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In melanoma, predicting which tumors will ultimately metastasize guides treatment decisions. Transcriptional signatures of primary tumors have been utilized to predict metastasis, but which among these are driver or passenger events remains unclear. We used data from the adjuvant AVAST-M trial to identify a predictive gene signature in localized tumors that ultimately metastasized. Using a zebrafish model of primary melanoma, we interrogated the top genes from the AVAST-M signature in vivo. This identified GRAMD1B, a cholesterol transfer protein, as a bona fide metastasis suppressor, with a majority of knockout animals rapidly developing metastasis. Mechanistically, excess free cholesterol or its metabolite 27-hydroxycholesterol promotes invasiveness via activation of an AP-1 program, which is associated with increased metastasis in humans. Our data demonstrate that the transcriptional seeds of metastasis are embedded within localized tumors, suggesting that early targeting of these programs can be used to prevent metastatic relapse. Significance: We analyzed human melanoma transcriptomics data to identify a gene signature predictive of metastasis. To rapidly test clinical signatures, we built a genetic metastasis platform in adult zebrafish and identified GRAMD1B as a suppressor of melanoma metastasis. GRAMD1B-associated cholesterol overload activates an AP-1 program to promote melanoma invasion. This article is highlighted in the In This Issue feature, p. 1

Validation of gramd1b depletion in zebrafish cells and tumors.

Supplementary Table S2. David GO analysis of AVAST-M signature

In melanoma, predicting which tumors will ultimately metastasize guides treatment decisions. Transcriptional signatures of primary tumors have been utilized to predict metastasis, but which among these are driver or passenger events remains unclear. We used data from the adjuvant AVAST-M trial to identify a predictive gene signature in localized tumors that ultimately metastasized. Using a zebrafish model of primary melanoma, we interrogated the top genes from the AVAST-M signature in vivo. This identified GRAMD1B, a cholesterol transfer protein, as a bona fide metastasis suppressor, with a majority of knockout animals rapidly developing metastasis. Mechanistically, excess free cholesterol or its metabolite 27-hydroxycholesterol promotes invasiveness via activation of an AP-1 program, which is associated with increased metastasis in humans. Our data demonstrate that the transcriptional seeds of metastasis are embedded within localized tumors, suggesting that early targeting of these programs can be used to prevent metastatic relapse.Significance:We analyzed human melanoma transcriptomics data to identify a gene signature predictive of metastasis. To rapidly test clinical signatures, we built a genetic metastasis platform in adult zebrafish and identified GRAMD1B as a suppressor of melanoma metastasis. GRAMD1B-associated cholesterol overload activates an AP-1 program to promote melanoma invasion.This article is highlighted in the In This Issue feature, p. 1

Supplementary Table S1 Phenotypic characteristics of IBD-CRC cases. Supplementary Table S2A Selected somatic mutations in IBD-CRC hypermutators. Supplementary Table S2B Selected somatic mutations in IBD-CRC non-hypermutators. Supplementary Table S3 Cosine similarities of IBD-CRC signatures and Alexandrov signatures. Supplementary Table S4 Genes implicated in susceptibility to colorectal cancer. Supplementary Table S5 Selected germline variants. Supplementary Table S6 Estimated cellularity, ploidy and SCNAs in IBD-CRC samples.

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.

Supplementary Materials and Methods

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/published

Funder: University of Sydney Medical Foundation, Funder: Department of Health | National Health and Medical Research Council (NHMRC); doi: https://doi.org/10.13039/501100000925, Adjuvant systemic therapies are now routinely used following resection of stage III melanoma, however accurate prognostic information is needed to better stratify patients. We use differential expression analyses of primary tumours from 204 RNA-sequenced melanomas within a large adjuvant trial, identifying a 121 metastasis-associated gene signature. This signature strongly associated with progression-free (HR = 1.63, p = 5.24 × 10−5) and overall survival (HR = 1.61, p = 1.67 × 10−4), was validated in 175 regional lymph nodes metastasis as well as two externally ascertained datasets. The machine learning classification models trained using the signature genes performed significantly better in predicting metastases than models trained with clinical covariates (pAUROC = 7.03 × 10−4), or published prognostic signatures (pAUROC < 0.05). The signature score negatively correlated with measures of immune cell infiltration (ρ = −0.75, p < 2.2 × 10−16), with a higher score representing reduced lymphocyte infiltration and a higher 5-year risk of death in stage II melanoma. Our expression signature identifies melanoma patients at higher risk of metastases and warrants further evaluation in adjuvant clinical trials.

