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Background Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment. Methods We performed TIL analysis and T-cell analysis by IHC on the pretreatment and ‘On-treatment’ samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation. Results In our sample cohort (n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10–3) but not TILs (p = 0.1) in their ‘On-treatment’ tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 + (p = 0.04, rho = 0.60) and CD4 + (p = 0.01, rho = 0.72) T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p Conclusions The immune system may be ‘primed’ prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.

Trastuzumab has significantly improved the overall survival of patients with HER2+ metastatic breast cancer (MBC). However, outcomes can vary, with patients progressing within 1 year of treatment or exceptional cases of complete response to trastuzumab for ≥10 years. Identification of the underlying genomic aberrations of “exceptional responders (ExRs)” compared to “rapid non-responders (NRs)” increases our understanding of the mechanisms involved in MBC progression and identification of biomarkers of trastuzumab response and resistance. Whole-exome sequencing was performed on six ExRs compared to five NR. The overall fraction of genome copy number alteration (CNA) burden was higher in NR patients (P = 0.07), while more significantly pronounced in copy number gains (P = 0.03) in NR compared to ExRs. Delineation of the distribution of CNA burden across the genome identified a greater degree of CNA burden in NR within Chr8 (P = 0.02) and in Chr17 (P = 0.06) and conferred a statistically significant benefit in overall survival. Clinical trial number: NCT01722890 [ICORG 12/09].

Background and objectives Mucinous adenocarcinoma is a distinct subtype of colorectal cancer (CRC) with a worse prognosis when compared with non-mucinous adenocarcinoma. The aim of this study was to compare somatic mutations and copy number alteration (CNA) between mucinous and non-mucinous CRC. Methods Data from The Cancer Genome Atlas-colon adenocarcinoma and rectum adenocarcinoma projects were utilized. Mucinous and non-mucinous CRC were compared with regard to microsatellite status, overall mutation rate, the most frequently mutated genes, mutations in genes coding for mismatch repair (MMR) proteins and genes coding for mucin glycoproteins. CNA analysis and pathway analysis was undertaken. Results Mucinous CRC was more likely to be microsatellite instability-high (MSI-H) and hypermutated. When corrected for microsatellite status the single-nucleotide variation and insertion-deletion rate was similar between the two cohorts. Mucinous adenocarcinoma was more likely to have mutations in genes coding for MMR proteins and mucin glycoproteins. Pathway analysis revealed further differences between the two histological subtypes in the cell cycle, RTK-RAS, transforming growth factor-β, and TP53 pathways. Conclusions Mucinous CRC has some distinct genomic aberrations when compared with non-mucinous adenocarcinoma, many of which are driven by the increased frequency of MSI-H tumors. These genomic aberrations may play an important part in the difference seen in response to treatment and prognosis in mucinous adenocarcinoma.

BackgroundMucinous differentiation occurs in 5–15 per cent of colorectal adenocarcinomas. This subtype of colorectal cancer responds poorly to chemoradiotherapy and has a worse prognosis. The genetic aetiology underpinning this cancer subtype lacks consensus. The aim of this study was to use meta-analytical techniques to clarify the molecular associations of mucinous colorectal cancer.MethodsThis study adhered to MOOSE guidelines. Databases were searched for studies comparing KRAS, BRAF, microsatellite instability (MSI), CpG island methylator phenotype (CIMP), p53 and p27 status between patients with mucinous and non-mucinous colorectal adenocarcinoma. A random-effects model was used for analysis.ResultsData from 46 studies describing 17 746 patients were included. Mucinous colorectal adenocarcinoma was associated positively with KRAS (odds ratio (OR) 1·46, 95 per cent c.i. 1·08 to 2·00, P = 0·014) and BRAF (OR 3·49, 2·50 to 4·87; P < 0·001) mutation, MSI (OR 3·98, 3·30 to 4·79; P < 0·001) and CIMP (OR 3·56, 2·85 to 4·43; P < 0·001), and negatively with altered p53 expression (OR 0·46, 0·31 to 0·67; P < 0·001).ConclusionThe genetic origins of mucinous colorectal adenocarcinoma are predominantly associated with BRAF, MSI and CIMP pathways. This pattern of molecular alterations may in part explain the resistance to standard chemotherapy regimens seen in mucinous adenocarcinoma.

