Search

Supplementary Figure S5 - PDF file 777K, Schematic representation of the scoring system applied to assess MET protein expression by immunohistochemistry

A recent systematic sequence analysis of well-annotated human protein coding genes or consensus coding sequences led to the identification of 189 genes displaying somatic mutations in breast and colorectal cancers. Based on their mutation prevalence, a subset of these genes was identified as cancer candidate (CAN) genes as they could be potentially involved in cancer. We evaluated the mutational profiles of 19 CAN genes in the highly aggressive tumors: glioblastoma, melanoma, and pancreatic carcinoma. Among other changes, we found novel somatic mutations in EPHA3, MLL3, TECTA, FBXW7, and OBSCN, affecting amino acids not previously found to be mutated in human cancers. Interestingly, we also found a germline nucleotide variant of OBSCN that was previously reported as a somatic mutation. Our results identify specific genetic lesions in glioblastoma, melanoma, and pancreatic cancers and indicate that CAN genes and their mutational profiles are tumor specific. Some of the mutated genes, such as the tyrosine kinase EPHA3, are clearly amenable to pharmacologic intervention and could represent novel therapeutic targets for these incurable cancers. We also speculate that similar to other oncogenes and tumor suppressor genes, mutations affecting OBSCN could be involved in cancer predisposition. [Cancer Res 2007;67(8):3545–50]

Supplementary Table 1 from Novel Somatic and Germline Mutations in Cancer Candidate Genes in Glioblastoma, Melanoma, and Pancreatic Carcinoma

Supplementary Tables 1-7 from Genome-Wide Significant Association Between a Sequence Variant at 15q15.2 and Lung Cancer Risk

Genome-wide association studies (GWAS) have identified 3 genomic regions, at 15q24–25.1, 5p15.33, and 6p21.33, which associate with the risk of lung cancer. Large meta-analyses of GWA data have failed to find additional associations of genome-wide significance. In this study, we sought to confirm 7 variants with suggestive association to lung cancer (P < 10−5) in a recently published meta-analysis. In a GWA dataset of 1,447 lung cancer cases and 36,256 controls in Iceland, 3 correlated variants on 15q15.2 (rs504417, rs11853991, and rs748404) showed a significant association with lung cancer, whereas rs4254535 on 2p14, rs1530057 on 3p24.1, rs6438347 on 3q13.31, and rs1926203 on 10q23.31 did not. The most significant variant, rs748404, was genotyped in an additional 1,299 lung cancer cases and 4,102 controls from the Netherlands, Spain, and the United States and the results combined with published GWAS data. In this analysis, the T allele of rs748404 reached genome-wide significance (OR = 1.15, P = 1.1 × 10−9). Another variant at the same locus, rs12050604, showed association with lung cancer (OR = 1.09, 3.6 × 10−6) and remained significant after adjustment for rs748404 and vice versa. rs748404 is located 140 kb centromeric of the TP53BP1 gene that has been implicated in lung cancer risk. Two fully correlated, nonsynonymous coding variants in TP53BP1, rs2602141 (Q1136K) and rs560191 (E353D) showed association with lung cancer in our sample set; however, this association did not remain significant after adjustment for rs748404. Our data show that 1 or more lung cancer risk variants of genome-wide significance and distinct from the coding variants in TP53BP1 are located at 15q15.2. Cancer Res; 71(4); 1356–61. ©2011 AACR.

