CD20 Mutations at the Rituximab Binding Site Are Rare and Are Not a Significant Cause of R-CHOP Resistance in Patients with De Novo Diffuse Large B-Cell Lymphoma.

Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma and is not cured in 40% of patients who receive combined Rituximab + CHOP (R-CHOP) immunochemotherapy. The mechanisms of resistance to R-CHOP therapy are poorly understood. Rituximab is a humanized monoclonal antibody directed against the CD20 antigen on B lymphocytes. Since its addition to CHOP chemotherapy in 2001, it has reduced the mortality of patients with DLBCL by 50% in British Columbia (BC). Given this significant improvement in survival, rituximab must contribute an important role in the neoplastic B cell death. Its precise binding site on the CD20 antigen has recently been elucidated (Binder et al. Blood 2006). We hypothesized that mutations at this site could be a cause of failure to cure the DLBCL with R-CHOP. If so, detection of CD20 mutations could help risk-stratify patients and identify a group who may not benefit from the addition of rituximab to their chemotherapy regimen.

Methods: We extracted DNA from 282 frozen DLBCL specimens (including 21 patients with Primary Mediastinal B cell lymphoma) at the BC Cancer Agency performed after March 2001, the date when the provincial treatment policy for advanced DLBCL was changed to R-CHOP. We amplified exon 6 of the CD20 gene which contains the rituximab epitope with the following primers: 5′-TGTAAAACGACGGCCAGTTTGGAATTCCCTCCCAGATT-3′ and 5′-CAGGAAACAGCTATGACGGATCCAGAGTTCATGCTCA-3′. In italics are the sequencing primers -21M13F and M13R. The purified 431 base pair product was bi-directionally sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit and a 3730 XL Applied Biosystems sequencer. The sequence reads where then analyzed using Polyphred/Consed.

Results: 264 patients had successful sequences for this analysis. The clinical characteristics were available on only 241 pts and were as follows: median age 63 yrs (range 16–101); 129 (62%) were male; 64% IPI 0–2, 36% IPI 3–5. 197 pts received R-CHOP chemotherapy and were evaluated for outcome. The remaining patients were recorded as not receiving rituximab either because, the information was not available; they had limited-stage disease (in 2001), or were too frail to receive chemotherapy. 20% of patients relapsed or progressed after R-CHOP after a median follow-up time of 2 years (range 0.1–6.3 years). The sequencing analysis revealed 2/264 (0.008%) cases of CD20 mutations in exon 6, both in R-CHOP treated pts. One indicates a heterozygous 4 base pair (bp) deletion in nucleotides 353–356, upstream of the epitope. Clinically, this patient progressed on R-CHOP therapy. The other indicates a heterozygous 13 bp deletion at position 722 which is downstream of the epitope. This patient achieved a complete remission with R-CHOP. Interestingly, there were no Single Nucleotide Polymorphisms (SNPs) in this 431 base pair sequence which could also potentially impact rituximab binding at this site.

Conclusions: The incidence of CD20 mutations at the rituximab binding site in 264 pts with de novo DLBCL is extremely low. Mutations at this site are therefore not a significant cause of R-CHOP resistance in this group of pts.