Ultraviolet radiation (UVR) increases the incidence of cutaneous melanoma1–4. The ageing, sun-exposed dermis accumulates UVR damage5, and older patients develop more melanomas at UVR-exposed sites4,6,7. As fibroblasts are functionally heterogeneous and play key roles in the stromal contribution to cancer8,9, we asked whether UVR modifies dermal fibroblast function. Here we confirmed the expression of collagen-cleaving matrix metalloprotein-1 (MMP1) by UVR-damaged fibroblasts was persistently upregulated to reduce local levels of collagen 1 (COL1A1), and found dermal COL1A1 degradation by MMP1 decreased melanoma invasion. Conversely, we show inhibiting extracellular matrix degradation and MMP1 expression restored melanoma invasion to UVR damaged dermis. We confirmed in vitro findings in a cohort of primary cutaneous melanomas of aged humans, showing more cancer cells invade as single cells at the invasive front of melanomas expressing and depositing more collagen. We found collagen and single melanoma cell invasion are robust predictors of poor melanoma-specific survival. These data indicate melanomas arising over UVR-damaged, collagen-poor skin of the elderly are less invasive, and this reduced invasion improves survival. Consequently, although UVR increases tumour incidence, it delays primary melanoma invasion by degrading collagen. However, we show melanoma-associated fibroblasts can restore invasion in low-collagen primary tumours by increasing collagen synthesis. Finally, we demonstrate high COL1A1 gene expression is a biomarker of poor outcome across a broad range of primary cancers.

Background KIT mutations are primarily associated with acral and mucosal melanoma, and have been reported to show higher prevalence in chronic sun-damaged (CSD) than non-CSD melanomas. Objectives To investigate the prevalence of KIT mutations in melanoma according to subtype, and determine the clinical role of such mutations. Material & methods We present results from a study of a Spanish population of 492 melanomas, classified according to the latest World Health Organization (WHO) guidelines. We analysed the mutational status of KIT and correlated with different clinical variables related to sun exposure and family history. Results KIT mutations were significantly more frequent in acral (3/36; 8.3%) and mucosal (4/8; 50%) melanomas than non-acral cutaneous melanomas. No significant difference was observed in KIT mutational status between CSD and non-CSD melanomas. Conclusion Our results suggest that KIT mutations in melanoma tumours are unrelated to the development of nevi or chronic sun damage, but their presence is associated with aggressive melanomas which show ulceration, vascular invasiveness, and increased Breslow thickness. These findings are consistent with those reported by The Cancer Genome Atlas network.

BackgroundMany cancer types display sex and age disparity in incidence and outcome. The mutational load of tumours, including melanoma, varies according to sex and age. However, there are no tools to systematically explore if clinical variables such as age and sex determine the genomic landscape of cancer.ObjectivesTo establish a mathematical approach using melanoma mutational data to analyze how sex and age shape the tumour genome.MethodsWe model how age‐related (clock‐like) somatic mutations that arise during cell division, and extrinsic (environmental ultraviolet light) mutations accumulate in cancer genomes.ResultsMelanoma is primarily driven by cell‐intrinsic age‐related mutations and extrinsic ultraviolet light‐induced mutations, and we show these mutation types differ in magnitude, chronology and by sex in the distinct molecular melanoma subtypes. Our model confirms age and sex are determinants of cellular mutation rate, shaping the final mutation composition. We show mathematically for the first time how, similar to non‐cancer tissues, melanoma genomes reflect a decline in cell division during ageing. We find clock‐like mutations strongly correlate with the acquisition of ultraviolet light‐induced mutations, but critically, males present a higher number and rate of cell division‐linked mutations.ConclusionsThese data indicate the contribution of environmental damage to melanoma likely extends beyond genetic damage to affect cell division. Sex and age determine the final mutational composition of melanoma.

Background & Aims: KH-type splicing regulatory protein (KHSRP) is a multifunctional nucleic acid binding protein. KHSRP regulates transcription, mRNA decay and translation, and miRNA biogenesis. These distinct functions are associated with key aspects of cancer cell biology: inflammation and cell-fate determination. However, the role KHSRP plays in colorectal cancer (CRC) tumorigenesis remains largely unknown. Methods: We investigated the oncogenic role of KHSRP in CRC using a combination of in silico analysis of large datasets, ex vivo analysis of protein expression in patient samples, and mechanistic studies using in vitro models of CRC. Results: KHSRP was expressed in the epithelial and stromal compartments of both primary and metastatic tumors. Elevated KHSRP expression was found in tumor versus matched normal tissue in a cohort of 62 patients. This finding was validated in larger independent cohorts in silico. KHSRP expression was a prognostic indicator of worse overall survival (HR=3.74, 95% CI = 1.43-22.97, p=0.0138). Mechanistic data in CRC cell line models supported a role of KHSRP in driving epithelial cell proliferation in both a primary and metastatic setting, through control of the G1/S transition. Additionally, epithelial KHSRP was involved in promoting a pro-angiogenic extracellular environment, as well as regulating the secretion of oncogenic proteins involved in diverse cellular processes such as migration and response to cellular stress. Conclusion: Our study has uncovered novel mechanistic-based data on the tumor- promoting effects of KHSRP in CRC.

