Biallelic EPCAM deletions induce tissue-specific DNA repair deficiency and cancer predisposition.

We report a case of Mismatch Repair Deficiency (MMRD) caused by germline homozygous EPCAM deletion leading to tissue-specific loss of MSH2. Through the use of patient-derived cells and organoid technologies, we performed stepwise in vitro differentiation of colonic and brain organoids from reprogrammed EPCAMdel iPSC derived from patient fibroblasts. Differentiation of iPSC to epithelial-colonic organoids exhibited continuous increased EPCAM expression and hypermethylation of the MSH2 promoter. This was associated with loss of MSH2 expression, increased mutational burden, MMRD signatures and MS-indel accumulation, the hallmarks of MMRD. In contrast, maturation into brain organoids and examination of blood and fibroblasts failed to show similar processes, preserving MMR proficiency. The combined use of iPSC, organoid technologies and functional genomics analyses highlights the potential of cutting-edge cellular and molecular analysis techniques to define processes controlling tumorigenesis and uncovers a new paradigm of tissue-specific MMRD, which affects the clinical management of these patients.

Data resources for the identification and interpretation of actionable mutations by clinicians.

Following initial characterization of the reference human genome, initiatives have evolved worldwide to identify genomic aberrations in cancer with the aim of deriving diagnostic, prognostic and predictive information. However, the functional and clinical relevance of many somatic variants in cancer are presently unknown and there is no consensus definition of 'actionability' for genomic aberrancies. Therefore, while robust detection of a variety of genetic aberrations in clinical specimens remains a technical hurdle, the greater challenge lies in the interpretation of these alterations. Critical evaluation of genomic variation in cancer requires the integration of available clinical and preclinical evidence related to their frequencies, functions and roles as therapeutic targets. Many publicly accessible data resources have compiled such evidence to facilitate the understanding of genomic results and ultimately translating results to clinical action. Information for these data resources is derived from various sources including large population genomic datasets, curation of published literature, and data sharing by the scientific community. Currently, there is no widely accepted guidance to definitively assess and integrate the diagnostic, prognostic and predictive information of somatic variants using these knowledge databases. This review will describe data resources pertinent to the identification and interpretation of actionable genomic aberrations by clinicians, and highlight relevant issues in the clinical application of tumor molecular profiling results.

Exome sequencing of pleuropulmonary blastoma reveals frequent biallelic loss of TP53 and two hits in DICER1 resulting in retention of 5p-derived miRNA hairpin loop sequences.

Pleuropulmonary blastoma is a rare childhood malignancy of lung mesenchymal cells that can remain dormant as epithelial cysts or progress to high-grade sarcoma. Predisposing germline loss-of-function DICER1 variants have been described. We sought to uncover additional contributors through whole exome sequencing of 15 tumor/normal pairs, followed by targeted resequencing, miRNA analysis and immunohistochemical analysis of additional tumors. In addition to frequent biallelic loss  of TP53 and mutations of NRAS or BRAF in some cases, each case had compound disruption of DICER1: a germline (12 cases) or somatic (3 cases) loss-of-function variant plus a somatic missense mutation in the RNase IIIb domain. 5p-Derived microRNA (miRNA) transcripts retained abnormal precursor miRNA loop sequences normally removed by DICER1. This work both defines a genetic interaction landscape with DICER1 mutation and provides evidence for alteration in miRNA transcripts as a consequence of DICER1 disruption in cancer.

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

A systematic approach to assessing the clinical significance of genetic variants.

Molecular genetic testing informs diagnosis, prognosis, and risk assessment for patients and their family members. Recent advances in low-cost, high-throughput DNA sequencing and computing technologies have enabled the rapid expansion of genetic test content, resulting in dramatically increased numbers of DNA variants identified per test. To address this challenge, our laboratory has developed a systematic approach to thorough and efficient assessments of variants for pathogenicity determination. We first search for existing data in publications and databases including internal, collaborative and public resources. We then perform full evidence-based assessments through statistical analyses of observations in the general population and disease cohorts, evaluation of experimental data from in vivo or in vitro studies, and computational predictions of potential impacts of each variant. Finally, we weigh all evidence to reach an overall conclusion on the potential for each variant to be disease causing. In this report, we highlight the principles of variant assessment, address the caveats and pitfalls, and provide examples to illustrate the process. By sharing our experience and providing a framework for variant assessment, including access to a freely available customizable tool, we hope to help move towards standardized and consistent approaches to variant assessment.

Factors associated with improved biochemical response to neoadjuvant androgen deprivation therapy before definitive radiation therapy in prostate cancer patients.

