Restoration of Rituximab Sensitivity in CD20 Negative B-Cell Lymphoma Cells; Epigenetic Change of CD20 Expression after Treatment of Rituximab.

Abstract CD20 surface antigen is widely expressed on normal B-lineage cells and B-lymphoid malignant cells. Rituximab, which is a chimeric monoclonal antibody specifically recognizing CD20 surface antigen, works as a molecular targeting drug against CD20 positive lymphoid malignancies. Although combination chemotherapy with Rituximab has significantly improved the survival of CD20 positive lymphoma patients, CD20 negative tumor regression and transformation becomes a considerable problem. Recently, we have identified a patient with CD20(−) diffuse large B-cell lymphoma (DLBCL) transformed from CD20(+) follicular lymphoma after chemotherapies including Rituximab. Here we established RRBL1 (Rituximab resistant B-cell lymphoma 1) cells from this patient’s CD20(−) B-cell lymphoma cells, and analyzed the mechanisms of negative CD20 surface antigen expression. CD20 was not expressed as determined by immunoblotting and FACS analysis, and resistance to Rituximab was observed after cell culture analysis with/without Rituximab. Expression of wild type CD20 mRNA was confirmed by RT-PCR, and no genetic mutation in coding sequence or promoter region was observed. Quantitative RT-PCR showed that CD20 mRNA expression level was almost 100 times lower than that of CD20 positive B-cell lymphoma cells obtained from DLBCL patients. These data suggest that lower expression level of CD20 mRNA is closely related to the negative CD20 surface antigen expression, and the aberrant transcription regulation by epigenetic mechanisms can be anticipated. With these data, we thought that CD20 expression could be restored by modulating the transcription regulation. As we expected, CD20 protein re-expression was confirmed by enhancing CD20 mRNA expression after the treatment with specific molecular targeting drugs, and the sensitivity for the Rituximab was significantly restored. Chromatin immunoprecipitation assay suggested that recruitment of co-repressor complexes to CD20 promoter might be related to the regulation of CD20 expression. Our data may provide us with a new therapeutic strategy, epigenetic therapy or differentiation therapy, combined with molecular targeting therapy using Rituximab, for Rituximab resistant CD20(−) B-cell malignancies.

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Epigenetic Regulation of CD20 Protein Expression in B-Cell Lymphoma Cells after Rituximab-Containing Chemotherapy.

Blood ◽

10.1182/blood.v110.11.3423.3423 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3423-3423

Author(s):

Akihiro Tomita ◽

Junji Hiraga ◽

Hitoshi Kiyoi ◽

Tomohiro Kinoshita ◽

Tomoki Naoe

Keyword(s):

Protein Expression ◽

B Cell ◽

Mrna Expression ◽

Histone Deacetylase ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Molecular Targeting ◽

Rt Pcr ◽

B Cell Malignancies ◽

Lymphoma Cells

Abstract For the past 10 years, rituximab has been used with some forms of conventional chemotherapy to treat CD20(+) B-cell malignancies as a new molecular targeting drug. Although significant improvements in disease-free survival and cure rate have been obtained, resistance to rituximab has been recognized as a considerable problem. Here we report that CD20(−) tumor transformation is one important mechanism of resistance to rituximab. We also demonstrate a possibility that CD20 protein expression is downregulated by some epigenetic mechanisms, and interestingly, the protein expression can be restored by treatment with certain epigenetic drugs. During the 5 years beginning in 2001, 124 patients with B-cell malignancies were treated with rituximab-containing induction, consolidation, or salvage chemotherapies in our hospital. Relapse or recurrence of disease were confirmed in 36 patients (29.0%), and re-biopsy of tumor tissues was performed in 19 patients. Five cases (DLBCL, 3 cases, and DLBCL transformed from FL, 2 cases) of those 19 patients (26.3%) indicated CD20 protein-negative transformation in flow cytometry (FCM) and/or immunohistochemistry (IH) analyses. We established a CD20(−) lymphoma cell line RRBL1 from a DLBCL patient with CD20(−) transformation from CD20(+) FL after repeated treatment with rituximab, and analyzed molecular mechanisms of the CD20 protein-negative phenotype. CD20 was not expressed by IH, FCM, and immunoblotting analyses, and resistance to rituximab was observed after cell culture with rituximab treatment. Expression of wild-type CD20 mRNA was confirmed by RT-PCR, and no genetic mutation in coding sequence or promoter region was observed. Quantitative RT-PCR showed that the CD20 mRNA expression level was almost 100 times lower than that of CD20(+) B-cell lymphoma cells typically obtained from DLBCL patients. These data suggest that aberrant down regulation of CD20 mRNA may be closely related to the negative CD20 surface antigen expression. Interestingly, CD20 protein re-expression was confirmed by enhancing CD20 mRNA expression using specific epigenetic drugs including histone deacetylase inhibitor Trichostatin A (TSA). In addition, after TSA treatment, sensitivity for rituximab was significantly restored. Chromatin immunoprecipitation assay suggested that recruitment of histone deacetylase complexes to CD20 promoter may be related to the down regulation of CD20 expression. Our data may provide the basis for a new therapeutic strategy, epigenetic therapy combined with molecular targeting therapy using rituximab, for rituximab-resistant CD20(−) transformed B-cell malignancies.