Mucinous colorectal cancer is a unique histological subtype that is known to respond poorly to cytotoxic chemotherapy and radiotherapy. There are a number of genes known to be associated with resistance to 5-fluorouracil (5-FU), oxaliplatin, and irinotecan. The aim of this study was to compare the somatic mutation frequency and copy number variation (CNV) in these genes between mucinous and non-mucinous colorectal cancer. A systematic search of PubMed was performed to identify papers investigating drug resistance in colorectal cancer. From this review, a list of 26 drug-resistance-associated genes was compiled. Using patient data from The Cancer Genome Atlas (TCGA), the somatic mutation rate and CNV was compared between patients with mucinous and non-mucinous colorectal cancer. Statistical analysis was carried out using GraphPad PRISM® version 5.00. Data were available on 531 patients (464 non-mucinous, 67 mucinous). A statistically significant difference in the somatic mutation rate between the two cohorts was identified in the TYMP (p = 0.0179), ATP7B (p = 0.0465), SRPK1 (p = 0.0135), ABCB1 (p = 0.0423), and ABCG2 (p = 0.0102) genes. A statistically significant difference in CNV was identified between the two cohorts in the GSTP1 (p = 0.0405), CCS (p = 0.0063), and TOP1 (p = 0.0048) genes. Differences in somatic mutation rate and CNV in genes associated with resistance to 5-FU, oxaliplatin, and irinotecan may partly account for the pattern of resistance observed in mucinous colorectal cancers. These genetic alterations may prove useful when deciding on a personalized approach to chemotherapy and may also represent potential therapeutic targets going forward.

The treatment landscape for metastatic melanoma has been revolutionised by the introduction of immunotherapy and targeted therapies. Despite these advances, some patients exhibit primary or acquired resistance to treatment. We present the case of a resected mucosal melanoma that on relapse underwent transformation to a dedifferentiated state. The relapsed tumour was phenotypically disparate and demonstrated loss of all typical melanoma-associated immunohistochemical markers. Furthermore, it demonstrated aggressive biological behaviour and immunotherapy resistance. We performed genomic profiling of the original and relapsed tumour to further elucidate the mechanisms underlying this rare phenomenon. Mass spectrometry-based single-nucleotide polymorphism genotyping technology was used to screen for mutations in the original and recurrent tumour. Whole-exome sequencing was performed on the original tumour, recurrent tumour and blood. Both the original and recurrent tumour shared a NRAS mutation, a similar aneuploidy profile and proportion of somatic single-nucleotide variants. However, in contrast to the original tumour, the recurrent tumour demonstrated a lower mutational burden and deletions in the CDKN2A/CDKN2B and CHEK2 genes. The genomic similarity between the original and recurrent tumour attests to a common ancestry and the possible existence of nongenomic drivers inciting phenotype plasticity. In contrast, the low mutational load and potential inactivation of tumour suppressor genes in the recurrent tumour may underlie its rapid proliferative rate and immunoresistance. Dynamic treatment models are desired in the future to track the genomic and epigenetic evolution of a tumour to guide optimal therapy choice and sequencing.

Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell–derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. Significance: Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments. Cancer Discov; 6(3); 286–99. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 217

The comprehensive identification of mutations contributing to the development of cancer is a priority of large cancer sequencing projects. To date, most studies have scrutinized mutations in coding regions of the genome, but several recent discoveries, including the identification of recurrent somatic mutations in the TERT promoter in multiple cancer types, support the idea that mutations in non-coding regions are also important in tumour development. Furthermore, analysis of whole-genome sequencing data from tumours has elucidated novel mutational patterns and processes etched into cancer genomes. Here, we present an overview of insights gleaned from the analysis of mutations from sequenced cancer genomes. We then review the mechanisms by which non-coding mutations can play a role in cancer. Finally, we discuss recent efforts aimed at identifying non-coding driver mutations, as well as the unique challenges that the analysis of non-coding mutations present in contrast to the identification of driver mutations in coding regions.

Cutaneous melanoma is epidemiologically linked to ultraviolet radiation (UVR), but the molecular mechanisms by which UVR drives melanomagenesis remain unclear. The most common somatic mutation in melanoma is a V600E substitution in BRAF, which is an early event. To investigate how UVR accelerates oncogenic BRAF-driven melanomagenesis, we used a BRAF(V600E) mouse model. In mice expressing BRAF(V600E) in their melanocytes, a single dose of UVR that mimicked mild sunburn in humans induced clonal expansion of the melanocytes, and repeated doses of UVR increased melanoma burden. Here we show that sunscreen (UVA superior, UVB sun protection factor (SPF) 50) delayed the onset of UVR-driven melanoma, but only provided partial protection. The UVR-exposed tumours showed increased numbers of single nucleotide variants and we observed mutations (H39Y, S124F, R245C, R270C, C272G) in the Trp53 tumour suppressor in approximately 40% of cases. TP53 is an accepted UVR target in human non-melanoma skin cancer, but is not thought to have a major role in melanoma. However, we show that, in mice, mutant Trp53 accelerated BRAF(V600E)-driven melanomagenesis, and that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma. Thus, we provide mechanistic insight into epidemiological data linking UVR to acquired naevi in humans. Furthermore, we identify TP53/Trp53 as a UVR-target gene that cooperates with BRAF(V600E) to induce melanoma, providing molecular insight into how UVR accelerates melanomagenesis. Our study validates public health campaigns that promote sunscreen protection for individuals at risk of melanoma.

