Convergent evolution of BRCA2 reversion mutations under therapeutic pressure by PARP inhibition and platinum chemotherapy.

Reversion mutations that restore wild-type function of the BRCA gene have been described as a key mechanism of resistance to Poly(ADP-ribose) polymerase (PARP) inhibitor therapy in BRCA-associated cancers. Here, we report a case of a patient with metastatic castration-resistant prostate cancer (mCRPC) with a germline BRCA2 mutation who developed acquired resistance to PARP inhibition. Extensive genomic interrogation of cell-free DNA (cfDNA) and tissue at baseline, post-progression, and postmortem revealed ten unique BRCA2 reversion mutations across ten sites. While several of the reversion mutations were private to a specific site, nine out of ten tumors contained at least one mutation, suggesting a powerful clonal selection for reversion mutations in the presence of therapeutic pressure by PARP inhibition. Variable cfDNA shed was seen across tumor sites, emphasizing a potential shortcoming of cfDNA monitoring for PARPi resistance. This report provides a genomic portrait of the temporal and spatial heterogeneity of prostate cancer under the selective pressure of a PARP inhibition and exposes limitations in the current strategies for detection of reversion mutations.

Overcoming Barriers to Tumor Genomic Profiling through Direct-to-Patient Outreach.

PURPOSE: To overcome barriers to genomic testing for patients with rare cancers, we initiated a program to offer free clinical tumor genomic testing worldwide to patients with select rare cancer subtypes. EXPERIMENTAL DESIGN: Patients were recruited through social media outreach and engagement with disease-specific advocacy groups, with a focus on patients with histiocytosis, germ cell tumors (GCT), and pediatric cancers. Tumors were analyzed using the MSK-IMPACT next-generation sequencing assay with the return of results to patients and their local physicians. Whole-exome recapture was performed for female patients with GCTs to define the genomic landscape of this rare cancer subtype. RESULTS: A total of 333 patients were enrolled, and tumor tissue was received for 288 (86.4%), with 250 (86.8%) having tumor DNA of sufficient quality for MSK-IMPACT testing. Eighteen patients with histiocytosis have received genomically guided therapy to date, of whom 17 (94%) have had clinical benefit with a mean treatment duration of 21.7 months (range, 6-40+). Whole-exome sequencing of ovarian GCTs identified a subset with haploid genotypes, a phenotype rarely observed in other cancer types. Actionable genomic alterations were rare in ovarian GCT (28%); however, 2 patients with ovarian GCTs with squamous transformation had high tumor mutational burden, one of whom had a complete response to pembrolizumab. CONCLUSIONS: Direct-to-patient outreach can facilitate the assembly of cohorts of rare cancers of sufficient size to define their genomic landscape. By profiling tumors in a clinical laboratory, results could be reported to patients and their local physicians to guide treatment. See related commentary by Desai and Subbiah, p. 2339.

Reversion mutations in germline BRCA1/2-mutant tumors reveal a BRCA-mediated phenotype in non-canonical histologies.

The association between loss of BRCA1/2 and a homologous recombination deficiency phenotype is lineage dependent. In BRCA-associated cancers such as breast, ovarian, pancreas and prostate, this phenotype confers sensitivity to PARP inhibitors and platinum-therapies. Somatic reversion mutations restoring BRCA1/2 function mediate resistance, and have exclusively been reported in BRCA-associated tumors. In this study, we analyze matched tumor and normal sequencing from 31,927 patients and identify 846 (2.7%) patients with germline BRCA1/2 variants across 43 different cancer types, including 11 with somatic reversion mutations. While nine are in BRCA-associated tumors, we find two reversion mutations in non-BRCA-associated histologies, namely lung and esophagogastric adenocarcinomas. Both were detected following platinum therapy. Whole exome sequencing confirms the homologous recombination deficiency phenotype of these tumors. While reversion mutations arise in all BRCA-associated cancer types, here we show that reversion mutations arising post-platinum in non-BRCA associated histologies, while rare, may indicate BRCA1/2 mediated tumorigenesis.

NeuroD1 Dictates Tumor Cell Differentiation in Medulloblastoma.

Tumor cells are characterized by unlimited proliferation and perturbed differentiation. Using single-cell RNA sequencing, we demonstrate that tumor cells in medulloblastoma (MB) retain their capacity to differentiate in a similar way as their normal originating cells, cerebellar granule neuron precursors. Once they differentiate, MB cells permanently lose their proliferative capacity and tumorigenic potential. Differentiated MB cells highly express NeuroD1, a helix-loop-helix transcription factor, and forced expression of NeuroD1 promotes the differentiation of MB cells. The expression of NeuroD1 in bulk MB cells is repressed by trimethylation of histone 3 lysine-27 (H3K27me3). Inhibition of the histone lysine methyltransferase EZH2 prevents H3K27 trimethylation, resulting in increased NeuroD1 expression and enhanced differentiation in MB cells, which consequently reduces tumor growth. These studies reveal the mechanisms underlying MB cell differentiation and provide rationales to treat MB (potentially other malignancies) by stimulating tumor cell differentiation.

Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA.

Multichange ISOthermal (MISO) mutagenesis is a new technique allowing simultaneous introduction of multiple site-directed mutations into plasmid DNA by leveraging two existing ideas: QuikChange-style primers and one-step isothermal (ISO) assembly. Inversely partnering pairs of QuikChange primers results in robust, exponential amplification of linear fragments of DNA encoding mutagenic yet homologous ends. These products are amenable to ISO assembly, which efficiently assembles them into a circular, mutagenized plasmid. Because the technique relies on ISO assembly, MISO mutagenesis is additionally amenable to other relevant DNA modifications such as insertions and deletions. Here we provide a detailed description of the MISO mutagenesis concept and highlight its versatility by applying it to three experiments currently intractable with standard site-directed mutagenesis approaches. MISO mutagenesis has the potential to become widely used for site-directed mutagenesis.