Distribution and functional impact of MS4A1 mutations in rrDLBCL. (A) Topology of MS4A1 transmembrane domains and extracellular loops, as annotated by Uniprot and elsewhere.47 MS4A1 mutations observed in the rrDLBCL cohort have been labeled, along with the predicted binding epitope of 4 different CD20 mAbs. (B) Comparison of antibody binding between CHO-S cells transfected with plasmids expressing either WT CD20 or 1 of 3 mutants (Tyr86His, Tyr86Cys, and Leu66Arg) for 4 different CD20 antibodies: rituximab (RTX), tositumomab (B1), obinutuzumab (BHH-2), or ofatumumab (OFA). The percent of positively stained cells was compared between mutants within each antibody (adjusted P values from 2-way analysis of variance of 3 replicates: *P > .1, **P > .01, ***P > .001, ****P > .0001). See also supplemental Figure 5. (C) Representative western blot (of 2 independent experiments performed) showing CD20 expression of CHO-S cells transfected with WT or mutant CD20 (Y86H, Y86C, and L66R) and a nontransfected (NT) control. (D) Immunohistochemistry of CD20 in a cell line and tumor tissue biopsy harboring WT CD20 as well as 2 patient-derived cell lines harboring G98R (PT255), and Y86H along with a frameshift mutation, respectively. CD20 is stained red using the L26 CD20 antibody and B-cell nuclei were stained purple using a Pax5 antibody, visualized at ×20 original magnification.