CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation

Abstract Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.info:eu-repo/semantics/publishe

Mutational Analysis Identifies Therapeutic Biomarkers in Inflammatory Bowel Disease-Associated Colorectal Cancers.

Purpose: Inflammatory bowel disease-associated colorectal cancers (IBD-CRC) are associated with a higher mortality than sporadic colorectal cancers. The poorly defined molecular pathogenesis of IBD-CRCs limits development of effective prevention, detection, and treatment strategies. We aimed to identify biomarkers using whole-exome sequencing of IBD-CRCs to guide individualized management.Experimental Design: Whole-exome sequencing was performed on 34 formalin-fixed paraffin-embedded primary IBD-CRCs and 31 matched normal lymph nodes. Computational methods were used to identify somatic point mutations, small insertions and deletions, mutational signatures, and somatic copy number alterations. Mismatch repair status was examined.Results: Hypermutation was observed in 27% of IBD-CRCs. All hypermutated cancers were from the proximal colon; all but one of the cancers with hypermutation had defective mismatch repair or somatic mutations in the proofreading domain of DNA POLE Hypermutated IBD-CRCs had increased numbers of predicted neo-epitopes, which could be exploited using immunotherapy. We identified six distinct mutation signatures in IBD-CRCs, three of which corresponded to known mechanisms of mutagenesis. Driver genes were also identified.Conclusions: IBD-CRCs should be evaluated for hypermutation and defective mismatch repair to identify patients with a higher neo-epitope load who may benefit from immunotherapies. Prospective trials are required to determine whether IHC to detect loss of MLH1 expression in dysplastic colonic tissue could identify patients at increased risk of developing IBD-CRC. We identified mutations in genes in IBD-CRCs with hypermutation that might be targeted therapeutically. These approaches would complement and individualize surveillance and treatment programs. Clin Cancer Res; 24(20); 5133-42. ©2018 AACR

The mutational landscape of melanoma brain metastases presenting as the first visceral site of recurrence.

Funder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440Brain metastases are a major cause of melanoma-related mortality and morbidity. We undertook whole-exome sequencing of 50 tumours from patients undergoing surgical resection of brain metastases presenting as the first site of visceral disease spread and validated our findings in an independent dataset of 18 patients. Brain metastases had a similar driver mutational landscape to cutaneous melanomas in TCGA. However, KRAS was the most significantly enriched driver gene, with 4/50 (8%) of brain metastases harbouring non-synonymous mutations. Hotspot KRAS mutations were mutually exclusive from BRAFV600, NRAS and HRAS mutations and were associated with a reduced overall survival from the resection of brain metastases (HR 10.01, p = 0.001). Mutations in KRAS were clonal and concordant with extracranial disease, suggesting that these mutations are likely present within the primary. Our analyses suggest that KRAS mutations could help identify patients with primary melanoma at higher risk of brain metastases who may benefit from more intensive, protracted surveillance

Multi-site clonality analysis uncovers pervasive heterogeneity across melanoma metastases

Abstract: Metastatic melanoma carries a poor prognosis despite modern systemic therapies. Understanding the evolution of the disease could help inform patient management. Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal evidence of diversification among metastatic lineages. UV-induced mutations dominate the trunk, whereas APOBEC-associated mutations are found in the branches of the evolutionary tree. Multi-sample analyses from a further seven patients confirmed that lineage diversification was pervasive, representing an important mode of melanoma dissemination. Our analyses demonstrate that joint analysis of cancer cell fraction estimates across multiple metastases can uncover previously unrecognised levels of tumour heterogeneity and highlight the limitations of inferring heterogeneity from a single biopsy

Multi-site clonality analysis uncovers pervasive heterogeneity across melanoma metastases

