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Klebsiella pneumoniae strains that produce Klebsiella pneumoniae Carbapenemase (KPC) variants displaying resistance to ceftazidime-avibactam (CZA) often remain susceptible to meropenem (MEM), suggesting a potential therapeutic use of this carbapenem antibiotic. However, in vitro studies indicate that these sorts of strains can mutate becoming MEM-resistant, raising concerns about the effectiveness of carbapenems as treatment option. We have studied mutation rates occurring from the reversion of MEM-susceptible KPC-114 to MEM-resistant KPC-2, in CZA-resistant K. pneumoniae belonging to ST11. Two-step fluctuation assays (FAs) were conducted. In brief, initial cultures of KPC-114-producing K. pneumoniae showing 1 µg/mL MEM MIC were spread on Mueller-Hinton agar plates containing 2-8 µg/mL MEM. A second step of FA, at 4-16 µg/mL MEM was performed from a mutant colony obtained at 2 µg/mL MEM. Mutation rates were calculated using maximum likelihood estimation. Parental and mutant strains were sequenced by Illumina NextSeq, and mutations were predicted by variant-calling analysis. At 8 µg/mL MEM, mutants derived from parental CZA-resistant (MIC ≥ 64 µg/mL)/MEM-susceptible (MIC = 1 µg/mL) KPC-114-positive K. pneumoniae exhibited an accumulative mutation rate of 3.05 × 10-19 mutations/cell/generation, whereas at 16 µg/mL MEM an accumulative mutation rate of 1.33 × 10-19 mutations/cell/generation resulted in the reversion of KPC-114 (S181_P182 deletion) to KPC-2. These findings highlight that the reversion of MEM-susceptible KPC-114 to MEM-resistant KPC-2, in CZA-resistant K. pneumoniae ST11 is related to low mutation rates suggesting a low risk of therapeutic failure. In vivo investigations are necessary to confirm the clinical potential of MEM against CZA-resistant KPC variants.IMPORTANCEThe emergence of ceftazidime-avibactam (CZA) resistance among carbapenem-resistant Klebsiella pneumoniae is a major concern due to the limited therapeutic options. Strikingly, KPC mutations mediating CZA resistance are generally associated with meropenem susceptibility, suggesting a potential therapeutic use of this carbapenem antibiotic. However, the reversion of meropenem-susceptible to meropenem-resistant could be expected. Therefore, knowing the mutation rate related to this genetic event is essential to estimate the potential use of meropenem against CZA-resistant KPC-producing K. pneumoniae. In this study, we demonstrate, in vitro, that under high concentrations of meropenem, reversion of KPC-114 to KPC-2 in CZA-resistant/meropenem-susceptible K. pneumoniae belonging to the global high-risk ST11 is related to low mutation rates.

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BRCA1/2 genes are part of homologous recombination (HR) DNA repair pathways in charge of error-free double-strand break (DSB) repair. Loss-of-function mutations of BRCA1/2 genes have been associated for a long time with breast and ovarian cancer hereditary syndrome. Recently, polyadenosine diphosphate-ribose polymerase inhibitors (PARPi) have revolutionized the therapeutic landscape of BRCA1/2-mutated tumors, especially of BRCA1/2 high-grade serous ovarian cancer (HGSC), taking advantage of HR deficiency through the synthetic lethality concept. However, PARPi efficiency differs among patients, and most of them will develop resistance, particularly in the relapse setting. In the current proposal, we aim to review primary and secondary resistance to PARPi in HGSC owing to BRCA1/2 alterations. Of note, as several mechanisms of primary or secondary resistance to PARPi have been described, BRCA1/2 reversion mutations that restore HR pathways are by far the most reported. First, the type and location of the BRCA1/2 primary mutation have been associated with PARPi and platinum-salt sensitivity and impact the probability of the occurrence and the type of secondary reversion mutation. Furthermore, the presence of multiple reversion mutations and the variation of allelic frequency under treatment underline the role of intratumor heterogeneity (ITH) in treatment resistance. Of note, circulating tumor DNA might help us to detect and characterize reversion mutations and ITH to finally refine the treatment strategy. Importantly, forthcoming therapeutic strategies, including combination with antiangiogenics or with targeted therapies, may help us delay and overcome PARPi resistance secondary to BRCA1/2 reversion mutations. Also, progression despite PARPi therapy does not preclude PARPi rechallenge in selected patients.