We used a combination of whole-genome sequencing and in vitro validation to show that mutations that activated at least two pro-growth/survival pathways mediated intrinsic resistance to BRAF inhibition in a melanoma patient. These data demonstrate how in-depth analysis can reveal intrinsic resistance to standard of care, providing an opportunity for alternative therapeutic strategies for patients who are likely to fail first-line treat-575 ment., Background BRAF is mutated in ∼42% of human melanomas (COSMIC. http://www.sanger.ac.uk/genetics/CGP/cosmic/) and pharmacological BRAF inhibitors such as vemurafenib and dabrafenib achieve dramatic responses in patients whose tumours harbour BRAFV600 mutations. Objective responses occur in ∼50% of patients and disease stabilisation in a further ∼30%, but ∼20% of patients present primary or innate resistance and do not respond. Here, we investigated the underlying cause of treatment failure in a patient with BRAF mutant melanoma who presented primary resistance. Methods We carried out whole-genome sequencing and single nucleotide polymorphism (SNP) array analysis of five metastatic tumours from the patient. We validated mechanisms of resistance in a cell line derived from the patient's tumour. Results We observed that the majority of the single-nucleotide variants identified were shared across all tumour sites, but also saw site-specific copy-number alterations in discrete cell populations at different sites. We found that two ubiquitous mutations mediated resistance to BRAF inhibition in these tumours. A mutation in GNAQ sustained mitogen-activated protein kinase (MAPK) signalling, whereas a mutation in PTEN activated the PI3 K/AKT pathway. Inhibition of both pathways synergised to block the growth of the cells. Conclusions Our analyses show that the five metastases arose from a common progenitor and acquired additional alterations after disease dissemination. We demonstrate that a distinct combination of mutations mediated primary resistance to BRAF inhibition in this patient. These mutations were present in all five tumours and in a tumour sample taken before BRAF inhibitor treatment was administered. Inhibition of both pathways was required to block tumour cell growth, suggesting that combined targeting of these pathways could have been a valid therapeutic approach for this patient.

An increased risk of developing Alzheimer's disease (AD) has previously been found to be associated with variants at the MS4A6A locus. We sought to identify which genes and transcripts in this region have altered expression in AD and mild cognitive impairment (MCI) and are influenced by the AD risk variant(s), as a first step to understanding the molecular basis of AD susceptibility at this locus. Common variants located within highly expressed MS4A6A transcripts were significantly associated with AD and MS4A6A expression levels in blood from MCI and AD subjects (p < 0.05, rs610932, rs7232, rs583791). More copies of the protective (minor) allele were associated with lower MS4A6A expression of each transcript (e.g., p = 0.019; rs610932-total MS4A6A). Furthermore, in heterozygous AD subjects, relative expression of the protective allele of V4-MS4A6A transcripts was lower (p < 0.008). Irrespective of genotype, MS4A6A transcripts were increased in blood from people with AD (p < 0.003), whereas lower expression of full length V1-MS4A6A (p = 0.002) and higher expression of V4-MS4A6A (p = 1.8 × 10−4) were observed in MCI, relative to elderly controls. The association between genotype and expression was less consistent in brain, although BA9 did have a similar genotype association with V4-MS4A6A transcripts as in blood. MS4A6A transcripts were widely expressed in tissues and cells, with the exception of V4-MS4A6A, which was not expressed in neuronal cells. Together these results suggest that high levels of MS4A6A in emerging AD pathology are detrimental. Persons with MCI may lower MS4A6A expression to minimize detrimental disease associated MS4A6A activity. However, those with the susceptibility allele appear unable to decrease expression sufficiently, which may explain their increased risk for developing AD. Inhibiting MS4A6A may therefore promote a more neuroprotective phenotype, although further work is needed to establish whether this is the case.

