplied by the manufacturer. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using M-MLV Reverse Transcriptase (Life Technologies, Grand Island, NY) and Pfu DNA polymerase (Promega, Madison, WI), according to the manufacturer’s protocol. The protocol and primer sequences of the VL region used in this study have been reported previously (9). Three different RT-PCR experiments produced a monoclonal band of the same size, using the following primer set: 5'-CAGTCTGT (C/G) (C/G/T)TGAC (G/T)CAGCC (A/G)CC (G/T)-3' (V
1, FR1 sense) and 5'-ACC (G/T)AGGACGGT (C/G)A (C/G)CT (G/T)GGT (C/G)CC-3' (JC, FR4 anti-sense). Following electrophoresis on agarose gels, RT-PCR products were cloned using a pGEM-T Easy Vector kit (Promega) and sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer Biosystems, Foster City, CA). Using the same methods, we also obtained the partial nucleotide sequence of the immunoglobulin VH genes, using previously reported primers (10). At least six clones of the VL and VH genes were examined in each RT-PCR product. Alignment of the DNA sequence was performed using Entrez [GenBank database: National Center for Biotechnology Information (NCBI), Bethesda, MD; http://www.ncbi.nlm. nih.gov/]. The sequence of CDRs (cording regions) or FRs (framework regions) in these germline genes were identified according to the IMGT database (11). Figures 5 and 6 show the nucleotide sequences of the VL and VH genes obtained from the leukemic cells in this case. We compared VL with its closest germline gene DPL10 (12) and compared VH with HSIGDP14 (13). One of the rearranged genes or proteins of the DPL10 is the NEI Bence–Jones protein (14). The VL and VH genes derived from the leukemic cells in this case had significantly mutated from the germline genes. Alterations included single base substitutions, deletions and insertions, resulting in both amino acid replacement (R) and silent (S) mutations. The degree of somatic mutation in the VL gene was higher than that of the VH gene. (86~89% homologous in VL and 95~96% homologous in VH). Calculation of the number of expected R mutation in the CDRs or FRs in the VL and VH gene was based on the binomial distribution model developed by Chang and Casali (15). In both VL and VH genes, the R/S ratio in the CDRs exceeded the expected values, whereas those in FRs were lower than expected (Table 1).
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Additionally, intraclonal heterogeneity was observed among the nucleotide sequences of the VL and VH genes. According to Lossos et al. (16), a nucleotide substitution in more than one clone was considered as a mutation. To determine whether the mutations in different clones arose from intraclonal variation or an error caused by the Pfu DNA polymerase during the RT-PCR procedure, we estimated the error rate by comparing the present results with those for a VH gene obtained from a case with precursor B-cell acute lymphoblastic leukemia, which was one out of 4190 (data not shown). Thus, the level of nucleotide variation in the VH and VL genes of the different clones could be interpreted as arising from a hypermutable state in the tumor cells, rather than RT-PCR artifacts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONCLINICAL SUMMARYPATHOLOGICAL FINDINGSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Morphological heterogeneity, including various immature features (4), in atypical plasma cells sometimes makes the diagnosis of PCL difficult and may require an immunohistochemical or electromicroscopic examination (3) to establish a definitive diagnosis. In the present case, most of the leukemic cells in the peripheral blood exhibited an unusual morphology similar to that of hairy-cell leukemia. An unusual ‘hairy-cell’ variant of plasma cell myeloma has been described (17). Moreover, a case of lymphoplasmacytoid leukemia with this unusual feature, which closely resembles that of our case, has also been reported (18). However, the plasma cells in that case reported by Loo et al. produced biclonal IgA but not Bence–Jones protein (18). The clinical signs and a detailed morphology of the leukemic cells were not reported in that case, so it is difficult to conclude whether the case was actually an example of hairy-cell leukemia or of splenic lymphoma with villous lymphocytes. Histochemical and immunohistochemical findings indicated that the leukemic cells arose from the plasma cells. Recently, Kawano et al. reported a series of variously matured myeloma cases, containing proliferative VLA-5-negative and MPC-1-negative myeloma cells and IL-6-responsive immature cells, found predominantly in the peripheral blood (5). We did not obtain MPC-1 data in the present case, but the leukemic cells are thought to be plasma cells of a more immature stage.