Summary Patients with acute myeloid leukemia (AML) carrying high-risk genetic lesions or high residual disease levels after therapy are particularly exposed to the risk of relapse. Here, we identified the long non-coding RNA CDK6-AS1 able to cluster an AML subgroup with peculiar gene signatures linked to hematopoietic cell differentiation and mitochondrial dynamics. CDK6-AS1 silencing triggered hematopoietic commitment in healthy CD34+ cells, whereas in AML cells the pathological undifferentiated state was rescued. This latter phenomenon derived from RUNX1 transcriptional control, responsible for the stemness of hematopoietic precursors and for the block of differentiation in AML. By CDK6-AS1 silencing in vitro, AML mitochondrial mass decreased with augmented pharmacological sensitivity to mitochondria-targeting drugs. In vivo, the combination of tigecycline and cytarabine reduced leukemia progression in the AML-PDX model with high CDK6-AS1 levels, supporting the concept of a mitochondrial vulnerability. Together, these findings uncover CDK6-AS1 as crucial in myeloid differentiation and mitochondrial mass regulation., Graphical abstract, Highlights • CDK6-AS1 acts in concert with CDK6 • High CDK6-AS1 levels trigger RUNX1 early differentiation arrest in myeloid cells • CDK6-AS1 controls mitochondrial mass of AML blasts • CDK6-AS1 levels impact on mitochondrial-targeted agents sensitivity, Molecular biology; Cell biology; Cancer

Neuroblastoma (NB) is the most common extra-cranial malignancy in preschool children. To portray the genetic landscape of an overly aggressive NB leading to a rapid clinical progression of the disease, tumor DNA collected pre- and post-treatment has been analyzed. Array comparative genomic hybridization (aCGH), whole-exome sequencing (WES), and pharmacogenetics approaches, respectively, have identified relevant copy number alterations (CNAs), single nucleotide variants (SNVs), and polymorphisms (SNPs) that were then combined into an integrated analysis. Spontaneously formed 3D tumoroids obtained from the recurrent mass have also been characterized. The results prove the power of combining CNAs, SNVs, and SNPs analyses to assess clonal evolution during the disease progression by evidencing multiple clones at disease onset and dynamic genomic alterations during therapy administration. The proposed molecular and cytogenetic integrated analysis empowers the disease follow-up and the prediction of tumor recurrence.

Chromosome instability has a pivotal role among the hallmarks of cancer, but its transcriptional counterpart is rarely considered a relevant factor in cell destabilization. To examine transcription instability (TIN) we first devised a metric we named TIN-index and used it to evaluate TIN on a dataset containing more than 500 neuroblastoma samples. We found that metastatic tumors from high-risk (HR) patients are characterized by significantly different TIN-index values compared to low-intermediate risk (LIR) patients. Our results indicate that the TIN-index is a good predictor of neuroblastoma patient's outcome, and a related TIN-index gene signature (TIN-signature) is also able to predict the neuroblastoma patient's outcome with high confidence. Interestingly, we find that TIN-signature genes have a strong positional association with super-enhancers in neuroblastoma tumors. Finally, we show that TIN is linked to chromatin structural domains and interferes with their integrity in HR neuroblastoma patients. This novel approach to gene expression analysis broadens the perspective of genome instability investigations to include functional aspects.

Minimal residual disease (MRD) analysis has become a powerful indicator to refine therapy in acute lymphoblastic leukemia (ALL). Here, we present an MRD detection based on the next-generation sequencing of PTEN exon 7 mutations (NGS-PTEN) in 30 pediatric T-cell ALL patients. By comparing the NGS-PTEN results with current quantitative PCR of antigen receptor gene rearrangements (qPCR-Ig/TR), an overall concordance of 80% was found between the two methods. However, the NGS-PTEN qualified a lower number of high-risk patients than qPCR-Ig/TR. These findings suggest that NGS-PTEN is a promising tool that could potentially be used to support current MRD methodologies for T-ALL patients.