Background The identification of mutations that play a causal role in tumour development, so called “driver” mutations, is of critical importance for understanding how cancers form and how they might be treated. Several large cancer sequencing projects have identified genes that are recurrently mutated in cancer patients, suggesting a role in tumourigenesis. While the landscape of coding drivers has been extensively studied and many of the most prominent driver genes are well characterised, comparatively less is known about the role of mutations in the non-coding regions of the genome in cancer development. The continuing fall in genome sequencing costs has resulted in a concomitant increase in the number of cancer whole genome sequences being produced, facilitating systematic interrogation of both the coding and non-coding regions of cancer genomes. Results To examine the mutational landscapes of tumour genomes we have developed a novel method to identify mutational hotspots in tumour genomes using both mutational data and information on evolutionary conservation. We have applied our methodology to over 1300 whole cancer genomes and show that it identifies prominent coding and non-coding regions that are known or highly suspected to play a role in cancer. Importantly, we applied our method to the entire genome, rather than relying on predefined annotations (e.g. promoter regions) and we highlight recurrently mutated regions that may have resulted from increased exposure to mutational processes rather than selection, some of which have been identified previously as targets of selection. Finally, we implicate several pan-cancer and cancer-specific candidate non-coding regions, which could be involved in tumourigenesis. Conclusions We have developed a framework to identify mutational hotspots in cancer genomes, which is applicable to the entire genome. This framework identifies known and novel coding and non-coding mutional hotspots and can be used to differentiate candidate driver regions from likely passenger regions susceptible to somatic mutation. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3420-9) contains supplementary material, which is available to authorized users.

// Sinead Toomey 1, * , Stephen F. Madden 2, * , Simon J. Furney 3 , Yue Fan 4 , Mark McCormack 5 , Carragh Stapleton 5 , Mattia Cremona 1 , Gianpiero L. Cavalleri 5 , Malgorzata Milewska 1 , Naomi Elster 1 , Aoife Carr 1 , Joanna Fay 6 , Elaine W. Kay 6 , Susan Kennedy 7 , John Crown 7 , William M. Gallagher 4 , Bryan T. Hennessy 1, 8, ** , Alex J. Eustace 1, ** 1 Medical Oncology Group, Department of Molecular Medicine, Royal College of Surgeons in Ireland, Ireland 2 Population Health Sciences Division, Royal College of Surgeons in Ireland, Ireland 3 School of Medicine, University College Dublin, Ireland 4 University College Dublin, UCD School of Biomolecular and Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, Ireland 5 Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Ireland 6 Department of Histopathology, Beaumont Hospital, Dublin, Ireland 7 St Vincent’s University Hospital, Dublin, Ireland 8 Department of Oncology, Beaumont Hospital, Dublin, Ireland * These authors contributed equally to this work ** Joint senior authors Correspondence to: Alex J. Eustace, email: alexeustace@rcsi.ie Keywords: ERBB-family germline mutations, single nucleotide polymorphisms, high depth sequencing, survival impact Received: May 03, 2016 Accepted: October 12, 2016 Published: October 20, 2016 ABSTRACT Background: Trastuzumab treatment for women with HER2-positive breast cancer (BC) resulted in the significant improvement of both relapse free survival (RFS) and overall survival (OS). However, many women who are classified as HER2-positive do not respond. Many studies have focused on the role of somatic mutations rather than germline polymorphisms in trastuzumab resistance. Results: We completed an Agena MassArray screen of 10 ERBB-family single nucleotide polymorphisms (SNPs) in 194 adjuvant trastuzumab treated HER2-positive BC patients. SNPs in EGFR genes have a significant association with RFS and OS. Patients with the minor allele of EGFR N158N had significantly worse OS (hazard ratio (HR) = 4.01, (confidence interval (CI) = 1.53– 10.69), p = 0.05) relative to those with either the heterozygous or wild-type (WT) allele. Patients with the minor allele of EGFR T903T (HR = 3.52, (CI = 1.38– 8.97), p = 0.05) had worse RFS relative to those with either the heterozygous or WT allele. Patients and methods: Using next generation sequencing (NGS) we identified ERBB-family (EGFR, HER2, HER3 and HER4) single nucleotide polymorphisms (SNPs) that occurred in 2 or more patients of a 32 HER2-positive BC patient cohort. Agena MassArray analysis confirmed the frequency of these SNPs in 194 women with HER2-positive BC who received trastuzumab in the adjuvant setting. Using Kaplan-Meier estimates and Cox regression analysis we correlated the presence of ERBB-family SNPs with both RFS and OS. Conclusions: The presence of germline ERBB-family SNPs may play an important role in how a patient responds to adjuvant trastuzumab, and clinical assessment of these SNPs by targeted genetic screening of patients’ blood may be important to stratify patients for treatment.

Alzheimer's disease (AD), like other dementias, is characterized by progressive neuronal loss and neuroinflammation in the brain. The peripheral leukocyte response occurring alongside these brain changes has not been extensively studied, but might inform therapeutic approaches and provide relevant disease biomarkers. Using microarrays, we assessed blood gene expression alterations occurring in people with AD and those with mild cognitive changes at increased risk of developing AD. Of the 2,908 differentially expressed probes identified between the three groups (p < 0.01), a quarter were altered in blood from mild cognitive impairment (MCI) and AD subjects, relative to controls, suggesting a peripheral response to pathology may occur very early. There was strong evidence for mitochondrial dysfunction with decreased expression of many of the respiratory complex I-V genes and subunits of the core mitochondrial ribosome complex. This mirrors changes previously observed in AD brain. A number of genes encoding cell adhesion molecules were increased, along with other immune-related genes. These changes are consistent with leukocyte activation and their increased the transition from circulation into the brain. In addition to expression changes, we also found increased numbers of basophils in people with MCI and AD, and increased monocytes in people with an AD diagnosis. Taken together this study provides both an insight into the functional response of circulating leukocytes during neurodegeneration and also identifies potential targets such as the respiratory chain for designing and monitoring future therapeutic interventions using blood.

