CD20 (MS4A1) mutations are strongly associated with prior chemoimmunotherapy exposure in Waldenström macroglobulinemia and have predicted functional consequences for the development of CD20-directed cellular therapies.

Abstract Introduction: CD20, encoded by MS4A1, is one of the earliest and most extensively used therapeutic targets in B-cell malignancies. In Waldenström macroglobulinemia (WM), rituximab-based chemoimmunotherapy (CIT) and BTK inhibitors are mainstays of many frontline regimens. Although resistance mutations to BTK inhibitors are well characterized, the mechanisms underlying resistance to CIT and the diminished efficacy observed upon rituximab re-exposure remain poorly understood. This knowledge gap is particularly relevant as novel CD20 therapies (e.g., bispecific T-cell engagers, CAR-T) are entering clinical trials for previously treated WM patients. We therefore performed the largest sequencing effort reported in previously treated WM patients, within which we focused on genomic alterations in MS4A1. Methods Whole-exome sequencing was performed in CD19+ sorted bone marrow mononuclear cells and CD19-depleted mononuclear cells from peripheral blood (germline control) for 247 previously treated WM patients. Clinical data were annotated to genomic findings. Structural localization of MS4A1 mutations was assessed using the CD20 crystal structure (PDB: 6Y90) and B-factors were plotted by residue number to annotate structural domains, including the four transmembrane regions (TM1-TM4), both extracellular loops, and therapeutic antibody binding sites. Mutations were modeled in 3D to assess structural consequences. Functional impact was assessed using in-silico tools with predefined deleterious thresholds: CADD (>20) and AlphaMissense (>70) for point mutations, and SpliceAI (>70) for splice-site mutations. Results We restricted our analysis to 164 patients who had a >5% MYD88 variant allele fraction to ensure adequate tumor content. The median age for these patients was 64.9 (36.7-88.5 years) and 61% were male. The median time from diagnosis and first therapy were 6.04 and 4.33 years, respectively. Patients received a median of 1.5 prior therapies (range 1-9), with 64 (38.4%) having received CIT with a CD20-containing antibody, 81 (49.4%) receiving a CD20 antibody without chemotherapy, and 20 (12.2%) not receiving any anti-CD20 antibody. At the time of study biopsy, 57.9% were progressing. CXCR4 mutations and TP53 alterations were identified in 89/164 (45.7%) and 19/163 (11.6%) of patients. Among the 164 MYD88 mutated, previously treated WM patients, 9 (5.5%) harbored somatic CD20 (MS4A1) mutations. 20 MS4A1 mutations (median 2 per patient; range 1-4) were detected among these 9 patients, with only one recurrent variant (p.Lys50Ter in 2 patients). Seven of nine (78%) patients harbored truncating mutations within TM1 and TM3 that were upstream of the rituximab epitope (aa 168-175) and predicted to eliminate rituximab's binding site. Another patient harbored two mutations: an in-frame insertion at amino acid 52 and a missense p.Gly60Val substitution within the α-helix of TM1 (B-factor <50 Ų), both with high predicted impact (CADD 28.8; AlphaMissense 0.75) and modeled to disrupt CD20 folding and surface expression. The ninth patient harbored a splice-site mutation (c.336+1G>T) predicted to result in altered protein structure (SpliceAI score 0.79; CADD 33). Importantly, MS4A1 mutations were observed exclusively in patients previously exposed to CD20-containing CIT (9/63; 14.3%) and were absent among those treated with CD20 antibody without chemotherapy (0/81) or never exposed to a CD20 antibody (0/20; p<0.001 for three-way comparison). All nine patients with MS4A1 mutations had progressed after treatment with a CD20-containing CIT regimen. Additionally, they had received more prior anti-CD20 containing lines of therapy (median 3 vs. 2; p=0.02) compared to CIT-exposed patients without MS4A1 mutations. Compared to all patients without MS4A1 mutations, those with mutations had received more prior lines of therapy (median 3 vs. 1; p=0.04) and had a higher frequency of co-occurring TP53 alterations (33.3% vs. 9%; p=0.01). Conclusion: CD20 (MS4A1) mutations are common in WM patients previously exposed to CD20 antibody containing chemotherapy and are predicted to impact rituximab as well as other CD20-targeting agents, including cellular therapies. Our findings provide clarity on mechanisms associated with resistance to rituximab in previously treated WM patients and inform efforts for the development and selection of candidates for CD20 strategies, including cell-based approaches, in the relapsed or refractory setting.