The members of the BCL-2 associated athanogene (BAG) family participate in the regulation of a variety of interrelated physiological processes, such as autophagy, apoptosis, and protein homeostasis. Under normal circumstances, the six BAG members described in mammals (BAG1-6) principally assist the 70 kDa heat-shock protein (HSP70) in protein folding; however, their role as oncogenes is becoming increasingly evident. Deregulation of the BAG multigene family has been associated with cell transformation, tumor recurrence, and drug resistance. In addition to BAG overexpression, BAG members are also involved in many oncogenic protein–protein interactions (PPIs). As such, either the inhibition of overloading BAGs or of specific BAG–client protein interactions could have paramount therapeutic value. In this review, we will examine the role of each BAG family member in different malignancies, focusing on their modular structure, which enables interaction with a variety of proteins to exert their pro-tumorigenic role. Lastly, critical remarks on the unmet needs for proposing effective BAG inhibitors will be pointed out.

Female fecundity is finely regulated by hormonal signaling, representing a potential target for endocrine-disrupting chemicals. Among the chemicals of most concern are the perfluoroalkyl substances (PFAS), widely used in consumer goods, that are associated with adverse effects on reproductive health. In this context, the endometrium clearly represents an important fertility determining factor. The aim of this study was to investigate PFAS interference on hormonal endometrial regulation. This study was performed within a screening protocol to evaluate reproductive health in high schools. We studied a cohort of 146 exposed females aged 18-21 from the Veneto region in Italy, one of the four areas worldwide heavily polluted with PFAS, and 1080 non-exposed controls. In experiments on Ishikawa cells included UV-Vis spectroscopy, microarray analysis and qPCR. We report a significant dysregulation of the genetic cascade leading to embryo implantation and endometrial receptivity. The most differentially-expressed genes upon PFOA coincubation were ITGB8, KLF5, WNT11, SULT1E1, ALPPL2 and G0S2 (all p 0.01). By qPCR, we confirmed an antagonistic effect of PFOA on all these genes, which was reversed at higher progesterone levels. Molecular interference of PFOA on progesterone was confirmed by an increase in the intensity of absorption spectra at 250 nm in a dose-dependent manner, but not in the presence of β-estradiol. Age at menarche (+164 days, p = 0.006) and the frequency of girls with irregular periods (29.5% vs 21.5%, p = 0.022) were significantly higher in the exposed group. Our results are indicative of endocrine-disrupting activity of PFAS on progesterone-mediated endometrial function.

The RNA-binding protein LIN28B regulates developmental timing and determines stem cell identity by suppressing the let-7 family of microRNAs. Postembryonic reactivation of LIN28B impairs cell commitment to differentiation, prompting their transformation. In this study, we assessed the extent to which ectopic lin28b expression modulates the physiological behavior of neural crest cells (NCC) and governs their transformation in the trunk region of developing embryos. We provide evidence that the overexpression of lin28b inhibits sympathoadrenal cell differentiation and accelerates NCC migration in two vertebrate models, Xenopus leavis and Danio rerio. Our results highlight the relevance of ITGA5 and ITGA6 in the LIN28B-dependent regulation of the invasive motility of tumor cells. The results also establish that LIN28B overexpression supports neuroblastoma onset and the metastatic potential of malignant cells through let-7a-dependent and let-7a-independent mechanisms.

