{'fraction_non_alphanumeric': 0.05279187817258883, 'fraction_numerical': 0.0640439932318105, 'mean_word_length': 4.834649555774926, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Cytogenetic Study and Analysis of Protein Expression in Plasma Cell Myeloma with t(11;14)(q13;q32): Absence of BCL6 and SOX11, and Infrequent Expression of CD20 and PAX5

The t(11;14)(q13;q32) translocation is the most common chromosomal translocation in plasma cell myeloma (PCM), but the cytogenetic and immunophenotypic features of PCM with t(11;14)(q13;q32) remain to be fully elucidated. To address the issue, we retrospectively analyzed 21 newly diagnosed PCM patients with the t(11;14)(q13;q32) translocation in our institute. CD20 is a B-cell-specific transmembrane protein that is the topic of much focus as a potential target in immunotherapy. We observed a low incidence of CD20 expression (2 of 21 patients, 11%), although the expression of CD20 was previously reported to be associated with t(11;14)(q13;q32). PAX5 is an essential transcriptional factor involved in B-cell development and commitment, and is down-regulated upon plasma cell differentiation. We observed one patient (6%) with expression of PAX5. The expression of CD19, CD56, and CD138 was detected in one (0.7%), nine (60%), and 13 patients (87%), respectively. Cyclin D1, CD38, and BCL2 were detected in all patients; on the other hand, neither BCL6 nor SOX11 was detected in any of the evaluated patients. Abnormalities of chromosome 13 were detected in six patients (38%), but deletion of TP53 was not observed in any of the evaluated patients. Our results suggest the absence of BCL6 and SOX11 expression, and infrequent expression of CD20, PAX5, and CD56 in PCM with t(11;14)(q13;q32), in contrast to the findings of earlier reports. 〔J Clin Exp Hematop 55(3) : 137-143, 2015〕


INTRODUCTION
Plasma cell myeloma (PCM) is a hematologic neoplasm characterized by monoclonal proliferation of plasma cells in the bone marrow. Nearly half of all patients with PCM carry chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at 14q32. 1 The t(11;14)(q13;q32) translocation is the most common IGH translocation in PCM, with a prevalence of approximately 15%. 1 As a result of this translocation, the coding region of cyclin D1 (CCND1) on 11q13 is juxtaposed to the transcriptionally active enhancers of IGH, resulting in upregulated expression of CCND1. 2 CD20 is a B-cell-specific transmembrane protein with four membrane-spanning domains. 3 Although the exact function of CD20 is not fully elucidated, it is believed that CD20 is involved in B-cell activation and proliferation, and regulation of transmembrane calcium transport. 4 CD20 is expressed in committed B-cells throughout their development, but is lost upon differentiation to plasma cells. 3 However, Robillard et al. reported that CD20 was expressed in 12 (18%) of 66 PCM patients. 5 Interestingly, they showed a significant correlation between immunophenotype and genotype; the expression of CD20 was strongly associated with t(11;14)(q13;q32) (10 of 12 patients, 88%). On the other hand, recent studies reported lower correlation between the expression of CD20 and t(11; 14)(q13;q32). 6,7 PAX5 is a transcriptional factor that is expressed in Bcells and the nervous system. 8 In B-cells, the expression of PAX5 is initiated in the pre-pro-B cells, maintained through out subsequent stages of B-cell development, and is silenced upon plasma cell differentiation. 9 In PAX5-defficient mice, B-cell development is arrested at a very early stage. 10 PAX5 activates B-cell specific genes, including Ebf1, CD19, and CD79a, and represses lineage-inappropriate genes, including Notch1, Csf1r, and Flt3. 9 Thus, PAX5 plays an essential role in B-cell development and commitment. Concurrently, PAX5 represses both Xbp1 and Blimp1, which are required for plasma cell differentiation. 9 Hence, down-regulation of PAX5 expression is required for differentiation to plasma cells, and consequently, PAX5 is not expressed in plasma cells. PCM cells have been assumed to not express PAX5 by analogy with normal plasma cells. In addition, Proulx et al. reported that PAX5 overexpression induces apoptosis in PCM cell lines. 11 However, Torlakovic et al. reported that 2 of 39 (5%) PCM patients showed unequivocal focal expression of PAX5, and two more patients were border-line positive. Notably, these four patients also exhibited weak expression of CD20. 12 Lin et al. also reported the expression of PAX5 in CD20expressing PCM (18 of 25 patients, 72%). 13 Accordingly, the expression patterns of CD20 and PAX5 in PCM with t(11;14)(q13;q32) remain unclear. To clarify the characteristics of PCM cells with t(11;14)(q13;q32), we retrospectively analyzed newly diagnosed PCM patients in our institute.