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with considerable evidence suggesting an initiation of disease in the entorhinal cortex and hippocampus and spreading thereafter to the rest of the brain. In this study, we combine genetics and imaging data obtained from the Alzheimer's Disease Neuroimaging Initiative and the AddNeuroMed study. To identify genetic susceptibility loci for AD, we conducted a genome-wide study of atrophy in regions associated with neurodegeneration in this condition. We identified one single-nucleotide polymorphism (SNP) with a disease-specific effect associated with entorhinal cortical volume in an intron of the ZNF292 gene (rs1925690; P-value=2.6 × 10(-8); corrected P-value for equivalent number of independent quantitative traits=7.7 × 10(-8)) and an intergenic SNP, flanking the ARPP-21 gene, with an overall effect on entorhinal cortical thickness (rs11129640; P-value=5.6 × 10(-8); corrected P-value=1.7 × 10(-7)). Gene-wide scoring also highlighted PICALM as the most significant gene associated with entorhinal cortical thickness (P-value=6.7 × 10(-6)).

Breast cancer is the leading cause of cancer related deaths in women worldwide and is classified into subtypes based on the cancer’s receptor status. Of these subtypes, those expressing the human epidermal growth factor receptor 2 (HER2) receptor were traditionally associated with poor prognosis. Several advances have been made in the treatment of HER2-positive breast cancer, yet issues of resistance and poor response to therapy remains prevalent. In this study we explored the impact of HER-family and homologous recombination deficiency SNPs on response to patients who received TCH-based (docetaxel (T), carboplatin (C), and trastuzumab (H)) treatment versus those who received other treatment regimens. Using Cox regression analysis, we identified 6 SNPs that correlate with recurrence free survival in our patients and supported our findings using support vector machines. We also used reverse phase protein array analysis to examine the impact ERBB3 SNPs may have on both the PI3K/AKT and MAPK/ERK signaling pathways. Finally, using cell line models, we correlated SNP status with sensitivity to platinum based drugs and docetaxel. We found that patients on a TCH based regimen with the minor allele of the ERBB3 (rs2229046 and rs773123) and BARD1 (rs2070096) SNPs, were significantly more likely to relapse than those women who were not. Additionally, we observed that patients with these ERBB3 SNPs had shown elevated protein expression/phosphorylation of Src kinase, c-MET (Y1234/1235), GSK-3β (S9) and p27, indicating that these SNPs are associated with non-PI3K/AKT signaling. Finally, using cell line models, we demonstrate that the BARD1 SNP (rs2229571) is associated with greater sensitivity to both carboplatin and cisplatin. The BARD1 and ERBB3 SNPs can potentially be used to determine those patients that will have a worse response to TCH based treatment, an effect that may arise from the SNPs impact on altered cellular signaling.

We used personalised medicine approaches for the first time in a tyrosine kinase inhibitor-resistant chronic myeloid leukaemia (CML) patient. Our whole genome sequencing data revealed the emergence of compound mutations in the BCR-ABL1 kinase domain and BCL2 amplification. Patient's CML cells showed increased sensitivity to the ponatinib/ABT-263 combination in vitro., Background Chronic myeloid leukaemia (CML) is characterised by the presence of a fusion driver oncogene, BCR-ABL1, which is a constitutive tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are the central treatment strategy for CML patients and have significantly improved survival rates, but the T315I mutation in the kinase domain of BCR-ABL1 confers resistance to all clinically approved TKIs, except ponatinib. However, compound mutations can mediate resistance even to ponatinib and remain a clinical challenge in CML therapy. Here, we investigated a ponatinib-resistant CML patient through whole-genome sequencing (WGS) to identify the cause of resistance and to find alternative therapeutic targets. Patients and methods We carried out WGS on a ponatinib-resistant CML patient and demonstrated an effective combination therapy against the primary CML cells derived from this patient in vitro. Results Our findings demonstrate the emergence of compound mutations in the BCR-ABL1 kinase domain following ponatinib treatment, and chromosomal structural variation data predicted amplification of BCL2. The primary CD34+ CML cells from this patient showed increased sensitivity to the combination of ponatinib and ABT-263, a BCL2 inhibitor with a negligible effect against the normal CD34+ cells. Conclusion Our results show the potential of personalised medicine approaches in TKI-resistant CML patients and provide a strategy that could improve clinical outcomes for these patients.