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Tumor sensitivity to platinum (Pt)-based chemotherapy and poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors is increased by homologous recombination deficiency-causing mutations; in particular, reversion mutations cause drug resistance by restoring protein function. Treatment response is predicted by breast cancer susceptibility gene 1/2 (BRCA1/2) mutations; however, BRCA1/2 reversion mutations have not been comprehensively studied in pan-cancer cohorts. We aimed to characterize BRCA1/2 reversion mutations in a large pan-cancer cohort of Japanese patients by retrospectively analyzing sequencing data for BRCA1/2 pathogenic/likely pathogenic mutations in 3738 patients with 32 cancer types. We identified somatic mutations in tumors or circulating cell-free DNA that could restore the ORF of adverse alleles, including reversion mutations. We identified 12 (0.32%) patients with somatic BRCA1 (n = 3) and BRCA2 (n = 9) reversion mutations in breast (n = 4), ovarian/fallopian tube/peritoneal (n = 4), pancreatic (n = 2), prostate (n = 1), and gallbladder (n = 1) cancers. We identified 21 reversion events-BRCA1 (n = 3), BRCA2 (n = 18)-including eight pure deletions, one single-nucleotide variant, six multinucleotide variants, and six deletion-insertions. Seven (33.3%) reversion deletions showed a microhomology length greater than 1 bp, suggesting microhomology-mediated end-join repair. Disease course data were obtained for all patients with reversion events: four patients acquired mutations after PARP-inhibitor treatment failure, two showed somatic reversion mutations after disease progression, following Pt-based treatment, five showed mutations after both treatments, one patient with pancreatic cancer and BRCA1 reversion mutations had no history of either treatment. Although reversion mutations commonly occur in BRCA-associated cancers, our findings suggest that reversion mutations due to Pt-chemotherapy might be correlated with BRCA1/2-mediated tumorigenesis even in non-BRCA-associated histologies.

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BACKGROUND: The BRCA2 gene is a well-known tumor suppressor gene implicated in breast and ovarian cancers. BRCA1/2 mutations can be sensitive to poly ADP-ribose polymerase (PARP) inhibitors such as olaparib. However, some of these patients develop resistance to this treatment and an essential factor contributing to acquired insensitivity is the occurrence of reversion mutations in the BRCA1/2 genes. CASE PRESENTATION: We report the case of a 65-year-old Brazilian female patient who had previously been diagnosed with metastatic lung carcinoma carrying a BRCA2 mutation that had extended to the central nervous system. Following disease progression, olaparib was administered, resulting in a stabilizing effect on her condition for ~ 30 months. During a routine follow-up, a new triple-negative breast tumor was found. Genetic testing revealed the presence of two distinct BRCA2 gene mutations in the breast tumor. The original mutation (p.Val220Ilefs4) led to a frameshift, culminating in the production of a truncated and non-functional BRCA2 protein; the second mutation, K437fs22, rectified the reading frame of exon 11. Consequently, Rad51 could properly bind to BRCA2-an essential protein crucial for DNA repair. This restoration resulted in a functional BRCA2 protein, effectively elucidating the clinical resistance observed in the new breast tumor in this case. CONCLUSIONS: This case report highlights the clinical significance of comprehensive next-generation sequencing analyses for lung adenocarcinomas, both at diagnosis and upon progression. Such analyses enable informed decisions regarding targeted therapies and facilitate a deeper comprehension of resistance mechanisms.

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Purpose: Mutations leading to homologous recombination deficiency (HRD) increase the tumor sensitivity to platinum-based chemotherapy and PARP inhibitors. However, reversion mutations often develop conferring acquired drug resistance. There is still a lack of comprehensive investigation on HRR reversion mutations in large pan-cancer cohorts, especially in the Eastern Asian population. This study aims to characterize reversion mutations in homologous recombination repair (HRR)-related genes in a large cohort of Chinese pan-cancer patients. Methods: Sequencing data from 23,375 patients across over 17 cancer types were retrospectively analyzed for pathogenic/likely pathogenic (P/LP) germline mutations in 15 HRR genes. Somatic mutations detected in tumor or circulating cell-free DNA predicted to restore the open reading frame of the deleterious allele were subsequently identified as reversion mutations. Results: 654 cases out of 23,375 (2.8%) unselected pan-cancer patients were identified with HRR germline mutations. Secondary somatic mutations were further analyzed in their matched tumor/plasma samples. The overall frequency of reversion mutation was 1.7% (11/654). The reversion mutations occurred only in 3 out of the 15 HRR genes: BRCA1 (3.8%), BRCA2 (3.5%) and PALB2 (2.0%) from 11 patients (6 breast cancers, 1 ovarian cancer, 1 pancreatic cancer, 1 lung cancer and 2 breast and ovarian dual cancers). We identified total 25 reversion events (BRCA1, n=9; BRCA2, n=8; PALB2, n=8), including 12 pure deletions, 10 missense single nucleotide variants, 2 insertions and 1 splice site mutation. Besides, we detected microhomology length >1bp in seven out of the reversion deletions (58.3%), suggestive of microhomology-mediated end-joining (MMEJ) repair signature. Intriguingly, a positive correlation (r=0.85, p=0.001) between the length of deletion and the microhomology length was also observed. We obtained disease courses from 6/11 patients with reversion events. Four acquired reversions after the failure of the PARP inhibitor treatment. Two patients had somatic reversion mutations identified after progressing on platinum-based treatment. Conclusion: This study comprehensively depicts the prevalence and characteristics of HRR reversion mutation of germline mutations in an unselected Chinese pan-cancer cohort. The reversion mutations predominantly occurred in BRCA1, BRCA2 and PALB2. The results revealed that reversion mutations frequently occurred after resistance to platinum-based chemotherapy and/or PARP inhibitor. Our study provides insight into the underlying mechanism of drug resistance in HRD tumors and suggests that monitoring HRR mutation status along the disease course could be beneficial especially for informing resistance mechanisms and guiding subsequent therapies.