Funder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440, Brain 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.

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., Metastatic melanoma is associated with a poor prognosis and understanding the genetic features of metastases may enable better treatment strategies. Here, the authors analyse multiple metastases from individual patients finding high levels of heterogeneity in metastases from different organs.

SUMMARYIn melanoma, transcriptional profiling has revealed multiple co-existing cell states, including proliferative versus invasive sub-populations that have been posited to represent a “go or grow” tradeoff. Both of these populations are maintained in tumors, but how they physically interact to promote metastasis is unknown. We demonstrate that these subpopulations form spatially structured heterotypic clusters that cooperate in the seeding of metastasis. We unexpectedly found that INV cells were tightly adherent to each other, and formed clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation between these populations, in which the INV cells facilitated the spread of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of both clustering and the PRO/INV states. Our data suggest a framework for the co-existence of these two divergent cell populations, in which differing cell states form heterotypic clusters that promote metastasis via cell-cell cooperation.

PurposePredicting outcomes after resection of primary melanoma remains crude, primarily based on tumour thickness. We explored gene expression signatures for their ability to better predict outcomes.MethodsDifferential expression analysis of 194 primary melanomas resected from patients who either developed distant metastasis (n=89) or did not (n=105) was performed. We identified 121 metastasis-associated genes that were included in our prognostic signature, “Cam_121”. Several machine learning classification models were trained using nested leave- one-out cross validation (LOOCV) to test the signature’s capacity to predict metastases, as well as regression models to predict survival. The prognostic accuracy was externally validated in two independent datasets.ResultsCam_121 performed significantly better in predicting distant metastases than any of the models trained with the clinical covariates alone (pAccuracy=4.92×10−3), as well as those trained with two published prognostic signatures. Cam_121 expression score was strongly associated with progression-free survival (HR=1.7, p=3.44×10−6), overall survival (HR=1.73, p=7.71×10−6) and melanoma-specific survival (HR=1.59, p=0.02). Cam_121 expression score also negatively correlated with measures of immune cell infiltration (ρ=−0.73, p−16), with a higher score representing reduced tumour lymphocytic infiltration and a higher absolute 5-year risk of death in stage II melanoma.ConclusionsThe Cam_121 primary melanoma gene expression signature outperformed currently available alternatives in predicting the risk of distant recurrence. The signature confirmed (using unbiased approaches) the central prognostic importance of immune cell infiltration in long-term patient outcomes and could be used to identify stage II melanoma patients at highest risk of metastases and poor survival who might benefit most from adjuvant therapies.Translational relevancePredicting outcomes after resection of primary melanoma is currently based on traditional histopathological staging, however survival outcomes within these disease stages varies markedly. Since adjuvant systemic therapies are now being used routinely, accurate prognostic information is needed to better risk stratify patients and avoid unnecessary use of high cost, potentially harmful drugs, as well as to inform future adjuvant strategies. The Cam_121 gene expression signature appears to have this capability and warrants evaluation in prospective clinical trials.