Ontogenetically, B-cell tumors can be regarded as B-cells that arrested at a certain stage of differentiation when the neoplastic transformation occurred. An analysis of the immunoglobulin variable region genes has provided invaluable information on the status of tumors in B-cell differentiation and has added an important dimension to the classification of B-cell tumors at a molecular level (9,10). Recently the germ-line repertoire of VL genes has been mapped (12,19) and its involvement in B-cell tumors has been studied (9,20). VL and VH genes obtained from leukemic cells in the peripheral blood of this case exhibited significant somatic hypermutation compared with that observed in germline genes. In both VL and VH genes, extensive R mutations tended to cluster in the CDRs, whereas the R/S ratio in FRs were lower than expected. These findings allowed us to speculate that the leukemic cells, with their unique hairy-cell-like morphology, were derived from B-cells that had been exposed to a hypermutation stage prior to their neoplastic transformation (21). A sequence analysis of V
gene in light chain-expressing multiple myeloma shows that somatic hypermutation occurs in the course of the disease, clustering in the CDR regions (22). Sahota et al. reported that the clustering of R mutation in CDR regions in the 10 cases with multiple myeloma is localized in either VH or VL, but not both (9). In our case, the clustering was observed in both VH and VL genes and the VL gene mutated more frequently than the VH gene and frame shift mutation occurred in the FR1 region within two of six clones. In addition to somatic hypermutation, our examination of the nucleotide sequences in the VL and VH genes of this case showed intraclonal nucleic acid sequence variations, i.e. intraclonal diversity, which is usually not found in multiple myelomas (9,23,24), but, interestingly, has been found in some cases of monoclonal gammopathy of undetermined significance (MGUS) (10). The PCR error rate resulting from the polymerase (mutation frequency/bp/duplication) may influence the estimation of intraclonal heterogeneity. However, the error rate of Pfu DNA polymerase estimated in our laboratory was low (~1 in 4200). In the medical literature, the average error rate of Pfu DNA polymerase is 1/1.3x106, which is much lower than that of the Taq polymerase (25). The nucleotide variation observed among the different clones of the VL and VH genes was greater than that attributable to the error rate of Pfu DNA polymerase. Intraclonal diversities in the immunoglobulin genes are indicative of ongoing mutation (26) and allow the discrimination between germinal center B-cells, which show ongoing mutations, and post-germinal center B-cells, which do not (27). This pattern is characteristic of tumors with a germinal center origin, such as follicular lymphoma (6,7), and is also seen in Burkitt’s lymphoma (28), mucosa-associated lymphoid tissue (MALT) lymphoma (29) and some cases of MGUS (10), but not in multiple myeloma (9,23,24). These molecular findings may indicate that the small lymphoid cells with a hairy-cell-like morphology may be more immature and antigen stimulation may be necessary for leukemic cell growth (9,29). Here, we report a case of plasma cell leukemia consisting of small lymphoid cells and lymphoplasmacytoid lymphocytes with cytoplasmic hairy projections. Molecular analysis revealed that these unique leukemic cells are ontogenetically different from myeloma cells in multiple myeloma. Not all cases with PCL are rare complications of multiple myeloma or leukemic forms of late-stage multiple myeloma. The origin of leukemic cells in some cases with PCL may have resulted from exposure to mutational mechanisms, both before and after the neoplastic transformation. The clinicopathological characterization of PCL requires further investigation. We suggest that the present case of PCL may be typical of cases in which the tumor cells consist of derivatives from the various time points in B-cell development.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONCLINICAL SUMMARYPATHOLOGICAL FINDINGSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The authors thank Ms Ihara and Ms Nagura for their assistance with the molecular analysis. This work was supported by a Grant-in-Aid from the MEXT of Japan, the Smoking Research Foundation and RSP.
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FOOTNOTES |
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+ For reprints and all correspondence: Haruhiko Sugimura, First Department of Pathology, Hamamatsu University School of Medicine, 1–20–1 Handayama, Hamamatsu 431-3192, Japan. E-mail: hsugimur{at}hama-med.ac.jp
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Received October 8, 2002; accepted April 1, 2003
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