Characterization of Aberrations in DNA Damage Repair Pathways in Gastrointestinal Stromal Tumors: The Clinicopathologic Relevance of γH2AX and 53BP1 in Correlation with Heterozygous Deletions of CHEK2, BRCA2, and RB1

SIMPLE SUMMARY: Aberrations of DNA damage repair (DDR) pathways enable the transformation of pre-neoplastic lesions, but their roles are barely understood in gastrointestinal stromal tumors (GISTs). The targeted next-generation sequencing of GISTs has demonstrated heterozygous deletions (HetDels) as frequent aberrations of DDR genes, usually lacking recurrent pathogenic single-nucleotide variants. As independently validated by multiplex ligation-dependent probe amplification, CHEK2-HetDel was the most prevalent, and similar to BRCA2-HetDel, was only related to older age groups. RB1-HetDel, albeit rare, was preferentially detected in the non-gastric, high-risk, and 53BP1-overexpressing GISTs, with occasional co-occurrence with BRCA2-HetDel. Being strongly correlated with immunofluorescence, immunohistochemistry reliably showed positive associations between the risk levels and expression levels of γH2AX and 53BP1, representative of DDR response, in two independent cohorts. Compared with those overexpressing either one or two biomarkers, low expressers of both γH2AX and 53BP1 displayed significantly longer disease-free survival in GISTs, indicating the early engagement of DDR aberrations in tumorigenesis. ABSTRACT: Genetic aberrations involving DNA damage repair (DDR) remain underexplored in gastrointestinal stromal tumors (GISTs). We characterized DDR abnormalities using targeted next-generation sequencing and multiplex ligation-dependent probe amplification, and performed immunofluorescence (IF) and immunohistochemistry (IHC) analyses of γH2AX and 53BP1. Consistent with IF-validated nuclear co-localization, γH2AX and 53BP1 showed robust correlations in expression levels, as did both biomarkers between IF and IHC. Without recurrent pathogenic single-nucleotide variants, heterozygous deletions (HetDels) frequently targeted DNA damage-sensing genes, with CHEK2-HetDel being the most prevalent. Despite their chromosomal proximity, BRCA2 and RB1 were occasionally hit by HetDels and were seldom co-deleted. HetDels of CHEK2 and BRCA2 showed a preference for older age groups, while RB1-HetDel predominated in the non-gastric, high-risk, and 53BP1-overexpressing GISTs. Higher risk levels were consistently related to γ-H2AX or 53BP1 overexpression (all p < 0.01) in two validation cohorts, while only 53BP1 overexpression was associated with the deletion of KIT exon 11 (KITex11-del) among genotyped GISTs. Low expressers of dual biomarkers were shown by univariate analysis to have longer disease-free survival (p = 0.031). However, higher risk levels, epithelioid histology, and KITex11-del retained prognostic independence. Conclusively, IHC is a useful surrogate of laborious IF in the combined assessment of 53BP1 and γ-H2AX to identify potential DDR-defective GISTs, which were frequently aberrated by HetDels and a harbinger of progression.