Purpose: Patient-specific molecular alterations leading to PARP inhibitor (PARPi) resistance are relatively unexplored. In this study, we analyzed serially collected circulating tumor DNA (ctDNA) from patients with BRCA1/2 mutations who received PARPis to investigate the resistance mechanisms and their significance in postprogression treatment response and survival. Experimental Design: Patients were prospectively enrolled between January 2018 and December 2021 (NCT05458973). Whole-blood samples were obtained before PARPi administration and serially every 3 months until progression. ctDNA was extracted from the samples and sequenced with a 531-gene panel; gene sets for each resistance mechanism were curated. Results: Fifty-four patients were included in this analysis. Mutation profiles of genes in pre-PARPi samples indicating a high tumor mutational burden and alterations in genes associated with replication fork stabilization and drug efflux were associated with poor progression-free survival on PARPis. BRCA hypomorphism and reversion were found in 1 and 3 patients, respectively. Among 29 patients with matched samples, mutational heterogeneity increased postprogression on PARPis, showing at least one postspecific mutation in 89.7% of the patients. These mutations indicate non-exclusive acquired resistance mechanisms—homologous recombination repair restoration (28%), replication fork stability (34%), upregulated survival pathway (41%), target loss (10%), and drug efflux (3%). We observed poor progression-free survival with subsequent chemotherapy in patients with homologous recombination repair restoration (P = 0.003) and those with the simultaneous involvement of two or more resistance mechanisms (P = 0.040). Conclusions: Analysis of serial ctDNAs highlighted multiple acquired resistance mechanisms, providing valuable insights for improving postprogression treatment and survival.

Gastric cancer (GC) is one of the most common deadly cancers in the world. Although patient genomic data have identified AT-rich interaction domain 1A (ARID1A), a key chromatin remodeling complex subunit, as the second most frequently mutated gene after TP53, its in vivo role and relationship to TP53 in gastric tumorigenesis remains unclear. Establishing a novel mouse model that reflects the ARID1A heterozygous mutations found in the majority of human GC cases, we demonstrated that Arid1a heterozygosity facilitates tumor progression through a global loss of enhancers and subsequent suppression of the p53 and apoptosis pathways. Moreover, mouse genetic and single-cell analyses demonstrated that the homozygous deletion of Arid1a confers a competitive disadvantage through the activation of the p53 pathway, highlighting its distinct dosage-dependent roles. Using this unique vulnerability of Arid1a mutated GC cells, our combined treatment with the epigenetic inhibitor, TP064, and the p53 agonist, Nutlin-3, inhibited growth of Arid1a heterozygous tumor organoids, providing a novel therapeutic option for GC.

Ultra-deep sequencing to detect low-frequency mutations in circulating tumor-derived DNA (ctDNA) increases the diagnostic value of liquid biopsy. The demand for large ctDNA panels for comprehensive genomic profiling and tumor mutational burden (TMB) estimation is increasing; however, few ctDNA panels for TMB have been validated. Here, we designed a ctDNA panel with 531 genes, named TMB500, along with a technical and clinical validation. Synthetic reference cell-free DNA materials with predefined allele frequencies were sequenced in a total of 92 tests in 6 batches to evaluate the precision, linearity, and limit of detection of the assay. We used clinical samples from 50 patients with various cancers, 11 healthy individuals, and paired tissue samples. Molecular barcoding and data analysis were performed using customized pipelines. The assay showed high precision and linearity (coefficient of determination, r² =0.87) for all single nucleotide variants, with a limit of detection of 0.24%. In clinical samples, the TMB500 ctDNA assay detected most variants present and absent in tissues, showing that ctDNA could assess tumor heterogeneity in different tissues and metastasis sites. The estimated TMBs correlated well between tissue and blood, except in 4 cases with extreme heterogeneity that showed very high blood TMBs compared to tissue TMBs. A pilot evaluation showed that the TMB500 assay could be used for disease monitoring. The TMB500 assay is an accurate and reliable ctDNA assay for many clinical purposes. It may be useful for guiding the treatment of cancers with diverse genomic profiles, estimating TMB in immune therapy, and disease monitoring.

The phenotype of the 5α-reductase type 2 deficiency (5αRD2) by the SRD5A2 gene mutation varies, and although there have been many attempts, the genotype-phenotype correlation still has not yet been adequately evaluated. Recently, the crystal structure of the 5α-reductase type 2 isozyme (SRD5A2) has been determined. Therefore, the present study retrospectively evaluated the genotype-phenotype correlation from a structural perspective in 19 Korean patients with 5αRD2. Additionally, variants were classified according to structural categories, and phenotypic severity was compared with previously published data. The p.R227Q variant, which belongs to the NADPH-binding residue mutation category, exhibited a more masculine phenotype (higher external masculinization score) than other variants. Furthermore, compound heterozygous mutations with p.R227Q mitigated phenotypic severity. Similarly, other mutations in this category showed mild to moderate phenotypes. Conversely, the variants categorized as structure-destabilizing and small to bulky residue mutations showed moderate to severe phenotypes, and those categorized as catalytic site and helix-breaking mutations exhibited severe phenotypes. Therefore, the SRD5A2 structural approach suggested that a genotype-phenotype correlation does exist in 5αRD2. Furthermore, the categorization of SRD5A2 gene variants according to the SRD5A2 structure facilitates the prediction of the severity of 5αRD2 and the management and genetic counseling of patients affected by it.

Yeeun Shim, M.S., Jieun Seo, Ph.D., Seung-Tae Lee, M.D., Ph.D., Jong Rak Choi, M.D., Ph.D., Young-Chul Choi, M.D., Ph.D., Saeam Shin, M.D., Ph.D., and Hyung Jun Park, M.D., Ph.D.. Ann Lab Med -0001;0:. https://doi.org/10.3343/alm.2023.0437