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Cancers with mutations in BRCA2 have defective DNA repair capacity which is potentially targetable with poly-ADP(ribose) polymera se (PARP) inhibitors such as olaparib and rucaparib. However, the development of a secondary mutation that restores BRCA2 function is a well-documented mechanism of resistance to PARP inhibitors. Here, we present a case report of a man with metastatic castration-resistant prostate cancer with a germline BRCA2 frameshift mutation. Treatment with olaparib resulted in an initial response but was followed by progression. Cell-free DNA testing after progression revealed the presence of polyclonal BRCA2 mutations that were estimated to restore it into the correct reading frame. We describe his treatment course and genetic testing results and then discuss the biological mechanisms driving this mechanism of resistance.

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PARP inhibitor (PARPi) therapies have been approved for treating multiple germline BRCA mutated (gBRCAm) advanced cancers including metastatic pancreatic cancer. Although significantly prolonged progression-free survival was observed in gBRCAm pancreatic cancer patients, there was no improved overall survival. The underlined resistant mechanism to PARPi therapy is worth pursuing. Here, we reported a patient with advanced pancreatic cancer harboring a germline deleterious BRCA2 V1804Kfs mutation as well as somatic mutations in KRAS, TP53 and PTEN. Stable disease was achieved with the combination therapy of cisplatin and PARPi olaparib, but the disease quickly progressed after 18 weeks of treatment. Next-generation sequencing (NGS)-based genomic profiling of the liver metastasis and liquid biopsy revealed four newly acquired BRCA2 indel mutations, including two reversion mutations that could potentially restore BRCA2 function in the PARPi-resistant tumor. Our case showed that although initial response to PARPi therapy can be achieved in advanced gBRCAm pancreatic cancer patient, the tumor rapidly evolved to acquire multiple secondary BRCA2 mutations to restore the integrity of DNA repair and confer drug resistance, which may contribute to the unimproved overall survival in pancreatic cancer patients.

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BACKGROUND: Myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (MIRAGE) syndrome is a recently described congenital disorder caused by heterozygous SAMD9 mutations. The phenotypic spectrum of the syndrome remains to be elucidated. METHODS AND RESULTS: We describe two unrelated patients who showed manifestations compatible with MIRAGE syndrome, with the exception of haematological features. Leucocyte genomic DNA samples were analysed with next-generation sequencing and Sanger sequencing, revealing the patients to have two de novoSAMD9 mutations on the same allele (patient 1 p.[Gln695*; Ala722Glu] and patient 2 p.[Gln39*; Asp769Gly]). In patient 1, p.Gln695* was absent in genomic DNA extracted from hair follicles, implying that the non-sense mutation was acquired somatically. In patient 2, with the 46,XX karyotype, skewed X chromosome inactivation pattern was found in leucocyte DNA, suggesting monoclonality of cells in the haematopoietic system. In vitro expression experiments confirmed the growth-restricting capacity of the two missense mutant SAMD9 proteins that is a characteristic of MIRAGE-associated SAMD9 mutations. CONCLUSIONS: Acquisition of a somatic nonsense SAMD9 mutation in the cells of the haematopoietic system might revert the cellular growth repression caused by the germline SAMD9 mutations (ie, second-site reversion mutations). Unexpected lack of haematological features in the two patients would be explained by the reversion mutations.

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