Genetic Testing Utilization: Discrepancies Between Somatic and Germline Results in Patients With Cancer Reviewed at the UW Health Precision Medicine Molecular Tumor Board.

PURPOSE: Somatic and germline testing are increasingly used to estimate risks for patients with cancer. Although both germline testing and somatic testing can identify genetic variants that could change a patient's care and eligible treatments, the aims of these tests and their technologies are fundamentally different and cannot be used interchangeably. This study examines the timing and results of somatic and germline genetic testing for patients with cancer at UW Health. METHODS: Eight hundred and seventy-seven participants underwent somatic genetic testing, which was reviewed by the Precision Medicine Molecular Tumor Board (PMMTB). Patients were diagnosed with cancers, including breast, colorectal, endometrial, pancreatic, or ovarian cancer, and met National Comprehensive Cancer Network criteria for germline genetic testing. Germline testing details were collected by medical record review. RESULTS: The results of this study found that only 310 patients (35%) had germline evaluation before PMMTB review. The percent of germline pathogenic/likely pathogenic variants identified in actionable genes was 28%. Most germline variants were identified in the BRCA1 (26%) and BRCA2 (28%) genes. In total, 65% (54/83) of germline variants were detected with both germline testing and somatic testing; however, 35% (29/83) of germline variants were not identified on somatic results. These results demonstrate the importance of combination germline and somatic testing. CONCLUSION: This study highlights the differences in genetic testing types and demonstrates that conducting germline testing at earlier stages of diagnoses is necessary to identify potentially actionable and treatment-specific variants in patients with cancer.

Culture media composition influences patient-derived organoid ability to predict therapeutic responses in gastrointestinal cancers.

BACKGROUNDA patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO's ability to predict clinical response to gastrointestinal (GI) cancers.METHODSWe generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments.RESULTSWe report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures.CONCLUSIONWhile we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers.TRIAL REGISTRATIONNot applicable.FUNDINGThe Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).

A rare germline CDKN2A variant (47T>G; p16-L16R) predisposes carriers to pancreatic cancer by reducing cell cycle inhibition.

Germline mutations in CDKN2A, encoding the tumor suppressor p16, are responsible for a large proportion of familial melanoma cases and also increase risk of pancreatic cancer. We identified four families through pancreatic cancer probands that were affected by both cancers. These families bore a germline missense variant of CDKN2A (47T>G), encoding a p16-L16R mutant protein associated with high cancer occurrence. Here, we investigated the biological significance of this variant. When transfected into p16-null pancreatic cancer cells, p16-L16R was expressed at lower levels than wild-type (WT) p16. In addition, p16-L16R was unable to bind CDK4 or CDK6 compared with WT p16, as shown by coimmunoprecipitation assays and also was impaired in its ability to inhibit the cell cycle, as demonstrated by flow cytometry analyses. In silico molecular modeling predicted that the L16R mutation prevents normal protein folding, consistent with the observed reduction in expression/stability and diminished function of this mutant protein. We isolated normal dermal fibroblasts from members of the families expressing WT or L16R proteins to investigate the impact of endogenous p16-L16R mutant protein on cell growth. In culture, p16-L16R fibroblasts grew at a faster rate, and most survived until later passages than p16-WT fibroblasts. Further, western blotting demonstrated that p16 protein was detected at lower levels in p16-L16R than in p16-WT fibroblasts. Together, these results suggest that the presence of a CDKN2A (47T>G) mutant allele contributes to an increased risk of pancreatic cancer as a result of reduced p16 protein levels and diminished p16 tumor suppressor function.

GLI1/GLI2 functional interplay is required to control Hedgehog/GLI targets gene expression.

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.