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Discrepancy of CD20 Protein Expression In IHC and FCM Analyses In Primary B-Cell Lymphoma: Relationship Between FCM-Negative Phenotype and Rituximab Binding with Lymphoma Cells

Blood ◽

10.1182/blood.v116.21.5087.5087 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5087-5087 ◽

Cited By ~ 1

Author(s):

Takashi Tokunaga ◽

Akihiro Tomita ◽

Kazuyuki Shimada ◽

Junji Hiraga ◽

Takumi Sugimoto ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Primary Lymphoma ◽

Newly Diagnosed ◽

Rt Pcr ◽

Lymphoma Cells ◽

Anti Cd20

Abstract Abstract 5087 Background Rituximab is an anti-CD20 chimeric-monoclonal antibody, and its effectiveness for treatment of CD20-positive B-cell lymphomas has been proven over the past 10 years. Although rituximab is now a key molecular targeting drug for CD20-positive lymphomas, some patients with rituximab resistance have emerged. We previously reported that the CD20-protein-negative phenotypic change after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009., Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009.). Recently, we have recognized that some newly-diagnosed B-cell lymphomas show CD20-protein-positive in immunohistochemistry (IHC) but -negative in flow cytometry (FCM) analyses. For these patients, so far, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance are clear. Thus, the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims Analyses of the molecular backgrounds of CD20 IHC(+)/FCM(−) phenotype in primary B-lymphoma cells, and confirmation of the effectiveness of rituximab therapy for the patients who show CD20 IHC(+)/FCM(−) phenotype. Results Primary B-cell lymphoma (diffuse large B-cell (DLBCL), follicular, MALT, mantle cell, and Burkitt) tissues and cells were analyzed by IHC and FCM. Four newly-diagnosed B-cell lymphoma patients showed IHC CD79(+)/CD20(+) and FCM CD19(+)/CD20(−) phenotype using anti-CD20 antibodies L26 for IHC and B1 for FCM, and all were diagnosed as DLBCL. Chromosomal analysis showed complex karyotypes in 3 out of 3 patients analyzed, and no shared abnormalities were confirmed. Primary lymphoma cells from 3 patients were available for further molecular analyses, and the genomic DNA, the total RNA, and the protein from whole cell lysate were obtained from these lymphoma cells. DNA sequencing analysis indicated no significant genetic mutations on the coding sequences (CDS) of MS4A1 (CD20) gene. Semi-quantitative and quantitative RT-PCR indicated that CD20 mRNA expression was almost normal in 2 patients and ≂~f10 times lower in 1 patient compared to the positive control B-lymphoma/leukemia cells. Almost the same expression tendency with RT-PCR was confirmed in immunoblot analysis using whole cell lysate and the two different anti-CD20 antibodies. The molecular weight of the CD20 protein in immunoblotting corresponded to the wild type in these patients. Rituximab binding assay in vitro was performed using primary lymphoma cells from a patient and the fluorescent-labeled rituximab (Alexa488-rituximab). Interestingly, rituximab binding on the surface of the CD19 positive lymphoma cells was confirmed in vitro. Rituximab containing combination chemotherapy was performed, resulting in complete response in all 4 cases after completing 4 to 8 courses. Conclusions and Discussion CD20 IHC(+)/FCM(−) phenotype was confirmed in newly-diagnosed DLBCL patients. Significant abnormalities in CD20 protein and mRNA expression in immunoblotting and RT-PCR were not confirmed, and genetic mutations on CDS of MS4A1 gene, resulting in the conformation change of CD20 protein, were not detected. The possibility of abnormal post-translational modification or aberrant localization of CD20 protein, leading to interference with antibody binding, can not be excluded. Rituximab binding with CD19-positive primary lymphoma cells was confirmed in a patient, suggesting that CD20 IHC(+)/FCM(-) phenotype does not directly indicate the ineffectiveness of rituximab for these cells. Further investigations, performing in vitro CDC and ADCC assay using primary lymphoma cells, are still warranted to show rituximab effectiveness and sensitivity to those cells. Disclosures: Kinoshita: Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

