Identification of CD20 Mutations in Malignant Lymphoma: Can They Be Predictors of Response to Rituximab?.

Purpose: Although rituximab is commonly used as induction and maintenance therapy for CD20+ malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance.

Methods: To investigate whether CD20 mutations affect the response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, peripheral blood or bone marrow of 48 patients with NHL using magnetic activated cell sorting (MACS). CD19+/CD20+ cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The resulting PCR products were sequenced, subcloned into the mammalian expression vector pTARGET, transfected into K562 cells and CD20 expression was examined by flow cytometry and laser scanning confocal microscopy.

Results: In all 48 patients, overall response rate (CR+CRu+PR) to rituximab was 93.8% (45/48), but two cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in two CD20 domains - the third transmembrane domain and the C-terminal cytoplasmic domain. One patient had point mutations in the transmembrane domain, three cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. Thirty-eight patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain.

Conclusions: This study suggests that point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab.