CD20-Negative Phenotypic Change In B-Cell Lymphoma Cells After Using Rituximab: Possibility of a Particular Clinicopathologic Phenomenon Post-Rituximab Extranodal CD20-Negative Lymphoma

Rituximab is an anti-CD20 mouse/human chimeric monoclonal antibody. Recent reports indicate that combination chemotherapy with rituximab for CD20(+) B-cell lymphoma has improved the clinical outcomes compared to conventional chemotherapy without rituximab. However, patients showing rituximab resistance have been also recognized. We previously reported observation of relapse (RD)/progression (PD) with CD20(-) phenotype after using rituximab, and CD20(-) phenotype is closely related to ritxuximab resistance (Hiraga J, et al., Blood, 2009., Sugimoto T, et al., Biochem Biophys Res Commun, 2009., Tomita A, et al., Int J Hematol, 2007.). We concluded that aberrant down-regulation of MS4A1 (CD20) gene expression was one of the critical mechanisms of CD20(-) phenotypic change. Moreover, all those patients died of disease progression within one year after CD20(-) change, indicating the tendency for a relatively poor prognosis. Since such patients occur less frequently, more patients are definitely needed to confirm the clinical features and molecular basis.

To confirm the clinical features of patients who show CD20-protein-negative phenotypic change after using rituximab. Analyses of the molecular backgrounds of CD20(-) phenotypes will be conducted using CD20(-)-transformed primary lymphoma cells.

CD20(+) B-cell lymphoma patients (DLBCL, FL, MALT, MCL, CLL/SLL, and Burkitt) treated with rituximab containing chemotherapy, were used for the analyses. CD20 protein expression was confirmed by immunohistchemistry (IHC) and flow cytometry (FCM) using primary lymphoma cells at the time points of the initial diagnosis and the RD/PD. All patients analyzed showed CD20 IHC(+)/FCM(+) phenotype at the initial diagnosis, and the CD20(-) change was confirmed by IHC(-)/FCM(-) at RD/PD period. Ten patients showed CD20(-) phenotypic change after using rituximab. Pathological diagnosis of those patients was DLBCL (8 out of 10 cases; 8/10) and FL (2/10) at the initial diagnosis, and DLBCL (10/10) at CD20(-) change in RD/PD period. TdT was negative by IHC in 6 out of 6 analyzed cases. Bone marrow (BM) invasion at diagnosis was confirmed in 9/10, and extranodal involvement (BM, CNS, skin, and peripheral blood) at CD20(-) RD/PD period was confirmed in all patients (10/10). Leukemic transformation at the terminal stage was seen in 6/10 patients, and LDH elevation seems to reflect their disease status. All 10 patients died of disease progression within 1 year after the diagnosis of CD20(-) phenotypic change in spite of salvage chemotherapy. Molecular backgrounds were analyzed in patients whose primary lymphoma cells were available for molecular analyses. Quantitative RT-PCR indicated that the CD20 mRNA expression level was significantly lower than that of CD20 positive controls in all analyzed cases (5/5). Almost the same result was obtained by immunoblot analysis using anti-CD20 C-terminus antibody. Genetic mutations on the coding sequence (CDS) of MS4A1 gene were suggested in 2 out of 7 cases, but the frequency of the mutated clone was less than 20%. CD79b ITAM mutation was confirmed in 1 out of 5 cases. In vitro rituximab binding assay using fluorescent-labeled-rituximab and primary lymphoma cells indicated that the rituximab binding was significantly lower on CD20(-) cells compared to the control cells.

All the patients showing CD20(-) phenotypic change were diagnosed as DLBCL, and extranodal infiltration was confirmed in all cases. Leukemic transformation was relatively common at the terminal stage, and LDH elevation seemed to reflect the disease progression. CD20 mRNA expression level was generally lower than that of positive control cells, and clinically significant MS4A1 gene mutations were not confirmed. All the patients died within 1 year after diagnosis of CD20(-) transformation, suggesting that the CD20(-) change may closely correlate with the poor prognosis. From these findings, the phenotypic change after using rituximab may be considered as a particular clinicopathologic group such as “post-rituximab extranodal CD20-negative lymphoma.” Further accumulation of patients is warranted.

Kinoshita:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.