Identification of Spliceogenic Variants beyond Canonical GT-AG Splice Sites in Hereditary Cancer Genes.

Pathogenic/likely pathogenic variants in susceptibility genes that interrupt RNA splicing are a well-documented mechanism of hereditary cancer syndromes development. However, if RNA studies are not performed, most of the variants beyond the canonical GT-AG splice site are characterized as variants of uncertain significance (VUS). To decrease the VUS burden, we have bioinformatically evaluated all novel VUS detected in 732 consecutive patients tested in the routine genetic counseling process. Twelve VUS that were predicted to cause splicing defects were selected for mRNA analysis. Here, we report a functional characterization of 12 variants located beyond the first two intronic nucleotides using RNAseq in APC, ATM, FH, LZTR1, MSH6, PALB2, RAD51C, and TP53 genes. Based on the analysis of mRNA, we have successfully reclassified 50% of investigated variants. 25% of variants were downgraded to likely benign, whereas 25% were upgraded to likely pathogenic leading to improved clinical management of the patient and the family members.

Genetic testing results in Slovenian male breast cancer cohort indicate the BRCA2 7806-2A > G founder variant could be associated with higher male breast cancer risk.

PURPOSE: To analyze the prevalence of pathogenic/likely pathogenic variants (P/LPVs) in BRCA1 and BRCA2 genes in the largest cohort of Slovenian male breast cancer (MBC) patients to date and to explore a possible correlation between the Slovenian founder variant BRCA2:c.7806-2A > G and predisposition to MBC. METHODS: We performed a retrospective analysis of 81 MBC cases who underwent genetic counseling and/or testing between January 1999 and May 2020. To explore a possible genotype-phenotype correlation, we performed additional analyses of 203 unrelated families with P/LPVs in BRCA2 and 177 cases of female breast cancer (FBC) in carriers of P/LPVs in BRCA2. RESULTS: Detection rate of P/LPVs in the BRCA1 and BRCA2 genes was 24.7% (20/81) with 95% of them in BRCA2 gene. The only two recurrent P/LPVs were BRCA2:c.7806-2A > G and BRCA2:c.3975_3978dupTGCT (9 and 5 MBC cases, respectively). In families with BRCA2:c.7806-2A > G, the incidence of MBC cases was higher compared to families with other P/LPVs in BRCA2; however, the difference did not reach statistical significance (17.8% vs. 8.9%, p = 0.105). BRCA2:c.7806-2A > G was detected in both families with multiple cases of MBC. This splice-site variant represented a significantly higher proportion of all BRCA2 P/LPVs detected in MBC carriers compared to FBC carriers (47.4% vs. 26%, p = 0.049). CONCLUSION: We observed a high mutation detection rate and conclude this may be due to the prevalent BRCA2:c.7806-2A > G variant in Slovenia. Our results indicate a possible association between this variant and higher risk of breast cancer in males compared to other identified P/LPVs in BRCA2.

Outcome of severe infections in afebrile neutropenic cancer patients.

BACKGROUND: In some neutropenic cancer patients fever may be absent despite microbiologically and/or clinically confirmed infection. We hypothesized that afebrile neutropenic cancer patients with severe infections have worse outcome as compared to cancer patients with febrile neutropenia. PATIENTS AND METHODS: We retrospectively analyzed all adult cancer patients with chemotherapy-induced neutropenia and severe infection, who were admitted to the Intensive Care Unit at our cancer center between 2000 and 2011. The outcome of interest was 30-day in-hospital mortality rate. Association between the febrile status and in-hospital mortality rate was evaluated by the Fisher's exact test. RESULTS: We identified 69 episodes of severe neutropenic infections in 65 cancer patients. Among these, 9 (13%) episodes were afebrile. Patients with afebrile neutropenic infection presented with hypotension, severe fatigue with inappetence, shaking chills, altered mental state or cough and all of them eventually deteriorated to severe sepsis or septic shock. Overall 30-day in-hospital mortality rate was 55.1%. Patients with afebrile neutropenic infection had a trend for a higher 30-day in-hospital mortality rate as compared to patients with febrile neutropenic infection (78% vs. 52%, p = 0.17). CONCLUSIONS: Afebrile cancer patients with chemotherapy-induced neutropenia and severe infections might have worse outcome as compared to cancer patients with febrile neutropenia. Patients should be informed that severe neutropenic infection without fever can occasionally occur during cancer treatment with chemotherapy.