IMPORTANCEBrain metastases occur in 60% of patients with advanced melanoma and are a major cause of melanoma-related mortality and morbidity. Although our understanding of the molecular alterations associated with melanoma progression is improving, there are currently no validated biomarkers which might help identify those patients at highest risk of developing brain metastases.OBJECTIVETo examine the somatic mutational and copy-number landscape of brain metastases that develop as the isolated first visceral site of recurrence – “early brain-metastasis” compared to extracranial melanoma metastases.DESIGN, SETTING AND PARTICIPANTSWhole-exome sequencing of 50 tumors from patients undergoing surgical resection of one or more brain metastasis occurring as the first site of visceral relapse were identified from prospectively maintained databases in Sydney, Wellington, New York and Cambridge. Whole exome sequencing analyses allowed mutational profiles to be compared to cutaneous melanomas in The Cancer Genome Atlas (SKCM-TCGA; n=358) and the Memorial Sloan Kettering (SKCM-MSK-IMPACT; n=186) datasets. An external dataset comprising a further 18 patients with surgically resected early brain metastasis from two additional academic centers served as an independent validation cohort.MAIN OUTCOMES AND MEASURESTo assess the frequency of driver mutations in early brain metastasis and their influence on survival.RESULTSIn concordance with the landmark melanoma sequencing studies, we identified mutations in BRAF (21/50, 42%), NRAS (14/50, 28%) and NF1 (11/50, 22%) as the most frequently mutated melanoma driver genes. When compared to the mutational landscape of cutaneous melanomas in TCGA (SKCM-TCGA), KRAS was the most significantly enriched driver gene, with 5/50 (10%) of brain metastases harboring non-synonymous mutations, of which 4/5 (80%) were in the hotspot positions of codons 12 and 61. This was significantly higher than the corresponding frequency ofKRAS-mutations within the entire SKCM-TCGA (2% (7/358), p=0.009, Fisher’s Exact Test) as well as the SKCM-MSK-IMPACT cohort (1.6% (3/186), p=0.016). Variants in KRAS were mutually exclusive fromBRAFV600,NRASandHRASmutations and were associated with a significantly reduced overall survival from resection of brain metastasis (relative toKRAS-wild type brain metastases) in multivariate Cox proportional hazard models (HR 1.80, 95% CI 1.46-24.89, p=0.013). Mutations inKRASwere also clonal and concordant with extracranial disease, which suggests these mutations are present within the primary tumorCONCLUSIONS AND RELEVANCEOur analysis, the largest to date, suggests that early metastases to the brain (presenting as the first site of visceral relapse) are characterized by significant enrichment of hotspotKRASmutations, potentially implicating constitutive RAS-driven cellular programs in neurotropic metastatic behavior in these cases. Based on these data, we suggest that screening forKRASmutations might help identify those patients with primary melanoma at higher risk of brain metastases or poor survival, and could help inform future surveillance strategies.Key PointsQuestionWhat is the frequency of driver mutations in early melanoma brain metastases?FindingsIn this study of 50 patients with melanoma metastasizing first to the brain,KRASmutations were the most significantly enriched driver gene (n=5, 10% of patients) when compared to landmark cutaneous melanoma studies. The highKRASmutation frequency was also observed in an external validation cohort of 18 patients with early brain metastases. Mutations inKRASwere mutually exclusive from mutations in the key RAS signaling genes and conferred a worse overall survival from resection of brain metastasis.MeaningHotspotKRASmutations could help identify those patients with primary melanoma at higher risk of brain metastases that may benefit from more intensive, protracted surveillance as well as earlier use of adjuvant therapy.

Independent groups have demonstrated that the pre-treatment gut microbiome of cancer patients impacts the subsequent response to Immune Checkpoint Inhibitor (ICIs) therapy [1-4]. However, each study identified different sets of bacteria linked to outcome, which has limited the development of drug response biomarkers and clinic-first design of novel microbiome-based therapeutics. The Cambridge (UK) MELRESIST study includes a cohort of advanced melanoma patients receiving approved ICIs. Pre-treatment stool samples from MELRESIST were analysed by Microbiotica using shotgun metagenomic sequencing. Microbiotica's platform comprises the leading Reference Genome Database to give the most comprehensive and precise mapping of the gut microbiome. A bioinformatic analysis identify a small discrete microbiome signature that was different between responders and non-responders. We extended this signature by reanalysing three published melanoma cohorts [1-3] using the Microbiotica platform. The resultant bacterial signature predicted whether or not a patient responded to anti-PD1-based therapy with an accuracy of 91% in all four studies combined and was also an effective biomarker for each cohort individually. We validated the signature using a NSCLC study [4] indicating that it has great potential as a clinical biomarker for a number of indications. The signature was strongly skewed towards species raised in abundance in responding patients, suggesting that the microbiome influences ICI treatment primarily through bacteria that enhance the efficacy of the drugs. At the core of the signature was nine species strongly associated a positive outcome, which we hypothesized to be a central driver of drug response. MB097 is a consortium comprised of all nine bacteria. In a syngeneic mouse model of cancer, MB097 was able inhibit tumor growth, but most strikingly was potently synergistic when dose with anti-PD1. To understand the mechanisms by which these bacteria drive an anti-tumor response, we have profiled the bacteria individually and as a consortium in multiple assays with primary human immune cells. The bacteria strongly activate dendritic cells with a number inducing high levels of IL-12 relative to IL-10. These bacteria-stimulated dendritic cells went on to trigger Cytotoxic T Lymphocytes (CTLs) to upregulate Granzyme B, Perforin and IFNg. Further, we have demonstrated that these primed CTLs are very effective at tumor cell killing in vitro. In summary, Microbiotica's precision microbiome profiling and the MELRESIST study has allowed us to identify a consortium of bacteria, MB097, strongly linked to response in multiple melanoma cohorts and a NSCLC study. The consortium drives immune-mediated tumor killing in vivo and in vitro. MB097 is being scaled up for manufacture as a novel co-therapy with ICIs. References 1 Matson V et al Science (2018) 359:104 2 Gopalakrishnan V Science (2018) 359:97 3 Frankel AE et al Neoplasia (2017) 19:848 4 Routy B et al Science (2018) 359:91 Citation Format: Matthew J. Robinson, Kevin Vervier, Simon Harris, Amy Popple, Dominika Klisko, Robyne Hudson, Ghaith Bakdash, Laure Castan, Clelia Villemin, David J. Adams, Doreen Milne, Catherine Booth, Christine Parkinson, Roy Rabbie, Sarah J. Welsh, Emily Barker, Katie Dalchau, Pippa Corrie, Trevor Lawley. MB097: A therapeutic consortium of bacteria clinically-defined by precision microbiome profiling of immune checkpoint inhibitor patients with potent anti-tumor efficacy in vitro and in vivo [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P074.

