Predictors of treatment benefit and prognosis in cutaneous malignant melanoma

This thesis describes work designed to identify genomic prognostic and predictive markers for melanoma using nucleic acids extracted from formalin-fixed (FFPE) tumour samples and emerging technologies for gene expression profiling. I report use of the cDNA-mediated annealing, selection, extension and ligation (DASL) assay using a 502-gene Human Cancer panel to identify prognostic markers. The study identified over-expression of SPP1 and DNA repair genes in primary tumours as associated with reduced relapse-free survival time. These genes remained associated with survival in analyses adjusted for current prognostic markers. I also report use of the DASL assay to study gene expression of tiny metastatic specimens, allowing assessment of gene expression in matched primary and metastatic tumours. I report studies of driver mutations in BRAF and NRAS as prognostic markers in which V600K BRAF mutations, were independently associated with survival in multivariate analysis. Gene expression profiling using the DASL assay identified a number of genes differentially expressed in BRAF and NRAS mutated tumours, for example ETV1 and TYRO3, providing biological insight into these tumours. Ulceration of a primary tumour is a poor prognostic sign, but paradoxically also predicts response to interferon- adjuvant therapy. I report greater numbers of macrophages in ulcerated tumours and both over-expression of interleukin-6 and deranged expression of genes in the interferon Jak-STAT signalling pathway. These factors may contribute to mechanisms of responsiveness to interferon- in ulcerated tumours. I report an investigation of predictive markers for dacarbazine therapy. The Fluidigm quantitative Real-time PCR system was used to identify over-expression of MGMT as independently associated with chemotherapy resistance and shorter survival after starting chemotherapy. Following validation of these findings, prognostic and predictive markers were identified which could potentially be used clinically to provide additional information to patients with melanoma, allowing a more personalised approach to melanoma treatment

The contribution of X-linked coding variation to severe developmental disorders

Abstract: Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders

Wilms tumour surveillance in at-risk children: Literature review and recommendations from the SIOP-Europe Host Genome Working Group and SIOP Renal Tumour Study Group.

Since previous consensus-based Wilms tumour (WT) surveillance guidelines were published, novel genes and syndromes associated with WT risk have been identified, and diagnostic molecular tests for previously known syndromes have improved. In view of this, the International Society of Pediatric Oncology (SIOP)-Europe Host Genome Working Group and SIOP Renal Tumour Study Group hereby present updated WT surveillance guidelines after an extensive literature review and international consensus meetings. These guidelines are for use by clinical geneticists, pediatricians, pediatric oncologists and radiologists involved in the care of children at risk of WT. Additionally, we emphasise the need to register all patients with a cancer predisposition syndrome in national or international databases, to enable the development of better tumour risk estimates and tumour surveillance programs in the future

Uncovering the burden of hidden cillopathies in the 100,000 genomes project: a reverse phenotyping approach

Background: the 100,000 Genomes Project (100K) recruited NHS patients with eligible rare diseases and cancer between 2016 and 2018. PanelApp virtual gene panels were applied to whole genome sequencing data according to Human Phenotyping Ontology (HPO) terms entered by recruiting clinicians to guide focussed analysis.Methods: we developed a reverse phenotyping strategy to identify 100K participants with pathogenic variants in nine prioritised disease genes (BBS1, BBS10, ALMS1, OFD1, DYNC2H1, WDR34, NPHP1, TMEM67, CEP290), representative of the full phenotypic spectrum of multi‐systemic primary ciliopathies. We mapped genotype data “backwards” onto available clinical data to assess potential matches against phenotypes. Participants with novel molecular diagnoses and key clinical features compatible with the identified disease gene were reported to recruiting clinicians.Results: we identified 62 reportable molecular diagnoses with variants in these nine ciliopathy genes. Forty‐four have been reported by 100K, five were previously unreported and 13 are new diagnoses. We identified 11 participants with un‐reportable, novel molecular diagnoses, who lacked key clinical features to justify reporting to recruiting clinicians. Two participants had likely pathogenic structural variants and one a deep intronic predicted splice variant. These variants would not be prioritised for review by standard 100K diagnostic pipelines.Conclusion: reverse phenotyping improves the rate of successful molecular diagnosis for unsolved 100K participants with primary ciliopathies. Previous analyses likely missed these diagnoses because incomplete HPO term entry led to incorrect gene panel choice, meaning that pathogenic variants were not prioritised. Better phenotyping data is therefore essential for accurate variant interpretation and improved patient benefit

UK consensus recommendations for clinical management of cancer risk for women with germline pathogenic variants in cancer predisposition genes; RAD51C, RAD51D, BRIP1 and PALB2