Uveal melanoma, the most common eye malignancy, causes severe visual morbidity and is fatal in approximately 50% of patients. Primary uveal melanoma can be cured by surgery or radiotherapy, but the metastatic disease is treatment refractory. To understand comprehensively uveal melanoma genetics, we conducted single-nucleotide polymorphism arrays and whole-genome sequencing on 12 primary uveal melanomas. We observed only approximately 2,000 predicted somatic single-nucleotide variants per tumor and low levels of aneuploidy. We did not observe an ultraviolet radiation DNA damage signature, but identified SF3B1 mutations in three samples and a further 15 mutations in an extension cohort of 105 samples. SF3B1 mutations were associated with good prognosis and were rarely coincident with BAP1 mutations. SF3B1 encodes a component of the spliceosome, and RNA sequencing revealed that SF3B1 mutations were associated with differential alternative splicing of protein coding genes, including ABCC5 and UQCC, and of the long noncoding RNA CRNDE. Significance: Our data show that despite its dismal prognosis, uveal melanoma is a relatively simple genetic disease characterized by recurrent chromosomal losses and gains and a low mutational burden. We show that SF3B1 is recurrently mutated in uveal melanoma, and the mutations are associated with aberrant alternative splicing. Cancer Discov; 3(10); 1122–9. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 1083

Mucosal melanoma displays distinct clinical and epidemiological features compared to cutaneous melanoma. Here we used whole genome and whole exome sequencing to characterize the somatic alterations and mutation spectra in the genomes of ten mucosal melanomas. We observed somatic mutation rates that are considerably lower than occur in sun-exposed cutaneous melanoma, but comparable to the rates seen in cancers not associated with exposure to known mutagens. In particular, the mutation signatures are not indicative of ultraviolet light- or tobacco smoke-induced DNA damage. Genes previously reported as mutated in other cancers were also mutated in mucosal melanoma. Notably, there were substantially more copy number and structural variations in mucosal melanoma than have been reported in cutaneous melanoma. Thus, mucosal and cutaneous melanomas are distinct diseases with discrete genetic features. Our data suggest that different mechanisms underlie the genesis of these diseases and that structural variations play a more important role in mucosal than in cutaneous melanomagenesis.

Although the mechanism of Aβ action in the pathogenesis of Alzheimer's disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aβ neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aβ/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aβ toxicity and DKK1 upregulation and, conversely, Aβ increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aβ mediates neurotoxicity, we measured the effects of Aβ and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt-planar cell polarity (PCP)-c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aβ neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aβ-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt-PCP-JNK pathway is active in an Aβ-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aβ induces a clusterin/p53/Dkk1/wnt-PCP-JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aβ in neurodegenerative diseases.Molecular Psychiatry advance online publication, 20 November 2012; doi:10.1038/mp.2012.163., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Progression of people presenting with Mild Cognitive Impairment (MCI) to dementia is not certain and it is not possible for clinicians to predict which people are most likely to convert. The inability of clinicians to predict progression limits the use of MCI as a syndrome for treatment in prevention trials and, as more people present with this syndrome in memory clinics, and as earlier diagnosis is a major goal of health services, this presents an important clinical problem. Some data suggest that CSF biomarkers and functional imaging using PET might act as markers to facilitate prediction of conversion. However, both techniques are costly and not universally available. The objective of our study was to investigate the potential added benefit of combining biomarkers that are more easily obtained in routine clinical practice to predict conversion from MCI to Alzheimer's disease. To explore this we combined automated regional analysis of structural MRI with analysis of plasma cytokines and chemokines and compared these to measures of APOE genotype and clinical assessment to assess which best predict progression. In a total of 205 people with MCI, 77 of whom subsequently converted to Alzheimer's disease, we find biochemical markers of inflammation to be better predictors of conversion than APOE genotype or clinical measures (Area under the curve (AUC) 0.65, 0.62, 0.59 respectively). In a subset of subjects who also had MRI scans the combination of serum markers of inflammation and MRI automated imaging analysis provided the best predictor of conversion (AUC 0.78). These results show that the combination of imaging and cytokine biomarkers provides an improvement in prediction of MCI to AD conversion compared to either datatype alone, APOE genotype or clinical data and an accuracy of prediction that would have clinical utility.