A Population-Based Study of Patients With Small Cell Carcinoma of the Ovary, Hypercalcemic Type, Encompassing a 30-Year Period.

CONTEXT.­: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and lethal tumor, characterized by hypercalcemia and early onset and associated with germline and somatic SMARCA4 variants. OBJECTIVE.­: To identify all known cases of SCCOHT in the Slovenian population from 1991 to 2021 and present genetic testing results, histopathologic findings, and clinical data for these patients. We also estimate the incidence of SCCOHT. DESIGN.­: We conducted a retrospective analysis of hospital medical records and data from the Slovenian Cancer Registry in order to identify cases of SCCOHT and obtain relevant clinical data. Histopathologic review of tumor samples with assessment of immunohistochemical staining for SMARCA4/BRG1 was undertaken to confirm the diagnosis of SCCOHT. Germline and somatic genetic analyses were performed using targeted next-generation sequencing. RESULTS.­: Between 1991 and 2021, we identified 7 cases of SCCOHT in a population of 2 million. Genetic causes were determined in all cases. Two novel germline loss-of-function variants in SMARCA4 LRG_878t1:c.1423_1429delTACCTCA p.(Tyr475Ilefs*24) and LRG_878t1:c.3216-1G>T were identified. At diagnosis, patients were ages 21 to 41 and had International Federation of Gynecology and Obstetrics, or FIGO, stage IA-III disease. Outcomes were poor, with 6 of 7 patients dying of disease-related complications within 27 months from diagnosis. One patient had stable disease for 12 months while receiving immunotherapy. CONCLUSIONS.­: We present genetic, histopathologic, and clinical characteristics for all cases of SCCOHT identified in the Slovenian population during a 30-year period. We report 2 novel germline SMARCA4 variants, possibly associated with high penetrance. We estimate the minimal incidence of SCCOHT to be 0.12 per 1 million per year.

Real-World Data on Detection of Germline and Somatic Pathogenic/Likely Pathogenic Variants in BRCA1/2 and Other Susceptibility Genes in Ovarian Cancer Patients Using Next Generation Sequencing.

Detection of germline and somatic pathogenic/likely pathogenic variants (PV/LPV) in BRCA genes is at the moment a prerequisite for use of PARP inhibitors in different treatment settings of different tumors. The aim of our study was to determine the most appropriate testing workflow in epithelial ovarian cancer (EOC) patients using germline and tumor genotyping of BRCA and other hereditary breast and/or ovarian cancer (HBOC) susceptibility genes. Consecutive patients with advanced non-mucinous EOC, who responded to platinum-based chemotherapy, were included in the study. DNA extracted from blood and FFPE tumor tissue were genotyped using NGS panels TruSightCancer/Hereditary and TruSight Tumor 170. Among 170 EOC patients, 21.8% had BRCA germline or somatic PV/LPV, and additionally 6.4% had PV/LPV in other HBOC genes. Sensitivity of tumor genotyping for detection of germline PV/LPV was 96.2% for BRCA genes and 93.3% for HBOC genes. With germline genotyping-only strategy, 58.8% of HBOC PV/LPV and 68.4% of BRCA PV/LPV were detected. By tumor genotyping-only strategy, 96.1% of HBOC PV/LPV and 97.4% of BRCA PV/LPV were detected. Genotyping of tumor first, followed by germline genotyping seems to be a reasonable approach for detection of PV/LPV in breast and/or ovarian cancer susceptibility genes in non-mucinous EOC patients.