Germline pathogenic variants (GPVs) in the cancer predisposition genes BRCA1, BRCA2, MLH1, MSH2, MSH6, BRIP1, PALB2, RAD51D and RAD51C are identified in approximately 15% of patients with ovarian cancer (OC). While there are clear guidelines around clinical management of cancer risk in patients with GPV in BRCA1, BRCA2, MLH1, MSH2 and MSH6, there are few guidelines on how to manage the more moderate OC risk in patients with GPV in BRIP1, PALB2, RAD51D and RAD51C, with clinical questions about appropriateness and timing of risk-reducing gynaecological surgery. Furthermore, while recognition of RAD51C and RAD51D as OC predisposition genes has been established for several years, an association with breast cancer (BC) has only more recently been described and clinical management of this risk has been unclear. With expansion of genetic testing of these genes to all patients with non-mucinous OC, new data on BC risk and improved estimates of OC risk, the UK Cancer Genetics Group and CanGene-CanVar project convened a 2-day meeting to reach a national consensus on clinical management of BRIP1, PALB2, RAD51D and RAD51C carriers in clinical practice. In this paper, we present a summary of the processes used to reach and agree on a consensus, as well as the key recommendations from the meeting

Expansion of the neurodevelopmental phenotype of individuals with EEF1A2 variants and genotype-phenotype study

Translation elongation factor eEF1A2 constitutes the alpha subunit of the elongation factor-1 complex, responsible for the enzymatic binding of aminoacyl-tRNA to the ribosome. Since 2012, 21 pathogenic missense variants affecting EEF1A2 have been described in 42 individuals with a severe neurodevelopmental phenotype including epileptic encephalopathy and moderate to profound intellectual disability (ID), with neurological regression in some patients. Through international collaborative call, we collected 26 patients with EEF1A2 variants and compared them to the literature. Our cohort shows a significantly milder phenotype. 83% of the patients are walking (vs. 29% in the literature), and 84% of the patients have language skills (vs. 15%). Three of our patients do not have ID. Epilepsy is present in 63% (vs. 93%). Neurological examination shows a less severe phenotype with significantly less hypotonia (58% vs. 96%), and pyramidal signs (24% vs. 68%). Cognitive regression was noted in 4% (vs. 56% in the literature). Among individuals over 10 years, 56% disclosed neurocognitive regression, with a mean age of onset at 2 years. We describe 8 novel missense variants of EEF1A2. Modeling of the different amino-acid sites shows that the variants associated with a severe phenotype, and the majority of those associated with a moderate phenotype, cluster within the switch II region of the protein and thus may affect GTP exchange. In contrast, variants associated with milder phenotypes may impact secondary functions such as actin binding. We report the largest cohort of individuals with EEF1A2 variants thus far, allowing us to expand the phenotype spectrum and reveal genotype-phenotype correlations.</p

Inherited variation in the PARP1 gene and survival from melanoma

We report the association of an inherited variant located upstream of the poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) gene (rs2249844), with survival in 11 BioGenoMEL melanoma cohorts. The gene encodes a protein involved in a number of cellular processes including single-strand DNA repair. Survival analysis was conducted for each cohort using proportional hazards regression adjusting for factors known to be associated with survival. Survival was measured as overall survival (OS) and, where available, melanoma-specific survival (MSS). Results were combined using random effects meta-analysis. Evidence for a role of the PARP1 protein in melanoma ulceration and survival was investigated by testing gene expression levels taken from formalin-fixed paraffin-embedded tumors. A significant association was seen for inheritance of the rarer variant of PARP1, rs2249844 with OS (hazard ratio (HR) = 1.16 per allele, 95% confidence interval (CI) 1.04-1.28, p=0.005, eleven cohorts) and MSS (HR=1.20 per allele, 95% CI 1.01-1.39, p=0.03, eight cohorts). We report bioinformatic data supportive of a functional effect for rs2249844. Higher levels of PARP1 gene expression in tumors were shown to be associated with tumor ulceration and poorer OS. What’s new? Although staging systems predict outcome fairly well for melanoma, survival still varies among individual patients. In this meta-analysis, the authors found that inheritance of a rare genetic variant of PARP1 was associated with improved survival of melanoma patients. Increased expression of PARP1 has been associated with poorer outcome, and depletion of PARP1 may reduce both melanoma growth and angiogenesis. The identification of this and other germline variants that affect survival may help to identify key biological pathways active in host/tumor interactions, which may lead to the discovery of new therapeutic targets for treating advanced melanoma. Epidemiolog