From Springer Nature via Jisc Publications RouterHistory: received 2019-11-18, accepted 2020-07-27, registration 2020-08-04, pub-electronic 2020-08-27, online 2020-08-27, collection 2020-12Publication status: PublishedAbstract: Metastatic melanoma carries a poor prognosis despite modern systemic therapies. Understanding the evolution of the disease could help inform patient management. Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal evidence of diversification among metastatic lineages. UV-induced mutations dominate the trunk, whereas APOBEC-associated mutations are found in the branches of the evolutionary tree. Multi-sample analyses from a further seven patients confirmed that lineage diversification was pervasive, representing an important mode of melanoma dissemination. Our analyses demonstrate that joint analysis of cancer cell fraction estimates across multiple metastases can uncover previously unrecognised levels of tumour heterogeneity and highlight the limitations of inferring heterogeneity from a single biopsy

The molecular landscape of cutaneous melanoma and its clinical implications

Cutaneous melanoma arises due to the uncontrolled proliferation of melanocytes. It is the deadliest form of skin cancer and its incidence, particularly in younger adults, is rapidly rising worldwide. Over the past decade, multiple large-scale sequencing studies have uncovered the molecular landscape of cutaneous melanoma. In particular, an improved understanding of the melanoma genome and regulation of the immune system have led to the development of effective targeted and immunotherapies that have revolutionised the treatment landscape. Nevertheless, only a subset of patients demonstrate durable responses to these therapies and identifying those patients most likely to benefit remains an important unmet need. Melanoma is an aggressive malignancy that often metastasises beyond its primary site. It has a particular propensity to metastasise to the brain and the mechanisms underlying this devastating complication remain incompletely understood. Melanoma risk stratification has traditionally relied on the examination of clinical and pathological features, however it has become clear that traditional staging may fall short in accurately assessing an individual patients’ risk. There is therefore a need for robust prognostic biomarkers capable of identifying truly high-risk patients, or patients with biologically indolent tumours, which could be used as an adjunct to conventional clinicopathologic assessments, and would afford a unique insight into the underlying tumour cell biology. In this thesis, I explore the mutational landscape of cutaneous melanoma focussing on four key clinical cohorts; adolescent cutaneous melanoma, patients with brain metastases, widespread lethal metastatic disease and patients with high-risk primary melanoma. I use a range of high-throughput sequencing technologies to identify the tumour-specific somatic alterations characterising these cohorts and apply detailed multi-site phylogenetic analyses to uncover some of the key evolutionary changes. In the final chapter, I apply deep RNA sequencing to primary melanomas embedded within a prospective phase III clinical trial, to define and validate a gene expression signature identifying primary melanoma patients at higher risk of adverse outcomes. Together, these studies demonstrate how detailed analyses of molecular sequencing can uncover novel biological insights. It is hoped that further prospective molecular analyses coupled with high-quality experimental validation will pave the way towards novel therapeutic strategies for these patient cohorts

Peer mentoring for core medical trainees:Uptake and impact

ABSTRACT Objective To assess the uptake and impact of a peer mentoring scheme for core medical trainees on both mentors and mentees. Method All second year core medical trainees in the Southwest London Training programme in September 2012 were invited to mentor a first year core medical trainee. In parallel, all first year core medical trainees were invited to be mentored. Both potential mentors and mentees were asked to submit personal statements, to attend a three-session mentoring training programme and to be matched into mentoring pairs. The impact of the mentoring scheme on trainees’ behaviour and outlook was assessed through questionnaires distributed at the start and at the end of the year. Results 31 of 72 (43%) core medical trainees submitted personal statements and 40 of 72 (56%) attended training sessions. 42 trainees (58%) participated in the scheme (21 mentor/mentee pairs were established). Of the trainees who participated, 23 of 42 (55%) completed the end of year questionnaire. Participating trainees viewed the scheme positively. Reported benefits included changes in their behaviour and acquiring transferable skills that might help them in later career roles, such as an educational supervisor. The end of year questionnaire was sent to all trainees and 10 responded who had not participated. They were asked why they had not participated and their reasons included lack of time, lack of inclination and a desire for more senior mentors. Their suggestions for improvement included more structured sessions to allow the mentor/mentee pairs to meet. Conclusions This simple peer mentoring scheme was popular despite busy workloads and benefited all concerned. It is a simple effective way of supporting doctors. More work is needed to improve training for mentors and to improve access to mentoring. </jats:sec