Patients
We retrospectively reviewed 21 PCM patients with t(11; 14)(q13;q32) who were treated at our institute between May 2003 and October 2013. The diagnosis of PCM was made according to the 2008 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. 14 Chromosome analysis and fluorescence in situ hybridization (FISH) were used to detect t(11;14)(q13;q32). The study protocol was approved by the Institutional Review Board of St. Marianna University School of Medicine.

Flow cytometry
Flow cytometric analysis of aspirated bone marrow cells was performed using antibodies against CD19, CD20, CD49e, CD56, CD138, and MPC1. Positivity was evaluated using a cut-off value of 20%.

Patient characteristics
Patient characteristics are summarized in Table 1. The median age at diagnosis was 69 years with a slight predominance of females (57%). Monoclonal serum paraprotein was detected in 20 of 21 patients with the following subtypes; IgG in seven patients (33%), IgA in five (24%), and light chain only (Bence Jones protein type, BJP type) in eight (38%). Only one patient had non-secretory PCM. The type of light chain was k in seven patients (33%) and l in 14 (67%). Nine patients (43%) were classified as stage III according to the International Staging System. 16 Laboratory data of all patients are shown in Table 2. The median percentage of plasma cells was 58.9%.
other hand, the deletion of TP53 was not detected in any of the evaluated patients.

Immunohistochemical analysis
The results of immunohistochemical analysis are shown in Table 4. Strong nuclear positivity of CCND1 was observed in all of the evaluated patients. Both CD38 and BCL2 were also positive in all evaluated patients. CD20 was positive in two patients (11%) (Fig. 2c, 2f), and CD20 expression in these patients was also detected by flow cytometry. May-Grünwald-Giemsa-stained PCM cells exhibited a lymphoplasmacytic cell morphology, as defined by Goasguen et al. 18 (Fig. 2a, 2d). PAX5 was positive in only one patient (0.06%), as discussed in our previous report (UPN 3). 19 The karyotype of this patient was normal at diagnosis, but t(9;14;11)(p13; q32;q13), a complex variant translocation of t(11;14)(q13; CD20/PAX5 in PCM with t(11;14)(q13;q32) 139 q32), was later detected. We performed two-color immunohistochemical analysis of PAX5 and CD138 in the current study, and confirmed definite expression of PAX5 in the PCM cells (Fig. 2i).