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Melanomas can have multiple co-existing cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a “go or grow” tradeoff; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other, and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.

Four independent international groups have demonstrated that the pre-treatment gut microbiome of cancer patients is associated with the subsequent response to treatment with Immune Checkpoint Inhibitors (ICI) [1-4]. However, each study identified different bacteria as being linked to outcome, which has limited the development of drug response biomarkers and novel microbiome-based therapeutics. Here we describe the identification of a microbial signature predictive of response to ICI across multiple melanoma studies, and a derived Live Bacterial Therapeutic with potent anti-tumour activity. MELRESIST is a single centre, prospective melanoma patient data and biosample collection research study. We collected longitudinal stool samples from 69 patients with advanced melanoma who received standard anti-PD-1+/- anti-CTLA-4 antibodies. Shotgun metagenomic sequencing analysis of the baseline stool microbiome was done using Microbiotica's platform, which comprises the world's leading Reference Genome Database to give the most comprehensive and precise mapping of gut microbiomes. Using 6 months progression-free survival as our cut-off for response, the analysis revealed a small but discrete microbiome signature that differentiated responders and non-responders with an accuracy of 93%. We extended this signature by reanalysing another 3 melanoma patient stool sample sequence datasets [1-3] using the Microbiotica platform, and a machine learning-based bioinformatic model. The resultant bacterial signature accurately predicted response when all 4 studies when combined (91%), as well as when the cohorts were analysed individually (82-100%). We validated the model using independent cohorts and the signature using NSCLC and Renal Cell Carcinoma (RCC) datasets [4]. The latter indicated the bacteria associated with response may differ slightly between indications. At the core of the signature was 9 bacteria that were all overrepresented in patients that responded to ICI treatment. Notably as a consortium, these 9 bacteria demonstrated tumor growth inhibition when dosed in a syngeneic mouse model. These strains also stimulate primary immune cells in vitro leading to tumor cell killing. In summary, we have identified a microbiome biomarker that is predictive of response to ICI treatment in multiple clinical studies from different countries. In addition, a unique set of bacteria derived from the signature has great therapeutic potential in combination with ICIs. References 1 Matson V et al Science (2018) 359:104 2 Gopalakrishnan V Science (2018) 359:97 3 Frankel AE et al Neoplasia (2017) 19:848 4 Routy B et al Science (2018) 359:91 Citation Format: Matthew J. Robinson, Kevin Vervier, Simon Harris, Roy Rabbie, Doreen Milne, Catherine Booth, Christine Parkinson, Sarah J. Welsh, David Bruce, Emily Barker, David Adams, Pippa Corrie, Trevor D. Lawley. Precision microbiome profiling identifies a novel biomarker predictive of Immune Checkpoint Inhibitor response in multiple cohorts and a potent therapeutic consortium of bacteria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1783.

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 that metastatic cells may depart the primary tumour very early in the disease course and follow a branched pattern of evolution. Truncal UV-induced mutations that often swamp downstream analyses of heterogeneity, were found to be replaced by APOBEC-associated mutations in the branches of the evolutionary tree. Multi-sample analyses from a further 7 patients confirmed that branched evolution was pervasive, representing an important mode of melanoma dissemination. Our analyses illustrate that combining cancer cell fraction estimates across multiple metastases provides higher resolution phylogenetic reconstructions relative to single sample analyses and highlights the limitations of accurately inferring inter-tumoural heterogeneity from a single biopsy.

