CD20 Protein Immunohistochemistry-Positive/Flow Cytometry-Negative Diffuse Large B-Cell Lymphoma—Analyses of the Molecular Mechanisms and Rituximab Effectiveness
Authors: Tokunaga Takashi , Tomita Akihiro , Hiraga Junji , Sugimoto Takumi , Shimada Kazuyuki , Sugimoto Keiki , Kinoshita Tomohiro , Naoe Tomoki 
Published: October 2019

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.