// Vito Alessandro Lasorsa 1,2 , Daniela Formicola 1,2 , Piero Pignataro 1,2 , Flora Cimmino 1,2 , Francesco Maria Calabrese 3 , Jaume Mora 4 , Maria Rosaria Esposito 5 , Marcella Pantile 5 , Carlo Zanon 5 , Marilena De Mariano 6 , Luca Longo 6 , Michael D. Hogarty 7 , Carmen de Torres 4 , Gian Paolo Tonini 5 , Achille Iolascon 1,2 and Mario Capasso 1,2,8 1 University of Naples Federico II, Department of Molecular Medicine and Medical Biotechnology, Naples, Italy 2 CEINGE Biotecnolgie Avanzate, Naples, Italy 3 University of Bari, Department of Biology, Bari, Italy 4 Hospital Sant Joan de Deu, Developmental Tumor Biology Laboratory and Department of Oncology, Esplugues de Llobregat, Barcelona, Spain 5 Pediatric Research Institute (IRP), Fondazione Citta della Speranza, Neuroblastoma Laboratory, Padua, Italy 6 U.O.C. Bioterapie, IRCCS AOU San Martino-IST, National Cancer Research Institute, Genoa, Italy 7 Children’s Hospital of Philadelphia, Division of Oncology, Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States of America 8 IRCCS SDN, Istituto di Ricerca Diagnostica e Nucleare, Naples, Italy Correspondence: Mario Capasso, email: // Flora Cimmino, email: // Keywords : NGS, neuroblastoma, high risk, somatic mutation, cancer driver genes Received : December 22, 2015 Accepted : February 09, 2016 Published : March 18, 2016 Abstract The spectrum of somatic mutation of the most aggressive forms of neuroblastoma is not completely determined. We sought to identify potential cancer drivers in clinically aggressive neuroblastoma. Whole exome sequencing was conducted on 17 germline and tumor DNA samples from high-risk patients with adverse events within 36 months from diagnosis (HR-Event3) to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in additional 48 germline and tumor pairs (62.5% HR-Event3 and high-risk patients), 17 HR-Event3 tumors and 17 human-derived neuroblastoma cell lines. We revealed 22 significantly mutated genes, many of which implicated in cancer progression. Fifteen genes (68.2%) were highly expressed in neuroblastoma supporting their involvement in the disease. CHD9 , a cancer driver gene, was the most significantly altered (4.0% of cases) after ALK . Other genes ( PTK2 , NAV3 , NAV1 , FZD1 and ATRX ), expressed in neuroblastoma and involved in cell invasion and migration were mutated at frequency ranged from 4% to 2%. Focal adhesion and regulation of actin cytoskeleton pathways, were frequently disrupted (14.1% of cases) thus suggesting potential novel therapeutic strategies to prevent disease progression. Notably BARD1 , CHEK2 and AXIN2 were enriched in rare, potentially pathogenic, germline variants. In summary, whole exome and deep targeted sequencing identified novel cancer genes of clinically aggressive neuroblastoma. Our analyses show pathway-level implications of infrequently mutated genes in leading neuroblastoma progression.

// Sanja Aveic 1 , Marcella Pantile 1 , Anke Seydel 1 , Maria Rosaria Esposito 1 , Carlo Zanon 1 , Gary Li 2 , Gian Paolo Tonini 1 1 Neuroblastoma Laboratory, Pediatric Research Institute, Citta della Speranza, Padua, Italy 2 Ignyta Inc., San Diego, California, USA Correspondence to: Sanja Aveic, e-mail: s.aveic@irpcds.org Keywords: neuroblastoma, autophagy, ALK inhibitors; drug combination, entrectinib Received: July 21, 2015 Accepted: December 22, 2015 Published: December 28, 2015 ABSTRACT Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological, and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells. Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03–5 μM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67 , and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib, and demonstrated strong activation of autophagy in SH-SY5Y F1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate, and PARP protein cleavage in SH-SY5Y F1174L cells. In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest, and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB, and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib.

The somatic translocation t(8;21)(q22;q22)/RUNX1-RUNX1T1 is one of the most frequent rearrangements found in children with standard-risk acute myeloid leukemia (AML). Despite the favorable prognostic role of this aberration, we recently observed a higher than expected frequency of relapse. Here, we employed an integrated high-throughput approach aimed at identifying new biological features predicting relapse among 34 t(8;21)-rearranged patients. We found that the DNA methylation status of patients who suffered from relapse was peculiarly different from that of children maintaining complete remission. The epigenetic signature, made up of 337 differentially methylated regions, was then integrated with gene and protein expression profiles, leading to a network, where cell-to-cell adhesion and cell-motility pathways were found to be aberrantly activated in relapsed patients. We identified most of these factors as RUNX1-RUNX1T1 targets, with Ras Homolog Family Member (RHOB) overexpression being the core of this network. We documented how RHOB re-organized the actin cytoskeleton through its downstream ROCK-LIMK-COFILIN axis: this increases blast adhesion by stress fiber formation, and reduces mitochondrial apoptotic cell death after chemotherapy treatment. Altogether, our data show an epigenetic heterogeneity within t(8;21)-rearranged AML patients at diagnosis able to influence the program of the chimeric transcript, promoting blast re-emergence and progression to relapse.

