Complement-Regulatory Proteins CFHR1 and CFHR3 and Patient Response to Anti-CD20 Monoclonal Antibody Therapy

Abstract

Purpose: Anti-CD20 mAb therapies, including rituximab and obinutuzumab (GA101), are common treatments for follicular lymphoma. In an effort to better understand the role of complement in mAb action, we recently performed germline SNP profiling on 142 follicular lymphoma patients and found rs3766404 genotype correlated with patient response to rituximab. To assess the role of three SNP-associated complement-regulatory proteins (CFH, CFHR1, and CFHR3) in clinical response to anti-CD20 mAb, we studied two cohorts of patients treated with anti-CD20 mAb.Experimental Design: Cohorts included the Iowa/Mayo Lymphoma SPORE observational cohort of subjects with a new diagnosis of follicular lymphoma treated with rituximab and the GAUSS prospective randomized trial cohort of follicular lymphoma subjects randomized to receive single-agent rituximab or obinutuzumab. Circulating protein expression was measured for CFH, CFHR1, and CFHR3 and correlated to clinical outcome.Results: rs3766404 genotype correlated with expression of the related downstream genes CFHR1 and CFHR3 Loss of CFHR1 expression correlated with inferior patient outcome in the observational cohort, but not in the GAUSS cohort. Loss of CFHR3 correlated with superior event-free survival in GAUSS subjects treated with obinutuzumab, but not rituximab.Conclusions: We conclude that the relationship between complement-regulatory proteins CFHR1 and CFHR3 and response to anti-CD20 mAb therapy varies based on the specific anti-CD20 mAb used. We propose that CFHR3 is a candidate biomarker for obinutuzumab response. Further studies are needed to validate these findings and to better understand how complement pathways and complement-regulatory proteins impact on the efficacy of anti-CD20 mAb therapy. Clin Cancer Res; 23(4); 954-61. ©2016 AACR.

Figure 1. SNP genotype and plasma CFH, CFHR1, and CFHR3 expression

A. Plasma collected from FL subjects in the UI/Mayo Lymphoma SPORE were analyzed by western blot using an anti-CFHR1 antibody that cross reacts with CFH to measure plasma protein levels. Individuals with the C/C genotype retained expression of CFH in their sera, while CFHR1 expression was lost. Asterisks indicate samples genotyped in panel B. Negative control sera, from which CFH and CFHR proteins were depleted, was purchased from CompTech (Tyler, TX). Purified CFH, also purchased from CompTech, was used as the positive control. B. Genomic DNA was isolated the FL subjects indicated in panel A. PCR amplification of CFHR1 or CFHR3 was performed, using Raji cells as a positive control. Individuals with homozygous minor allele (C/C) also exhibited homozygous deletion of CFHR1 and CFHR3. Note this approach cannot detect a heterozygous deletion. The negative control was identified based on lack of plasma CFHR1 and CFHR3 expression. C. Plasma protein expression in FL subjects was analyzed by western blot as shown in panel A, and expression was quantified using band densitometry. A T/T reference plasma sample from a healthy donor and expressing both CFH and CFHR1 was included on every gel, and protein levels were normalized to these before graphing. While plasma CFH and CFHR3 levels do not vary based on SNP genotype, CFHR1 levels are reduced in individuals with at least one C allele (P<0.001, unpaired t test). Dots represent individual patients and the horizontal bars represent the mean.

Figure 2. Event free survival by rs3766404 genotype in GAUSS clinical cohort

Kaplan-Meier survival curves of FL subjects participating in the GAUSS clinical trial based on SNP genotype. A. FL patients treated with rituximab had similar event free survival (EFS) regardless of SNP genotype (P=0.8635, log-rank test). (T/T n=22, T/C and C/C n=8.) B. FL patients treated with obinutuzumab also had similar EFS regardless of SNP genotype (P=0.1985, log-rank test). (T/T n=23, T/C and C/C n=8.).