e21546 Background: Four independent groups have demonstrated that the pre-treatment gut microbiome of cancer patients impacts the subsequent response to Immune Checkpoint Inhibitor (ICIs) therapy [1-4]. However, the patient’s outcome was linked to different bacteria in each study, which has limited the development of drug response biomarkers and clinic-first design of novel microbiome-based therapeutics. Methods: The Cambridge (UK) MELRESIST study includes a cohort of advanced melanoma patients receiving approved ICIs. Pretreatment stool samples from MELRESIST were analysed by Microbiotica using shotgun metagenomic sequencing. Microbiotica’s platform comprises the world’s leading Reference Genome Database to give the most comprehensive and precise mapping of the gut microbiome. Results: MELRESIST samples showed an overall difference in the microbiome composition between advanced melanoma patients and healthy donors, but not between patients who did or did not respond to ICIs. However, we did identify a discrete microbiome signature that differentiated responders and non-responders with an accuracy of 93%. We extended this signature by reanalysing three published melanoma cohorts [1-3] using the Microbiotica platform, and a propriety bioinformatic model. The resultant bacterial signature was very accurate at predicting response in all 4 published studies combined (91%), and each cohort individually (82-100%). We validated the model using independent validation cohorts and the signature using lung and renal cancer studies [4]. At the core of our microbiome signature was 9 bacteria most significantly associated with ICI efficacy. All 9 were overrepresented in patients who responded to immunotherapy suggesting high abundance of these bacteria is a central driver of ICI response. A consortium comprised of all 9 strains had very potent anti-tumor efficacy in a cancer syngeneic mouse model. The bacteria also demonstrate multiple interactions with primary human immune cells in vitro leading to dendritic cells activation, Cytotoxic T lymphocyte activation and tumor cell killing. These validate the potential of this consortium as a novel therapy for use in combination with ICIs. Conclusions: We have identified a unique microbiome signature predictive of ICI response in 4 independent melanoma cancer cohorts. This removes a major challenge to the field, and could represent a new highly accurate biomarker with clinical application. Nine core bacteria appear to be driving response, and demonstrate anti-tumor activity in vivo and in vitro. This consortium holds great potential as a co-therapy with ICIs. References:1 Matson V et al, Science (2018) 359:104; 2 Gopalakrishnan V et al, Science (2018) 359:97; 3 Frankel AE et al, Neoplasia (2017) 19:848; 4 Routy B et al, Science (2018) 359:91.

Purpose: Adjuvant therapies prolong survival in patients with stage III melanoma. However, biomarkers are needed to stratify patients with primary melanoma at highest risk for metastases which could help minimize exposure to potentially irreversible toxicities and allow for rational clinical trial designs in the adjuvant setting. Methods: We analyzed data from 194 RNA-sequenced primary cutaneous melanomas from patients with stage IIB-IIIC disease recruited to the multicenter AVAST-M phase III randomized trial. By undertaking covariate-corrected differential expression between patients experiencing distant metastasis (n=89) versus no-metastases (n=105), we identified metastasis-associated genes of which 121 were externally validated and made up our predictive signature, “Cam_121”. Several machine learning classification models were trained using nested leave-one-out cross validation (LOOCV) to test the signature's capacity to predict metastases. Univariate and multivariate Cox proportional hazard regression survival analyses were performed. The signatures' predictive accuracy was further externally validated in an independent population-controlled cohort study measuring melanoma-specific survival (Leeds Melanoma Cohort, n=687). Results: The signature distinguished patients with distant recurrence from those without across multiple machine learning models (sensitivity=0.64, specificity=0.79, accuracy=0.72, kappa=0.43) and performed significantly better than any of the models trained with the clinical covariates alone (pAccuracy =4.92x10-3), as well as those trained with predictive signatures selected from two published datasets (Decision-Dx MelanomaTM and Leeds Melanoma Cohort 150 genes). The signature also correlated with progression-free survival (PFS), overall survival (OS) and melanoma-specific survival (MSS) while retaining its predictive accuracy following multivariate correction (PFS: HR=0.49 (0.35-0.69), p=2.8x10-5, OS: HR=0.6 (0.42-0.86), p=0.005 and MSS: HR=0.57, p=8x10-5). Importantly, we found that the median signature expression score positively correlated with measures of immune cell infiltration, with a lower score representing a poorer tumor lymphocytic infiltration and worse long-term prognosis. Conclusions: We have identified Cam_121 a primary melanoma expression signature that outperforms currently available predictive signatures. The signature confirms (using unbiased approaches) the central prognostic importance of immune cell infiltration in long-term patient outcomes and could help identify primary melanoma patients at highest risk of metastases and poor survival who might benefit most from adjuvant therapies. Citation Format: Manik Garg, Dominique-Laurent Courturier, Nuno A. Fonseca, Matthew Wongchenko, Yibing Yan, Jeremie Nsengimana, Tim Bishop, Julia Newton-Bishop, Mark Middleton, Pippa Corrie, David J. Adams, Alvis Brazma, Roy Rabbie. Primary tumor gene expression signature predicts long-term outcomes in primary melanoma: A prospective multicenter study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-274.

