Genomic analyses of germline and somatic variation in high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 557 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations (SCNA). RESULTS: Approximately one-third of tumors had loss-of-function (LOF) germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM, and PALB2. LOF germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP, and NF1. In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of LOF variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536, and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival. CONCLUSIONS: From 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 557 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Genomic Analyses of Germline and Somatic Variation in High-Grade Serous Ovarian Cancer.

Background High-grade serous ovarian cancers (HGSCs) display a high degree of complex genetic alterations. In this study, we identified germline and somatic genetic alterations in HGSC and their association with relapse-free and overall survival. Using a targeted capture of 577 genes involved in DNA damage response and PI3K/AKT/mTOR pathways, we conducted next-generation sequencing of DNA from matched blood and tumor tissue from 71 HGSC participants. In addition, we performed the OncoScan assay on tumor DNA from 61 participants to examine somatic copy number alterations. Results Approximately one-third of tumors had loss-of-function germline (18/71, 25.4%) or somatic (7/71, 9.9%) variants in the DNA homologous recombination repair pathway genes BRCA1, BRCA2, CHEK2, MRE11A, BLM , and PALB2 . Loss-of-function germline variants also were identified in other Fanconi anemia genes and in MAPK and PI3K/AKT/mTOR pathway genes. Most tumors harbored somatic TP53 variants (65/71, 91.5%). Using the OncoScan assay on tumor DNA from 61 participants, we identified focal homozygous deletions in BRCA1, BRCA2, MAP2K4, PTEN, RB1, SLX4, STK11, CREBBP , and NF1 . In total, 38% (27/71) of HGSC patients harbored pathogenic variants in DNA homologous recombination repair genes. For patients with multiple tissues from the primary debulking or from multiple surgeries, the somatic mutations were maintained with few newly acquired point mutations suggesting that tumor evolution was not through somatic mutations. There was a significant association of loss-of-function variants in homologous recombination repair pathway genes and high-amplitude somatic copy number alterations. Using GISTIC analysis, we identified NOTCH3, ZNF536 , and PIK3R2 in these regions that were significantly associated with an increase in cancer recurrence and a reduction in overall survival. Conclusions From 71 patients with HGCS, we performed targeted germline and tumor sequencing and provided a comprehensive analysis of these 577 genes. We identified germline and somatic genetic alterations including somatic copy number alterations and analyzed their associations with relapse-free and overall survival. This single-site long-term follow-up study provides additional information on genetic alterations related to occurrence and outcome of HGSC. Our findings suggest that targeted treatments based on both variant and SCNA profile potentially could improve relapse-free and overall survival.

Neuroimaging in infants and children in select neurocutaneous disorders.

The role of neuroimaging in neurocutaneous disorders is an evolving field. Research can be inconsistent and inconclusive, leading to divergent practice for some disorders. This study provides an overview of the current role of magnetic resonance imaging (MRI) of the brain in select neurocutaneous disorders, namely Sturge-Weber syndrome, congenital melanocytic naevus syndrome, neurofibromatosis type 1, tuberous sclerosis complex, incontinentia pigmenti and basal cell naevus syndrome. Future research assessing new targeted treatments and novel MRI techniques may change current practice.

Occurrence and determinants of enterococcal bloodstream infections: a population-based study.

Background: Although enterococci are common causes of bloodstream infections (BSIs), few studies have examined their epidemiology in non-selected populations.Objective: To examine the incidence and risk factors for development of enterococcal BSI.Methods: Surveillance for incident enterococcal BSI was conducted among all residents of the western interior of British Columbia, Canada during 2011-2018.Results: The overall annual incidence was 10.0 per 100,000 and was 6.6 and 2.7 per 100,000 for E. faecalis and E. faecium, respectively. Among the overall cohort of 145 incident cases of enterococcal BSI, 22 (15.2%) were community-associated, 63 (43.5%) were healthcare associated and 60 (41.4%) were hospital-onset. Enterococcal BSI was predominantly a disease of older adults with rare cases occurring among those aged less than 40 years. Males showed significantly increased risk compared to females (14.3 vs. 5.6 per 100,000; incidence rate ratio; IRR; 2.6; 95% confidence interval; CI; 1.8-3.8; p < .0001) and this was most pronounced with advanced age. Several co-morbid illnesses were associated with increased risk (IRR; 95% CI) for development of enterococcal BSI most importantly cancer (8.8; 6.0-12.9; p < .0001), congestive heart failure (5.7; 3.1-9.7; p < .0001), diabetes mellitus (4.4; 3.0-6.3; p < .0001) and stroke (3.7; 1.9-6.5; .0001). As compared to patients with E. faecalis, patients with E. faecium BSI were more likely to be of hospital-onset, more likely to have an intra-abdominal/pelvic focus, and trended towards higher 30-day case-fatality rate.Conclusions: Enterococci are relatively common causes of BSI. Although E faecalis and E faecium share commonalities they are epidemiologically distinguishable on several criteria.

Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300.

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

Outpatient clinic numbers: a review of standards and current practice.

This article explores current recommendations for the safe and efficient running of outpatient clinics, why non-adherence exists, the consequences of non-adherence, and what can be done to improve standards. It promotes adherence to recommendations by both clinical and managerial staff.

Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia.

The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.

Measles virus causes immunogenic cell death in human melanoma.

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host antitumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.

A KRAS variant is a biomarker of poor outcome, platinum chemotherapy resistance and a potential target for therapy in ovarian cancer.

Germline variants in the 3' untranslated region (3'UTR) of cancer genes disrupting microRNA (miRNA) regulation have recently been associated with cancer risk. A variant in the 3'UTR of the KRAS oncogene, referred to as the KRAS variant, is associated with both cancer risk and altered tumor biology. Here, we test the hypothesis that the KRAS variant can act as a biomarker of outcome in epithelial ovarian cancer (EOC), and investigate the cause of altered outcome in KRAS variant-positive EOC patients. As this variant seems to be associated with tumor biology, we additionally test the hypothesis that this variant can be directly targeted to impact cell survival. EOC patients with complete clinical data were genotyped for the KRAS variant and analyzed for outcome (n=536), response to neoadjuvant chemotherapy (n=125) and platinum resistance (n=306). Outcome was separately analyzed for women with known BRCA mutations (n=79). Gene expression was analyzed on a subset of tumors with available tissue. Cell lines were used to confirm altered sensitivity to chemotherapy associated with the KRAS variant. Finally, the KRAS variant was directly targeted through small-interfering RNA/miRNA oligonucleotides in cell lines and survival was measured. Postmenopausal EOC patients with the KRAS variant were significantly more likely to die of ovarian cancer by multivariate analysis (hazard ratio=1.67, 95% confidence interval: 1.09-2.57, P=0.019, n=279). Perhaps explaining this finding, EOC patients with the KRAS variant were significantly more likely to be platinum resistant (odds ratio=3.18, confidence interval: 1.31-7.72, P=0.0106, n=291). In addition, direct targeting of the KRAS variant led to a significant reduction in EOC cell growth and survival in vitro. These findings confirm the importance of the KRAS variant in EOC, and indicate that the KRAS variant is a biomarker of poor outcome in EOC likely due to platinum resistance. In addition, this study supports the hypothesis that these tumors have continued dependence on such 3'UTR lesions, and that direct targeting may be a viable future treatment approach.

The role of molecular microsatellite identity testing to detect sampling errors in prenatal diagnosis.

OBJECTIVE: The objective was to determine the risk of sampling error in amniocentesis and chorionic villus sampling (CVS) in singleton and multiple pregnancies. Data from this and other published studies were used to discuss current practice guidelines for molecular identity testing. METHOD: Clinical and laboratory records of all patients undergoing molecular-based identity testing in our clinical laboratory from July 2002 until March 2008 were reviewed. DNA microsatellite testing was performed to determine zygosity in multiple pregnancies and maternal cell contamination (MCC) in both singleton and multiple pregnancies. RESULTS: MCC was detected in 6/148 (4%) CVS and 1/87 (1%) amniotic fluids from singleton pregnancies. In two of the CVS, only maternal cells were found. In 2/24 (8%) twin pregnancies, the same fetus was tested twice. In a total of 285 pregnancies (235 singleton, 24 twin, 26 with >or= 3 fetuses), without molecular identity testing, four women would have received erroneous results. CONCLUSION: Current guidelines recommend molecular identity testing for MCC in conjunction with molecular diagnostic testing, but not for cytogenetic testing. No published guidelines were found for zygosity testing in multiple pregnancies. We suggest that identity testing be considered for all prenatal testing of multiple pregnancies, especially if CVS is performed.