DISCUSSION
We evaluated 21 PCM patients with t(11;14)(q13;q32). The light chain-only (BJP type) was the most prevalent subtype (38%) in the current study, while a previous study has reported that the incidence of this subtype accounts for only 20% of all PCM patients. 14 Robillard et al. reported a significantly high correlation between the expression of CD20 and t(11;14)(q13;q32) in PCM patients. 5 However, Mateo et al. reported a lower incidence of CD20 expression in PCM with t(11;14)(q13;q32) (21 of 66 patients, 38%) in a larger study. 6 We observed a lower incidence of CD20 expression (2 of 21 patients, 11%), which was in accordance with a report by Grigoriadis et al. (2 of 19 patients, 11%). 7 Notably, the plasma cells of these patients exhibited lymphoplasmacytic cell morphology, which is a feature of PCM with t(11;14)(q13;q32). 5 The down-regulation of PAX5 is essential for terminal differentiation into plasma cells, 9 but the expression of PAX5 in PCM has also been previously reported in the literature. 12,13 Torlakovic et al. reported focal or border-line expression of PAX5 in PCM, in which CD20 was also expressed. 12 Lin et Fig. 1. Flow cytometry (1a, 1b, UPN 7; 1c, 1d, UPN  17). CD38 and side scatter identification of plasma cell myeloma cells (ellipse) (1a, 1c). A distinct population of plasma cell myeloma cells expressed CD20 (1b, 1d ).     al. also described a high correlation between PAX5 and CD20 expression in PCM. 13 In the current study, only one patient expressed PAX5. We have previously reported this PCM patient as having t(9;14;11)(p13;q32;q13), which is a complex variant translocation of t(11;14)(q13;q32). 19 CD20 was not detected in the PCM cells by flow cytometry or immunohistochemical analysis, while PAX5 positivity in the PCM cells was definitively detected by two-color immunohistochemical analysis for PAX5 and CD138. We postulate that the strong enhancers or promoters of IGH drove PAX5 expression as a result of the translocation, because t(9;14;11)(p13;q32;q13) is a complex variant translocation not only of t(11;14)(q13;q32) but also t(9;14)(p13;q32). BCL6 is a transcriptional repressor that is expressed in germinal center B-cells, and plays a crucial role in the formation of germinal centers. 8 In the current study, the expression of BCL6 was not detected in PCM with t(11;14)(q13;q32). BCL2 is a protein that inhibits the apoptosis pathway, 20 and is expressed in most PCM cells. 21 CD56 is an adhesion molecule involved in cell-to-cell and cell-to-matrix interactions. 22 Normal plasma cells lack CD56, whereas PCM cells express CD56 in 70-80% PCM patients. 23 On the other hand, PCM with t(11;14)(q13;q32) is correlated with the lack of CD56 (82%). 24 The expression of BCL2 was detected in all patients, and the expression of CD56 was observed in only 40% of patients in the current study.
The t (11; 14) (q13; q32) translocation is commonly observed in PCM and mantle cell lymphoma (MCL). 14 Abnormality of -13/13q-is a common additional cytogenetic change in these diseases. 14 In the current study, the frequency of -13/13q-was 38%, which was in accordance with a report by An et al.; 25 they reported that the frequency of -13/13q-in PCM with t(11;14)(q13;q32) (20 of 57 patients, 35%) was lower than that of PCM without t(11;14)(q13;q32) (99 of 191 patients, 52%). Furthermore, the allelic loss of 13q14-q34 was observed in 43-51% of MCL patients. 14 The abnormality of del(17p) is also a common cytogenetic change in both PCM and MCL, 14 the relevant gene of which is TP53, located on 17p13. 26 The deletion of TP53 has been reported in 21-45% of MCL patients 14 and 5-10% of PCM. 27 We did not detect the deletion of TP53 in any of the four patients evaluated. The deletion of TP53 is infrequent in newly diagnosed PCM patients, and is may be regarded as a later event in this disease. 21 SOX11 is a transcriptional factor expressed in the developing nervous system and plays a crucial role in neurogenesis, 28 but is absent in many adult tissues. 29 Recent studies showed that SOX11 is strongly expressed in the majority of MCL patients. In the current study, the expression of SOX11 was not detected in any PCM patients with t(11;14) (q13;q32). The t(11;14)(q13;q32) translocation is common in MCL and PCM with t(11;14)(q13;q32), but the expression of SOX11 is independent of this translocation and the resulting expression of CCND1, as mentioned by Dictor et al. 30 Although the number of patients was limited in the current study, the results suggest the absence of BCL6 and SOX11 expression, and infrequent expression of CD20, PAX5, and CD56 in PCM with t(11;14)(q13;q32), in contrast to the findings of earlier reports.