67 Olaparib in patients with biallelic BRCA1/2 inactivation gies had CB, which may in part contribute to the success of the cohort. Seven of 24 patients had non-BRCA associated tumor types, of whom four (57%) had clinical benefit (Supplementary Table 1). These results indicate that patients with tumor types other than the known BRCA associated histologies can benefit from treatment with PARPi, provided that they have bi-allelic LoF of BRCA, resulting in HRD. It also emphasizes the importance of extensive molecular tumor profiling by means of WGS or large panel sequencing for all patients. Small tumor-specific sequencing panels would, in all seven patients in this cohort, not have identified the BRCA mutations, as BRCA diagnostics is not part of the regular reimbursed care for these tumor types. An important limitation of this study is the small sample size of 24 patients. Nine different tumor types were enrolled in this histology-agnostic cohort, resulting in a heterogeneous population with large variations in biological tumor features and previous treatments. The number of patients per tumor type is low, there is a relative underrepresentation of patients with non-BRCA associated tumor types and since some important tumor types (i.e. non-Small Cell Lung Cancer) are not represented in our cohort, the results cannot simply be extrapolated to all patients with cancer. Though we find a clinically relevant signal of activity here, confirmation of our findings in a larger cohort is essential, with special emphasis on patients with non-BRCA tumor types. Six patients ultimately did not have bi-allelic BRCA loss (mono-allelic loss: N=4; no BRCA variant: N=2). In two patients with prostate cancer, the qualifying BRCA variant could not be re-identified in the baseline biopsy WGS data, the exact reason for this discordance is unclear. No evidence for reversion of HRD (for example due to platinum-based chemotherapy) was found in the WGS data. A possible explanation in both cases could be inter- or intra-tumor heterogeneity. Alternatively, in the first patient the low VAF of 24% may suggest that BRCA2 LoF was not the major driver of tumorigenesis and that the variant was lost in clonal evolution. However, the short time between pre-enrollment biopsy and baseline study biopsy did not support this. In the other patient with a BRCA1 exon 1 deletion, an alternative explanation could be that the deletion of exon 1, which is located outside the open reading frame and contains the BRCA1 promoter, could not be picked up by the bioinformatics pipeline. However, the low HRD-score suggests that there was no functional HRD, which points towards the more likely hypothesis of tumor heterogeneity. In three other patients, the information regarding bi-allelic status of BRCA could not be retrieved. In the early days of the trial, confirmation of complete LoF of BRCA was not a requirement for eligibility. The initial inclusion of patients without complete LoF of BRCA1 or BRCA2 in this cohort may be considered a weakness but we regard it as an unintentional strength, as it underlines the importance of a sharply defined biomarker. Our data illustrate the contrast between the groups with and without complete LoF, in terms of CB to PARPi treatment (73% versus 17%). Clearly, this study is not powered to demonstrate a significant difference between these subgroups within the cohort due to the small number of patients. However, we noted this as an interesting signal that warrants confirmation in a larger independent cohort. Currently, pathologists and molecular biologists struggle to reliably call loss-of-heterogeneity and bi-allelic BRCA LoF using the available standard large 3
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