Retargeted adenoviral cancer gene therapy for tumour cells overexpressing epidermal growth factor receptor or urokinase-type plasminogen activator receptor.

We have assessed the ability of bispecific fusion proteins to improve adenovirus-mediated transfer of therapeutic and marker transgenes. We constructed an expression vector that can be easily modified to synthesize a variety of fusion proteins for retargeting adenoviral gene therapy vectors to cell surface markers, which are differentially expressed between normal and cancer cells. Adenoviral transduction can be improved in a number of tumour cell lines which overexpress EGFR (epidermal growth factor receptor) or uPAR (urokinase-type plasminogen activator receptor), but which have only low levels of endogenous hCAR (human coxsackie B and adenovirus receptor) expression. Up to 40-fold improvement in beta-galactosidase transgene expression was seen using an EGFR retargeting protein, and up to 16-fold using a second fusion protein targeting uPAR. In vitro, our uPAR retargeting fusion protein improved the sensitivity to adenoviral herpes simplex virus thymidine kinase/ganciclovir by an order of magnitude, whereas in vivo, our EGFR retargeting protein is able to significantly delay tumour growth in rodent animal models in a dose-dependent manner. The 'cassette' design of our fusion protein constructs offers a flexible method for the straightforward synthesis of multiple adenoviral retargeting proteins, directed against a variety of tumour-associated antigens, for use in clinical trials.

Inflammatory tumour cell killing by oncolytic reovirus for the treatment of melanoma.

Reovirus is a promising unmodified double-stranded RNA (dsRNA) anti-cancer oncolytic virus, which is thought to specifically target cells with activated Ras. Although reovirus has been tested in a wide range of preclinical models and has entered early clinical trials, it has not previously been tested for the treatment of human melanoma. Here, we show that reovirus effectively kills and replicates in both human melanoma cell lines and freshly resected tumour; intratumoural injection also causes regression of melanoma in a xenograft in vivo model. Reovirus-induced melanoma death is blocked by caspase inhibition and is dependent on constituents of the Ras/RalGEF/p38 pathway. Reovirus melanoma killing is more potent than, and distinct from, chemotherapy or radiotherapy-induced cell death; a range of inflammatory cytokines and chemokines are released by infected tumour cells, while IL-10 secretion is abrogated. Furthermore, the inflammatory response generated by reovirus-infected tumour cells causes bystander toxicity against reovirus-resistant tumour cells and activates human myeloid dendritic cells (DC) in vitro. Hence, reovirus is suitable for clinical testing in melanoma, and may provide a useful danger signal to reverse the immunologically suppressive environment characteristic of this tumour.

New chromosome 11p15 epigenotypes identified in male monozygotic twins with Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome demonstrating heterogeneous molecular alterations of two imprinted domains on chromosome 11p15. The most common molecular alterations include loss of methylation at the proximal imprinting center, IC2, paternal uniparental disomy (UPD) of chromosome 11p15 and hypermethylation at the distal imprinting center, IC1. An increased incidence of female monozygotic twins discordant for BWS has been reported. The molecular basis for eleven such female twin pairs has been demonstrated to be a loss of methylation at IC2, whereas only one male monozygotic twin pair has been reported with this molecular defect. We report here two new pairs of male monozygotic twins. One pair is discordant for BWS; the affected twin exhibits paternal UPD for chromosome 11p15 whereas the unaffected twin does not. The second male twin pair is concordant for BWS and both twins of the pair demonstrate hypermethylation at IC1. Thus, this report expands the known molecular etiologies for BWS twins. Interestingly, these findings demonstrate a new epigenotype-phenotype correlation in BWS twins. That is, while female monozygotic twins with BWS are likely to show loss of imprinting at IC2, male monozygotic twins with BWS reflect the molecular heterogeneity seen in BWS singletons. These data underscore the need for molecular testing in BWS twins, especially in view of the known differences among 11p15 epigenotypes with respect to tumor risk.