Neuroblastoma (NB) is an embryonal tumor with low cure rate for patients classified as high-risk. This class of NB tumors shows a very complex genomic background and requires aggressive treatment strategies. In this work we evaluated the efficacy of the novel multi-kinase inhibitor TP-0903 in impairing NB cells' growth, proliferation and motility. In vitro studies were performed using cell lines with different molecular background, and in vivo studies were done using the zebrafish experimental model. Our results confirmed a strong cytotoxicity of TP-0903 already at the sub-micro molar concentrations. The observed cytotoxicity of TP-0903 was irreversible and the resulting apoptosis was caspase dependent. In addition, TP-0903 impaired colony formation and neurosphere creation. Depending on the molecular background of the selected NB cell lines, TP-0903 influenced either their capacity to migrate, to complete their cell cycle or both. Likewise, TP-0903 reduced NB cells intravasation in vitro and in vivo. Importantly, TP-0903 showed remarkable pharmacological efficacy not only as a mono-treatment, but also in combination with conventional chemotherapy drugs (ATRA, cisplatin, and VP16) in different types of NB cells. In conclusion, the multi-kinase activity of TP-0903 allowed the impairment of several biological processes required for expansion of NB cells, making them more vulnerable to the conventional chemotherapeutics. Altogether, our results support the eligibility of TP-0903 for further (pre)clinical assessments in NB.

AimsCurative surgery remains the primary form of treatment for locally advanced rectal cancer (LARC). Recent data support the use of preoperative chemoradiotherapy (pCRT) to improve the prognosis of LARC with a significant reduction of local relapse and an increase of overall survival. Unfortunately, only 20% of the patients with LARC present complete pathological response after pCRT, whereas in 20%–40%, the response is poor or absent.MethodsWe investigated the expression level of miR-194 in n=38 patients with LARC using our public microRNA (miRNA) expression dataset. miR-194 expression was further validated by real-time quantitative PCR (qRT-PCR) and in situ hybridisation (ISH). Protein–protein interaction network and pathway enrichment analysis were performed on miR-194 targets.Results and discussionUsing biopsy samples collected at diagnosis, mir-194 was significantly upregulated in patients responding to treatment (p value=0.016). The data was confirmed with qRT-PCR (p value=0.0587) and ISH (p value=0.026). Protein–protein interaction network and pathway enrichment analysis reveal a possible mechanism of susceptibility to pCRT involving Wnt pathway via its downstream mediator TRAF6. Finally, we interrogated the Comparative Toxicogenomics Database database in order to identify those chemical compounds able to mimic the biological effects of miR-194 as new possible therapeutic option in LARC treatment. The present study combining miRNA expression profiling with integrative computational biology identified miR-194 as predictive biomarker of response to pCRT. Using known and predicted drug mechanism of action, we then identified possible chemical compounds for further in vitro validation.