CONTEXT: Blood-based analytes may be indicators of pathological processes in Alzheimer disease (AD). OBJECTIVE: To identify plasma proteins associated with AD pathology using a combined proteomic and neuroimaging approach. DESIGN: Discovery-phase proteomics to identify plasma proteins associated with correlates of AD pathology. Confirmation and validation using immunodetection in a replication set and an animal model. SETTING: A multicenter European study (AddNeuroMed) and the Baltimore Longitudinal Study of Aging. PARTICIPANTS: Patients with AD, subjects with mild cognitive impairment, and healthy controls with standardized clinical assessments and structural neuroimaging. MAIN OUTCOME MEASURES: Association of plasma proteins with brain atrophy, disease severity, and rate of clinical progression. Extension studies in humans and transgenic mice tested the association between plasma proteins and brain amyloid. RESULTS: Clusterin/apolipoprotein J was associated with atrophy of the entorhinal cortex, baseline disease severity, and rapid clinical progression in AD. Increased plasma concentration of clusterin was predictive of greater fibrillar amyloid-beta burden in the medial temporal lobe. Subjects with AD had increased clusterin messenger RNA in blood, but there was no effect of single-nucleotide polymorphisms in the gene encoding clusterin with gene or protein expression. APP/PS1 transgenic mice showed increased plasma clusterin, age-dependent increase in brain clusterin, as well as amyloid and clusterin colocalization in plaques. CONCLUSIONS: These results demonstrate an important role of clusterin in the pathogenesis of AD and suggest that alterations in amyloid chaperone proteins may be a biologically relevant peripheral signature of AD.

Understanding the mechanism of regulation of cancer genes and the constraints on their coding sequences is of fundamental importance in understanding the process of tumour development. Here we test the hypothesis that tumour suppressor genes and proto-oncogenes, due to their involvement in tumourigenesis, have distinct patterns of regulation and coding selective constraints compared to non-cancer genes. Indeed, we found significantly greater conservation in the promoter regions of proto-oncogenes, suggesting that these genes are more tightly regulated, i.e. they are more likely to contain a higher density of cis-regulatory elements. Furthermore, proto-oncogenes appear to be preferentially targeted by microRNAs and have longer 3' UTRs. In addition, proto-oncogene evolution appears to be highly constrained, compared to tumour suppressor genes and non-cancer genes. A number of these trends are confirmed in breast and colon cancer gene sets recently identified by mutational screening.

The discovery of the genes involved in genetic diseases is a very important step towards the understanding of the nature of these diseases. In-lab identification is a difficult, time consuming task, where computational methods can be very useful. In silico identification algorithms can be used as a guide in future studies. Previous works in this topic have not taken into account that no reliable sets of negative examples are available, as it is not possible to ensure that a given gene is not related to any genetic disease. In this paper, this feature of the nature of the problem is considered, and identification is approached as a partially supervised classification problem. In addition, we have performed a more specific method to identify disease genes by classifying, for the first time, genes causing dominant and recessive diseases independently. We base this separation on previous results that show that these two types of genes present differences in their sequence properties. In this paper, we have applied a new model averaging algorithm to the identification of human genes associated with both dominant and recessive Mendelian diseases.

Background One of the main goals of cancer genetics is to identify the causative elements at the molecular level leading to cancer. Results We have conducted an analysis of a set of genes known to be involved in cancer in order to unveil their unique features that can assist towards the identification of new candidate cancer genes. Conclusion We have detected key patterns in this group of genes in terms of the molecular function or the biological process in which they are involved as well as sequence properties. Based on these features we have developed an accurate Bayesian classification model with which human genes have been scored for their likelihood of involvement in cancer.

Identification of functional variants from whole-exome sequencing, combined with neuroimaging genetics

Today, cancer is viewed as a genetic disease and many genetic mechanisms of oncogenesis are known. The progression from normal tissue to invasive cancer is thought to occur over a timescale of 5–20 years. This transformation is driven by both inherited genetic factors and somatic genetic alterations and mutations, and it results in uncontrolled cell growth and, in many cases, death. In this article, we review the main types of genomic and genetic alterations involved in cancer, namely copy-number changes, genomic rearrangements, somatic mutations, polymorphisms, and epigenomic alterations in cancer. We then discuss the transcriptomic consequences of these alterations in tumor cells. The use of “next-generation” sequencing methods in cancer research is described in the relevant sections. Finally, we discuss different approaches for candidate prioritization and integration and analysis of these complex data.

Background Global analyses of human disease genes by computational methods have yielded important advances in the understanding of human diseases. Generally these studies have treated the group of disease genes uniformly, thus ignoring the type of disease-causing mutations (dominant or recessive). In this report we present a comprehensive study of the evolutionary history of autosomal disease genes separated by mode of inheritance. Results We examine differences in protein and coding sequence conservation between dominant and recessive human disease genes. Our analysis shows that disease genes affected by dominant mutations are more conserved than those affected by recessive mutations. This could be a consequence of the fact that recessive mutations remain hidden from selection while heterozygous. Furthermore, we employ functional annotation analysis and investigations into disease severity to support this hypothesis. Conclusion This study elucidates important differences between dominantly- and recessively-acting disease genes in terms of protein and DNA sequence conservation, paralogy and essentiality. We propose that the division of disease genes by mode of inheritance will enhance both understanding of the disease process and prediction of candidate disease genes in the future.