In the last decade treatment for colorectal cancer (CRC) has evolved with the addition of contemporary chemotherapy drugs and targeted therapies. Despite this progress, our drug armamentarium is by no means complete and modern molecular biology techniques have led to the identification of a number of 'druggable' targets. One of the most important current drug targets is the phosphatidyl-inositol 3-kinase (PI3K) pathway, which is frequently deregulated in patients with CRC. In vitro and in vivo data strongly support the clinical development of compounds affecting signal transduction via the PI3K pathway. In this review we outline the role of PI3K in the development and progression of CRC and discuss data from current and ongoing clinical trials targeting this pathway. In addition we make suggestions toward the optimization of future research in order to derive the maximum benefit for patients with CRC.

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.

Introduction Inflammatory bowel disease-associated colorectal cancers (IBD-CRCs) occur infrequently but are associated with a higher mortality than sporadic CRCs. The poorly defined genetic and molecular pathogenesis of IBD-CRCs limits our ability to develop effective prevention, detection and treatment strategies. Since molecular changes identified at the time of surgery are most likely to represent informative biomarkers we elected to study primary IBD-CRC. Method 6The study was approved by the Lothian NHS Research Scotland BioResource (ref:15/ES/0094). Whole-exome sequencing and a comprehensive mutational analysis was performed on 35 FFPE IBD-CRCs and matched normal lymph node pairs from 32 patients (16 CD). Two of the patients had synchronous cancers. Results Ten (29%) of 35 IBD-CRCs were hypermutated and had a significantly higher 10 year survival than the non-hypermutator cancers (p=0.04). All hypermutator cancers were in the proximal colon; seven had loss of expression of MLH1, of which 5 had MLH1 promoter methylation. Two had somatic mutations in the proof-reading domain of DNA POLE. The hypermutated IBD-CRCs had a predicted increased neo-epitope load suggesting that these cancers may benefit from immune checkpoint blockade. Six distinct IBD-CRC mutational signatures were identified with four corresponding to known mutational mechanisms. Five genes including TP53, APC, PIK3CA, and KRAS were significantly mutated in the non-hypermutator IBD-CRCs and fourteen genes in the hypermutator IBD-CRCs, including RNF43 and AIM2, which are clinically actionable. Eleven of the 32 patients (34%) had germline mutations that may confer an inherited susceptibility to colorectal cancer; only two of these specific variants have a clinical pathogenic effect. Conclusion The hypermutated IBD-CRCs are associated with defects in MMR and DNA POLE; with a predicted higher neo-epitope load, which could be exploited using immunotherapies. Loss of MLH1 expression could be evaluated in potential colonic dysplastic or cancerous lesions detected in IBD patients. The identification of novel significantly mutated genes in hypermutated IBD-CRCs could be used to stratify therapy. These approaches would complement and individualise current surveillance and treatment programs for IBD-CRC. Disclosure of Interest None Declared

Summary Melanoma in young children is rare; however, its incidence in adolescents and young adults is rising. We describe the clinical course of a 15‐year‐old female diagnosed with AJCC stage IB non‐ulcerated primary melanoma, who died from metastatic disease 4 years after diagnosis despite three lines of modern systemic therapy. We also present the complete genomic profile of her tumour and compare this to a further series of 13 adolescent melanomas and 275 adult cutaneous melanomas. A somatic BRAFV 600E mutation and a high mutational load equivalent to that found in adult melanoma and composed primarily of C>T mutations were observed. A germline genomic analysis alongside a series of 23 children and adolescents with melanoma revealed no mutations in known germline melanoma‐predisposing genes. Adolescent melanomas appear to have genomes that are as complex as those arising in adulthood and their clinical course can, as with adults, be unpredictable.

Treatment of metastatic and locally advanced pancreatic cancer has made slow progress during the last decade. Single agent gemcitabine or in combination with capecitabine or erlotinib remained the preferred systemic treatment options until 2010 when the ACCORD study demonstrated significantly improved outcomes achieved with FOFIRINOX compared with gemcitabine monotherapy. Since 2010, use of FOLFIRINOX has increased both in metastatic and locally advanced cancer. Despite its gaining popularity among oncologists, unanswered questions remain. Do the often necessary dose modifications affect its efficacy? Are the toxicities manageable and how applicable are the results of the ACCORD study in the general population of patients with newly diagnosed pancreatic cancer? In the present manuscript, we review the published literature regarding the use of FOLFIRINOX, the challenges associated with its use and how it will be optimally incorporated into the management of patients with different stages of pancreatic cancer and ultimately, in a more biomarker-driven pathway algorithm.