In acute myeloid leukemia (AML), the assessment of post-induction minimal residual disease (MRD) is largely utilized for choosing post-remission therapies aimed at maintaining complete remission (CR) and preventing relapse. This latter is still the major cause of treatment failure in pediatric AML, and even if several efforts have been spent to validate MRD as a prognostic marker, numerous studies demonstrated that MRD negativity cannot be considered a completely reliable surrogate biomarker predicting outcome, since it does not exclude a relapse. The current interpretation is that disease relapse is due to mechanisms leading to therapy resistance mainly depending on driver chimeric or oncogenic protein-coding genes, which are monitored during treatment, and does not consider that chemotherapy resistance may arise from other genetic markers, phenomenon linked to methylation and non-coding RNAs genomic pressure. We, thus, hypothesized that other markers need to be explored to re-interpret leukemia progression. We showed an overall hyper-expression of the lncRNA BALR2 in 132 de novo AML bone marrow samples collected at diagnosis and analyzed the gene expression profile (GEP) of 58 cases. By unsupervised clustering analysis, we produced important advances in identifying BALR2 as a robust novel molecular marker of a new subgroup of AML characterized by a high rate of resistance to induction therapy, independently from the genetic lesions detected at diagnosis and any other prognostic clinical and genetic features. We demonstrated in vitro that BALR2 has a direct role in controlling bi-directionally its own and of its neighbor gene CDK6 promoter activity. This latter finding of high CDK6 expression was shown to sustain its complex with RUNX1 in order to inhibit RUNX1 binding to its target promoters, thus preventing the process of hematopoietic differentiation progression. To support BALR2 as a new proto-oncogene involved in the control of the myeloid differentiation program, we ranked the genes across the expression profile obtaining a signature of 337 transcripts able to cluster CD34+ human stem cell precursors (HSCPs) separately from more mature CD14+ cells. These in silico findings were validated in vitro by showing that, after BALR2 depletion, CD34+ cells had a skewed myeloid differentiation. Furthermore, we found that AML differentiation toward mature myeloid cells with increased phagocytic capacity was obtained through BALR2 level reduction, and enhanced by combinatorial differentiation stimuli. Our findings attribute a distinct role to BALR2 in the block of myeloid stem cell differentiation occurring during leukemogenesis. At the same time, we interrogated GEP ontology, finding that enrichments of genes involved in mitochondrial synthesis pathways were significantly correlated to patients with highest BALR2 levels, and confirmed the same mitochondriogenesis profile in the immature CD34+ HSCPs. We moved to deconvolute this feature and demonstrated that BALR2, by controlling mitochondria gene balance, was directly controlling the mitochondrial mass, which dramatically decreased after BALR2 silencing, this supporting the hypothesis that BALR2 would maintain mitochondrial functions to confer AML resistance to cytotoxicity. Consistently with this line of reasoning, we inhibited mitochondria by tigecycline, demonstrating that its activity was dramatically strengthened in BALR2 depleted cells, when used either alone or in combination with cytosine-arabinoside (Ara-C). Concomitantly, tigecycline treatment in BALR2 silenced AML cells reduced mitochondria depolarization, and increased the number of differentiated M-CFU colonies formation, confirming that BALR2, together with CDK6, forms novel transcriptional networks to create a circuit able to impair myeloid differentiation and to lower chemo-sensitivity in AML. We speculate that a novel therapeutic window of mitochondrial targeting in defined AML subgroups, identified through assessment of BALR2 levels at diagnosis or persistent MRD levels, could be envisaged to optimize the outcome of childhood AML. Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Contains fulltext : 97981.pdf (Publisher’s version ) (Closed access) We tested 16 million SNPs, identified through whole-genome sequencing of 457 Icelanders, for association with gout and serum uric acid levels. Genotypes were imputed into 41,675 chip-genotyped Icelanders and their relatives, for effective sample sizes of 968 individuals with gout and 15,506 individuals for whom serum uric acid measurements were available. We identified a low-frequency missense variant (c.1580C>G) in ALDH16A1 associated with gout (OR = 3.12, P = 1.5 x 10(-16), at-risk allele frequency = 0.019) and serum uric acid levels (effect = 0.36 s.d., P = 4.5 x 10(-21)). We confirmed the association with gout by performing Sanger sequencing on 6,017 Icelanders. The association with gout was stronger in males relative to females. We also found a second variant on chromosome 1 associated with gout (OR = 1.92, P = 0.046, at-risk allele frequency = 0.986) and serum uric acid levels (effect = 0.48 s.d., P = 4.5 x 10(-16)). This variant is close to a common variant previously associated with serum uric acid levels. This work illustrates how whole-genome sequencing data allow the detection of associations between low-frequency variants and complex traits.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field. Three genome-wide association studies in Europe and the USA have reported eight urinary bladder cancer (UBC) susceptibility loci. Using extended case and control series and 1000 Genomes imputations of 5 340 737 single-nucleotide polymorphisms (SNPs), we searched for additional loci in the European GWAS. The discovery sample set consisted of 1631 cases and 3822 controls from the Netherlands and 603 cases and 37 781 controls from Iceland. For follow-up, we used 3790 cases and 7507 controls from 13 sample sets of European and Iranian ancestry. Based on the discovery analysis, we followed up signals in the urea transporter (UT) gene SLC14A. The strongest signal at this locus was represented by a SNP in intron 3, rs17674580, that reached genome-wide significance in the overall analysis of the discovery and follow-up groups: odds ratio = 1.17, P = 7.6 × 10(-11). SLC14A1 codes for UTs that define the Kidd blood group and are crucial for the maintenance of a constant urea concentration gradient in the renal medulla and, through this, the kidney's ability to concentrate urine. It is speculated that rs17674580, or other sequence variants in LD with it, indirectly modifies UBC risk by affecting urine production. If confirmed, this would support the 'urogenous contact hypothesis' that urine production and voiding frequency modify the risk of UBC. info:eu-repo/grantAgreement/EC/FP7/018827 info:eu-repo/grantAgreement/EC/FP7/218071 Radboud University Nijmegen Medical Centre (RUNMC) Prinses Beatrix Fonds VSB Fonds National Institute of Mental Health (NIH/NIMH) MH078075 Cancer Research UK Yorkshire Cancer Research European Union 513943 Compagnia di San Paolo-Human Genetics Foundation (HuGeF) Italian Association for Cancer Research, Italy Piedmont Region Progetti di Ricerca Sanitaria Finalizzata Flemish government Belgian province of Limburg Swedish Cancer Society Swedish Research Council Shiraz Institute for Cancer Research Shiraz, Iran ICR-87-502 Red Tematica de Investigacion Cooperativa en Cancer RD06/0020/1054