BACKGROUND: In prostate cancer patients treated with androgen deprivation therapy (ADT) and radiation therapy (RT), a pre-RT PSA level 0.5 ng ml(-1), determined after neoadjuvant ADT and before RT, predicts for worse survival measures. The present study sought to identify patient, tumor and treatment characteristics associated with the pre-RT PSA in prostate cancer patients. METHODS: We reviewed the charts of all patients diagnosed with intermediate- and high-risk prostate cancer and treated with a combination of neoadjuvant (median, 2.2 and 2.5 months, respectively), concurrent, and adjuvant ADT and RT between 1990 and 2011. RESULTS: A total of 170 intermediate- and 283 high-risk patients met inclusion criteria. On multivariate analysis, both intermediate- and high-risk patients with higher pre-treatment PSA (iPSA) were significantly less likely to achieve a pre-RT PSA <0.5 ng ml(-1) (iPSA 10.1-20 ng ml(-1): P=0.005 for intermediate risk; iPSA 10.1-20 ng ml(-1): P=0.005, iPSA >20 ng ml(-1): P<0.001 for high risk). High-risk patients undergoing total androgen blockade were more likely to achieve a pre-RT PSA <0.5 ng ml(-1) (P=0.031). We observed an interaction between race and type of neoadjuvant ADT (P=0.074); whereas African-American men on total androgen blockade reached pre-RT PSA <0.5 ng ml(-1) as frequently as other men on total androgen blockade (P=0.999), African-American men on luteinizing hormone-releasing hormone (LH-RH) agonist monotherapy/orchiectomy were significantly less likely to reach pre-RT PSA <0.5 ng ml(-1) compared with other men on LH-RH monotherapy/orchiectomy (P=0.001). CONCLUSIONS: Our findings suggest that total androgen blockade in the neoadjuvant period may be beneficial compared with LH-RH monotherapy for achieving a pre-RT PSA <0.5 ng ml(-1) in African-American men with high-risk prostate cancer. In addition, men with higher iPSA are more likely to have a pre-RT PSA greater than 0.5 ng ml(-1) in response to neoadjuvant ADT and are therefore candidates for clinical trials testing newer, more aggressive hormone-ablative therapies.

Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer.

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.

Addition of short-term androgen deprivation therapy to dose-escalated radiation therapy improves failure-free survival for select men with intermediate-risk prostate cancer.

BACKGROUND: Dose-escalated (DE) radiation therapy (RT) and androgen deprivation therapy (ADT) improve prostate cancer outcomes over standard-dose RT. The benefit of adding ADT to DE-RT for men with intermediate-risk prostate cancer (IR-PrCa) is uncertain. PATIENTS AND METHODS: We identified 636 men treated for IR-PrCa with DE-RT (>75Gy). The adult comorbidity evaluation-27 index classifed comorbidity. Kaplan-Meier and log-rank tests compared failure-free survival (FFS) with and without ADT. RESULTS: Forty-five percent received DE-RT and 55% DE-RT with ADT (median 6 months). On Cox proportional hazard regression that adjusted for comorbidity and tumor characteristics, ADT improved FFS (adjusted hazard ratio 0.36; P = 0.004). Recursive partitioning analysis of men without ADT classified Gleason 4 + 3 = 7 or ≥50% positive cores as unfavorable disease. The addition of ADT to DE-RT improved 5-year FFS for men with unfavorable disease (81.6% versus 92.9%; P = 0.009) but did not improve FFS for men with favorable disease (96.3% versus 97.4%; P = 0.874). When stratified by comorbidity, ADT improved FFS for men with unfavorable disease and no or mild comorbidity (P = 0.006) but did not improve FFS for men with unfavorable disease and moderate or severe comorbidity (P = 0.380). CONCLUSION: The addition of ADT to DE-RT improves FFS for men with unfavorable IR-PrCa, especially those with no or minimal comorbidity.

Impact of whole genome amplification on analysis of copy number variants.

Large-scale copy number variants (CNVs) have recently been recognized to play a role in human genome variation and disease. Approaches for analysis of CNVs in small samples such as microdissected tissues can be confounded by limited amounts of material. To facilitate analyses of such samples, whole genome amplification (WGA) techniques were developed. In this study, we explored the impact of Phi29 multiple-strand displacement amplification on detection of CNVs using oligonucleotide arrays. We extracted DNA from fresh frozen lymph node samples and used this for amplification and analysis on the Affymetrix Mapping 500k SNP array platform. We demonstrated that the WGA procedure introduces hundreds of potentially confounding CNV artifacts that can obscure detection of bona fide variants. Our analysis indicates that many artifacts are reproducible, and may correlate with proximity to chromosome ends and GC content. Pair-wise comparison of amplified products considerably reduced the number of apparent artifacts and partially restored the ability to detect real CNVs. Our results suggest WGA material may be appropriate for copy number analysis when amplified samples are compared to similarly amplified samples and that only the CNVs with the greatest significance values detected by such comparisons are likely to be representative of the unamplified samples.