Homologous Recombination Deficiency: Concepts, Definitions, and Assays.

BACKGROUND: Homologous recombination deficiency (HRD) is a phenotype that is characterized by the inability of a cell to effectively repair DNA double-strand breaks using the homologous recombination repair (HRR) pathway. Loss-of-function genes involved in this pathway can sensitize tumors to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy, which target the destruction of cancer cells by working in concert with HRD through synthetic lethality. However, to identify patients with these tumors, it is vital to understand how to best measure homologous repair (HR) status and to characterize the level of alignment in these measurements across different diagnostic platforms. A key current challenge is that there is no standardized method to define, measure, and report HR status using diagnostics in the clinical setting. METHODS: Friends of Cancer Research convened a consortium of project partners from key healthcare sectors to address concerns about the lack of consistency in the way HRD is defined and methods for measuring HR status. RESULTS: This publication provides findings from the group's discussions that identified opportunities to align the definition of HRD and the parameters that contribute to the determination of HR status. The consortium proposed recommendations and best practices to benefit the broader cancer community. CONCLUSION: Overall, this publication provides additional perspectives for scientist, physician, laboratory, and patient communities to contextualize the definition of HRD and various platforms that are used to measure HRD in tumors.

Unconventional sequence requirement for viral late gene core promoters of murine gammaherpesvirus 68.

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.

Validation of a Targeted Next-Generation Sequencing Panel for Tumor Mutation Burden Analysis: Results from the Onconetwork Immuno-Oncology Consortium.

Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r2 = 0.998) and phase 2 (r2 = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research.