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.

Background Epithelial ovarian cancer (EOC) is often diagnosed at an advanced stage, requiring primary cytoreductive surgery and combination chemotherapy for its first-line management. Currently, the recommended standard first-line chemotherapy is platinum-based, usually consisting of carboplatin and paclitaxel (PAC/carbo). Pegylated liposomal doxorubicin (PLD) is an improved formulation of doxorubicin that is associated with fewer and less severe side effects than are seen with non-modified doxorubicin. In combination with carboplatin, PLD has recently been shown to improve progression-free survival compared with PAC/carbo in women with relapsed, platinum-sensitive EOC. It is therefore important to know whether any survival benefit can be attributed to PLD when it is used in the first-line setting. Objectives To evaluate the role of PLD, alone or in combination, in first-line chemotherapy for women with EOC. Search methods We searched The Cochrane Gynaecological Cancer Group's Trial Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE and EMBASE from January 1990 to February 2013. In addition, we searched online trial registries for ongoing trials and abstracts of studies presented at relevant scientific meetings from 2000 onwards. Selection criteria We included all randomised controlled trials (RCTs) that compared PLD alone or in combination with other agent/s (e.g. carboplatin) versus other agent/s for first-line chemotherapy in women with EOC who may or may not have undergone primary cytoreductive surgery. Data collection and analysis Two review authors independently selected trials, extracted data and assessed the risk of bias for each included trial. We obtained updated trial data when possible. Main results We included two large trials. One trial compared three-weekly PLD and carboplatin (PLD/carbo) with PAC/carbo. The other trial included four experimental arms, one of which was PLD plus PAC/carbo, that were compared with the standard PAC/carbo regimen. We did not combine results of these two trials in the meta-analysis. We considered the two studies to be at low risk of bias. For the comparison PLD/carbo versus PAC/carbo (820 women; stages Ic to IV), no statistically significant differences in progression-free survival (PFS) (hazard ratio [HR] 1.01, 95% confidence interval [CI] 0.85 to 1.19) or overall survival (OS) (HR 0.94, 95% CI 0.78 to 1.13) were noted between study arms. Severe anaemia (risk ratio [RR] 2.74, 95% CI 1.54 to 4.88) and thrombocytopenia (RR 8.09, 95% CI 3.93 to 16.67) were significantly more common with PLD/carbo, whereas alopecia (RR 0.09, 95% CI 0.06 to 0.14) and severe neurotoxicity (RR 0.09, 95% CI 0.01 to 0.66) were significantly more common with PAC/carbo. Quality of life scores were not significantly different. For the comparison PLD/PAC/carbo versus PAC/carbo (1726 women; stage III/IV), it is important to note that PLD was given for alternate cycles only (i.e. every 6 weeks). No statistically significant difference in PFS (HR 0.98, 95% CI 0.88 to 1.09) or OS (HR 0.95, 95% CI 0.84 to 1.08) between these two treatment arms was reported. However, women in the triplet arm experienced significantly more severe haematological adverse events (anaemia, thrombocytopenia, neutropenia and febrile neutropenia) compared with those given standard treatment. No RCTs evaluated single-agent PLD for first-line treatment of EOC. Authors' conclusions PLD/carbo is a reasonable alternative to PAC/carbo for the first-line treatment of EOC. Although three-weekly PLD/carbo may be associated with increased dose delays and discontinuations compared with the standard PAC/carbo regimen, it might be more acceptable to women who wish to avoid alopecia or those at high risk of neurotoxicity. No survival benefits appear to be associated with the alternating triplet regimen, and the additional toxicity associated with adding PLD to PAC/carbo limits further investigation. Further studies are needed to establish the safest, most effective PLD/carbo regimen for newly diagnosed disease.