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Bi20 (fBTA05), a novel trifunctional bispecific antibody (anti‐CD20 × anti‐CD3), mediates efficient killing of B‐cell lymphoma cells even with very low CD20 expression levels

International Journal of Cancer ◽

10.1002/ijc.23626 ◽

2008 ◽

Vol 123 (5) ◽

pp. 1181-1189 ◽

Cited By ~ 51

Author(s):

Michael Stanglmaier ◽

Margot Faltin ◽

Peter Ruf ◽

Annette Bodenhausen ◽

Petra Schröder ◽

...

Keyword(s):

B Cell ◽

Bispecific Antibody ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cells ◽

Expression Levels ◽

Cd20 Expression ◽

Anti Cd20

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CD20 Protein Immunohistochemistry-Positive/Flow Cytometry-Negative Diffuse Large B-Cell Lymphoma—Analyses of the Molecular Mechanisms and Rituximab Effectiveness

Blood ◽

10.1182/blood.v118.21.2659.2659 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2659-2659

Author(s):

Takashi Tokunaga ◽

Akihiro Tomita ◽

Junji Hiraga ◽

Takumi Sugimoto ◽

Kazuyuki Shimada ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

Molecular Mechanisms ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Positive Control ◽

Cell Populations ◽

Rt Pcr ◽

Lymphoma Cells

Abstract Abstract 2659 Background: Rituximab is a key molecular targeting drug for CD20(+) B-cell lymphoma, and CD20 protein expression is essential in the treatment mechanism of rituximab. We previously reported that the change to the CD20(−) phenotypic after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009; Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009). Recently, we encountered newly diagnosed patients with diffuse large B-cell lymphomas (DLBCL) which are CD20(+) on immunohistochemistry (IHC) but CD20(−) on flow cytometry (FCM). For these patients, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance have been clarified, and the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims: To confirm the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype using primary DLBCL cells, and to confirm the effectiveness of rituximab in vitro. Methods: Genomic DNA, mRNA, and total protein were extracted from primary DLBCL cells showing the CD20 IHC(+)/FCM(−) phenotype, and subjected to DNA sequencing, quantitative RT-PCR, and immunoblotting. L26 and B1 anti-CD20 antibodies were used in IHC and FCM analyses, respectively. Primary DLBCL cells were transplanted into NOD-SCID mice, and the engrafted lymphoma cells were used for in vitro CDC assay using rituximab. Results and Discussion: Ten patients with primary DLBCL had CD79a(+)/L26(+) results on IHC and CD19(+)/CD20(−) results on FCM. Quantitative RT-PCR indicated that CD20 mRNA expression that was significantly lower (almost 10 times) than that of positive control primary DLBCL cells. Immunoblotting analysis showed that CD20 protein expression was relatively lower than that of positive control cells, and no differences in length could be detected. No genetic mutations in the coding sequence of CD20 gene were detected in all samples examined in DNA sequencing analysis, suggesting that CD20 partial deletion is not the main reason for this phenotype. Interestingly, FCM analyses using fluorescent-labeled rituximab indicated that rituximab could partially recognize FCM CD20-B1(−)/CD19(+) B-cells, suggesting that the binding sensitivity of rituximab is much stronger than that of the B1 antibody. Lymphoma cells showing CD20 L26-IHC(+)/B1-FCM(−) phenotype proliferated in NOD-SCID mice and were partially killed by rituximab on in vitro CDC assay. These data suggest that rituximab is effective even for B-lymphoma cells with low CD20 expression if FCM using rituximab can be made to recognize those cell populations. Conclusion: Lower expression of CD20 mRNA may be one of the critical reasons for the CD20 L26-IHC(+)/B1-FCM(−) phenotype. FCM analysis using rituximab may be helpful to detect cell populations that are sensitive to rituximab treatment. Thus, rituximab therapy may be recommended if we can confirm the existence of cell populations recognized by rituximab on FCM. Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo.: Research Funding; Otsuka Pharma.: Research Funding.

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