Germline and Tumor Sequencing as a Diagnostic Tool To Resolve Suspected Lynch Syndrome

Abstract

Patients in whom mismatch repair (MMR)-deficient cancer develops in the absence of pathogenic variants of germline MMR genes or somatic hypermethylation of the MLH1 gene promoter are classified as having suspected Lynch syndrome (SLS). Germline whole-genome sequencing (WGS) and targeted and genome-wide tumor sequencing were applied to identify the underlying cause of tumor MMR deficiency in SLS. Germline WGS was performed on samples from 14 cancer-affected patients with SLS, including two sets of first-degree relatives. MMR genes were assessed for germline pathogenic variants, including complex structural rearrangements and noncoding variants. Tumor tissue was assessed for somatic MMR gene mutations using targeted, whole-exome sequencing or WGS. Germline WGS identified pathogenic MMR variants in 3 of the 14 cases (21.4%), including a 9.5-megabase inversion disrupting MSH2 in a mother and daughter. Excluding these 3 MMR carriers, tumor sequencing identified at least two somatic MMR gene mutations in 8 of 11 tumors tested (72.7%). In a second mother-daughter pair, a somatic cause of tumor MMR deficiency was supported by the presence of double somatic MSH2 mutations in their respective tumors. More than 70% of SLS cases had double somatic MMR mutations in the absence of germline pathogenic variants in the MMR or other DNA repair-related genes on WGS, and, therefore, were confidently assigned a noninherited cause of tumor MMR deficiency.

Figure 1

Family pedigrees of two sets of mother–daughter pairs. A: SLS11 and SLS12. B: SLS9 and SLS10. Probands SLS11 (Family A) and SLS9 (Family B) are indicated with arrows. In Family A, an inversion-specific PCR test was used for genotyping 14 additional relatives, identifying four additional carriers (two CRC affected, and one in whom adenomas developed at age 34 years) and two additional obligate carriers (both unaffected). HP, hyperplastic polyp; TA, tubular adenoma; TV, tubulovillous adenoma

Figure 2

A summary of testing approach and outcomes and the final classification of each SLS case from the germline whole-genome sequencing (WGS) and tumor-sequencing analyses in this study. ^†SLS2: kidney tumor (T2A) failed tumor sequencing. ^‡Additional tumor WGS information: no MMR deficiency–related mutational signatures, microsatellite stable by microsatellite instability sensor, tumor mutation burden not considered hypermutated; therefore, tumor not considered to have MMR deficiency and likely false-positive loss of MSH6 expression on IHC. ACCFR, Australasian Colorectal Cancer Family Registry; ANECS, Australian National Endometrial Cancer Study.

Figure 3

The somatic mutational signature components for tumor samples T5, T9A, T9B, T10, and T13 indicated by the signature number, suggested etiology, and the percentage contribution to the overall mutational composition for each sample. APOBEC, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like.