BACKGROUND: This is an updated version of the original review published in Issue 10, 2010 (Rabbie 2010). Migraine is a common, disabling condition and a burden for the individual, health services and society. Many sufferers do not seek professional help, relying instead on over‐the‐counter analgesics. Co‐therapy with an antiemetic should help to reduce symptoms commonly associated with migraine headaches. OBJECTIVES: To determine efficacy and tolerability of ibuprofen, alone or in combination with an antiemetic, compared to placebo and other active interventions in the treatment of acute migraine headaches in adults. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, the Oxford Pain Relief Database, ClinicalTrials.gov, and reference lists for studies through 22 April 2010 for the original review and to 14 February 2013 for the update. SELECTION CRITERIA: We included randomised, double‐blind, placebo‐ or active‐controlled studies using self‐administered ibuprofen to treat a migraine headache episode, with at least 10 participants per treatment arm. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial quality and extracted data. Numbers of participants achieving each outcome were used to calculate relative risk and number needed to treat (NNT) or harm (NNH) compared to placebo or other active treatment. MAIN RESULTS: No new studies were found for this update. Nine included studies (4373 participants, 5223 attacks) compared ibuprofen with placebo or other active comparators; none combined ibuprofen with a self‐administered antiemetic. All studies treated attacks with single doses of medication. For ibuprofen 400 mg versus placebo, NNTs for 2‐hour pain‐free (26% versus 12% with placebo), 2‐hour headache relief (57% versus 25%) and 24‐hour sustained headache relief (45% versus 19%) were 7.2, 3.2 and 4.0, respectively. For ibuprofen 200 mg versus placebo, NNTs for 2‐hour pain‐free (20% versus 10%) and 2‐hour headache relief (52% versus 37%) were 9.7 and 6.3, respectively. The higher dose was significantly better than the lower dose for 2‐hour headache relief. Soluble formulations of ibuprofen 400 mg were better than standard tablets for 1‐hour, but not 2‐hour headache relief. Similar numbers of participants experienced adverse events, which were mostly mild and transient, with ibuprofen and placebo. Ibuprofen 400 mg did not differ from rofecoxib 25 mg for 2‐hour headache relief or 24‐hour headache relief. AUTHORS' CONCLUSIONS: We found no new studies since the last version of this review. Ibuprofen is an effective treatment for acute migraine headaches, providing pain relief in about half of sufferers, but complete relief from pain and associated symptoms for only a minority. NNTs for all efficacy outcomes were better with 400 mg than 200 mg in comparisons with placebo, and soluble formulations provided more rapid relief. Adverse events were mostly mild and transient, occurring at the same rate as with placebo.

BACKGROUND: This review is an update of a previously published review in Issue 2, 2012 (Derry 2012a). Migraine is a common, disabling condition and a burden for the individual, health services and society. Many sufferers choose not to, or are unable to, seek professional help and rely on over‐the‐counter (OTC) analgesics. Diclofenac is an established analgesic, and new formulations using the potassium or epolamine salts, which can be dissolved in water, have been developed for rapid absorption, which may be beneficial in acute migraine. Co‐therapy with an antiemetic should help to reduce the nausea and vomiting commonly associated with migraine. OBJECTIVES: To determine the efficacy and tolerability of diclofenac, alone or in combination with an antiemetic, compared to placebo and other active interventions in the treatment of acute migraine headaches in adults. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, the Oxford Pain Relief Database, ClinicalTrials.gov, and reference lists for studies through 27 September 2011 for the original review and 15 February 2013 for the update. SELECTION CRITERIA: We included randomised, double‐blind, placebo‐controlled or active‐controlled studies, or both, using self administered diclofenac to treat a migraine headache episode, with at least 10 participants per treatment arm. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed trial quality and extracted data. We used numbers of participants achieving each outcome to calculate relative risk (or 'risk ratio') and numbers needed to treat to benefit (NNT) or harm (NNH) compared to placebo or a different active treatment. MAIN RESULTS: Five studies (1356 participants, 2711 attacks) compared oral diclofenac with placebo, and one also compared it with sumatriptan; none combined diclofenac with a self administered antiemetic. Four studies treated attacks with single doses of medication, and two allowed an optional second dose for inadequate response. Only two studies, with three active treatment arms, provided data for pooled analysis of primary outcomes. For single doses of diclofenac potassium 50 mg versus placebo (two studies), the NNTs were 8.9, 6.2, and 9.5 for pain‐free at two hours, headache relief at two hours, and pain‐free responses at 24 hours, respectively. Similar numbers of participants experienced adverse events, which were mostly mild and transient, with diclofenac and placebo. There were insufficient data to evaluate other doses of oral diclofenac, or to compare different formulations or different dosing regimens; only one study compared oral diclofenac with an active comparator (oral sumatriptan 100 mg). AUTHORS' CONCLUSIONS: Oral diclofenac potassium 50 mg is an effective treatment for acute migraine, providing relief from pain and associated symptoms, although only a minority of patients experience pain‐free responses. Adverse events are mostly mild and transient and occur at the same rate as with placebo.