Cancer Risk C (CR-C), a functional genomics test is a sensitive and rapid test for germline mismatch repair deficiency

Abstract

Purpose: Heritable pathogenic variants in the DNA mismatch repair (MMR) pathway cause Lynch syndrome, a condition that significantly increases risk of colorectal and other cancers. At least half of individuals tested using gene panel sequencing have a variant of uncertain significance or no variant identified leading to no diagnosis. To fill this diagnostic gap, we developed Cancer Risk C (CR-C), a flow variant assay test.

Methods: In response to treatment with an alkylating agent, individual assays of the nuclear translocation of MLH1, MSH2, BARD1, PMS2, and BRCA2 proteins and the nuclear phosphorylation of the ATM and ATR proteins distinguished pathogenic/likely pathogenic (P/LP) from benign/likely benign variants in MMR genes.

Results: A risk classification score based on MLH1, MSH2, and ATR assays was 100% sensitive and 98% specific. Causality of MMR P/LP variants was shown through gene editing and rescue. In individuals with suspected Lynch syndrome but no P/LP, CR-C identified most (73%) as having germline MMR defects. Direct comparison of CR-C on matched blood samples and lymphoblastoid cell lines yielded comparable results (r² > 0.9).

Conclusion: For identifying germline MMR defects, CR-C provides augmentation to traditional panel sequencing through greater accuracy, shorter turnaround time (48 hours), and performance on blood with minimal sample handling.

Keywords: Colorectal cancer; Functional genomics; Genetic testing; Lynch syndrome; Variant of uncertain significance.

Figure 1. Development of CR-C based on systematic evaluation of FVAs in discovery cohort.

Boxplots for group 1 (n = 20) and group 3 (n = 20) subjects’ FVAs for MMR pathway: MLH1 nuclear localization (A), MSH2 nuclear localization (B), BARD1 nuclear localization (C), PMS nuclear localization (D), ATR nuclear phosphorylation (E), and ATM nuclear phosphorylation (F). Boxplots for group 1 (n = 20) and group 3 (n = 20) subjects’ FVAs for DSBR pathway: BRCA2 nuclear translocation (G); p53 phosphorylation after MNNG treatment (H); risk classification score based on combination of MLH1, MSH2, and ATR FVAs (I). Colors represent gene where variant was identified through sequencing. CR-C, Cancer Risk C; FVA, flow variant assay; MNNG, methylnitronitrosoguanidine.

Figure 2. CR-C applied to a larger replication cohort shows similar high accuracy for discriminating individuals with P/LP and B/LB variants and diagnoses most MSI-H VUS subjects with LS.

Boxplots for CR-C FVAs for subjects with P/LP variants (group 1; n = 40), MSI-H, VUS (group 2; n = 60), and subjects negative for familial P/LP variants (group 3; n = 40). FVAs of MLH1 nuclear localization (A), MSH2 nuclear localization (B), ATR nuclear phosphorylation (C), and RCS (D). Colors represent gene where variant was identified through sequencing. CR-C, Cancer Risk C; FVA, flow variant assay; MSI-H, microsatellite instability-high; LP, likely pathogenic; LS, Lynch syndrome; P, pathogenic; RCS, risk classification score; VUS, variants of uncertain significance.

Figure 3. Transfection of wild-type cDNA expression plasmid restores benign FVA results in edited cells with P/LP variants.

Boxplots for CRISPR/Cas9 edited cells with either benign or P/LP variants in MLH1 (n = 4 each) or MSH2 (n = 4 each) and transfected with empty vector or wild-type expression plasmid containing cDNA of the corresponding edited variant: MLH1 (A), MSH2 (B), and ATR FVAs of cells with edits in MLH1 (C); MLH1 (D), MSH2 (E), and ATR FVAs of cells with edits in MSH2 (F); RCS of transfected cells with edits in MLH1 (G), and RCS of transfected cells with edits in MSH2 (H). Rescue is shown to be significant by pairwise comparison (P < .05). cDNA, complementary DNA; FVA, flow variant assay; P/LP, pathogenic/likely pathogenic; RCS, risk classification score.

Figure 4. CR-C shows similar results when comparing LCL and corresponding PBMCs.

Linear regression comparing matched PBMCs and LCLs (n = 20) from the same subjects for MLH1 FVAs (A), MSH2 FVAs (B), ATR FVAs (C), and RCS (D). CR-C, Cancer Risk C; FVA, flow variant assay; LCL, lymphoblastoid cell line; PBMC, peripheral blood mononuclear cell; RCS, risk classification score.