Characterization of discordance between mismatch repair deficiency and microsatellite instability testing may prevent inappropriate treatment with immunotherapy.

In the Drug Rediscovery Protocol (DRUP), patients with cancer are treated based on their tumor molecular profile with approved targeted and immunotherapies outside the labeled indication. Importantly, patients undergo a tumor biopsy for whole-genome sequencing (WGS) which allows for a WGS-based evaluation of routine diagnostics. Notably, we observed that not all biopsies of patients with dMMR/MSI-positive tumors as determined by routine diagnostics were classified as microsatellite-unstable by subsequent WGS. Therefore, we aimed to evaluate the discordance rate between routine dMMR/MSI diagnostics and WGS and to further characterize discordant cases. We assessed patients enrolled in DRUP with dMMR/MSI-positive tumors identified by routine diagnostics, who were treated with immune checkpoint blockade (ICB) and for whom WGS data were available. Patient and tumor characteristics, study treatment outcomes, and material from routine care were retrieved from the patient medical records and via Palga (the Dutch Pathology Registry), and were compared with WGS results. Initially, discordance between routine dMMR/MSI diagnostics and WGS was observed in 13 patients (13/121; 11%). The majority of these patients did not benefit from ICB (11/13; 85%). After further characterization, we found that in six patients (5%) discordance was caused by dMMR tumors that did not harbor an MSI molecular phenotype by WGS. In six patients (5%), discordance was false due to the presence of multiple primary tumors (n = 3, 2%) and misdiagnosis of dMMR status by immunohistochemistry (n = 3, 2%). In one patient (1%), the exact underlying cause of discordance could not be identified. Thus, in this group of patients limited to those initially diagnosed with dMMR/MSI tumors by current routine diagnostics, the true assay-based discordance rate between routine dMMR/MSI-positive diagnostics and WGS was 5%. To prevent inappropriate ICB treatment, clinicians and pathologists should be aware of the risk of multiple primary tumors and the limitations of different tests. © 2024 The Pathological Society of Great Britain and Ireland.

Proper genomic profiling of (BRCA1-mutated) basal-like breast carcinomas requires prior removal of tumor infiltrating lymphocytes.

INTRODUCTION: BRCA1-mutated breast carcinomas may have distinct biological features, suggesting the involvement of specific oncogenic pathways in tumor development. The identification of genomic aberrations characteristic for BRCA1-mutated breast carcinomas could lead to a better understanding of BRCA1-associated oncogenic events and could prove valuable in clinical testing for BRCA1-involvement in patients. METHODS: For this purpose, genomic and gene expression profiles of basal-like BRCA1-mutated breast tumors (n = 27) were compared with basal-like familial BRCAX (non-BRCA1/2/CHEK2*1100delC) tumors (n = 14) in a familial cohort of 120 breast carcinomas. RESULTS: Genome wide copy number profiles of the BRCA1-mutated breast carcinomas in our data appeared heterogeneous. Gene expression analyses identified varying amounts of tumor infiltrating lymphocytes (TILs) as a major cause for this heterogeneity. Indeed, selecting tumors with relative low amounts of TILs, resulted in the identification of three known but also five previously unrecognized BRCA1-associated copy number aberrations. Moreover, these aberrations occurred with high frequencies in the BRCA1-mutated tumor samples. Using these regions it was possible to discriminate BRCA1-mutated from BRCAX breast carcinomas, and they were validated in two independent cohorts. To further substantiate our findings, we used flow cytometry to isolate cancer cells from formalin-fixed, paraffin-embedded, BRCA1-mutated triple negative breast carcinomas with estimated TIL percentages of 40% and higher. Genomic profiles of sorted and unsorted fractions were compared by shallow whole genome sequencing and confirm our findings. CONCLUSION: This study shows that genomic profiling of in particular basal-like, and thus BRCA1-mutated, breast carcinomas is severely affected by the presence of high numbers of TILs. Previous reports on genomic profiling of BRCA1-mutated breast carcinomas have largely neglected this. Therefore, our findings have direct consequences on the interpretation of published genomic data. Also, these findings could prove valuable in light of currently used genomic tools for assessing BRCA1-involvement in breast cancer patients and pathogenicity assessment of BRCA1 variants of unknown significance. The BRCA1-associated genomic aberrations identified in this study provide possible leads to a better understanding of BRCA1-associated oncogenesis.