Contains fulltext : 95597.pdf (Publisher’s version ) (Closed access) Through complementary application of SNP genotyping, whole-genome sequencing and imputation in 38,384 Icelanders, we have discovered a previously unidentified sick sinus syndrome susceptibility gene, MYH6, encoding the alpha heavy chain subunit of cardiac myosin. A missense variant in this gene, c.2161C>T, results in the conceptual amino acid substitution p.Arg721Trp, has an allelic frequency of 0.38% in Icelanders and associates with sick sinus syndrome with an odds ratio = 12.53 and P = 1.5 x 10(2). We show that the lifetime risk of being diagnosed with sick sinus syndrome is around 6% for non-carriers of c.2161C>T but is approximately 50% for carriers of the c.2161C>T variant.

Previously, we reported germline DNA variants associated with risk of urinary bladder cancer (UBC) in Dutch and Icelandic subjects. Here we expanded the Icelandic sample set and tested the top 20 markers from the combined analysis in several European case-control sample sets, with a total of 4,739 cases and 45,549 controls. The T allele of rs798766 on 4p16.3 was found to associate with UBC (odds ratio = 1.24, P = 9.9 × 10 12). rs798766 is located in an intron of TACC3, 70 kb from FGFR3, which often harbors activating somatic mutations in low-grade, noninvasive UBC. Notably, rs798766[T] shows stronger association with low-grade and low-stage UBC than with more aggressive forms of the disease and is associated with higher risk of recurrence in low-grade stage Ta tumors. The frequency of rs798766[T] is higher in Ta tumors that carry an activating mutation in FGFR3 than in Ta tumors with wild-type FGFR3. Our results show a link between germline variants, somatic mutations of FGFR3 and risk of UBC. © 2010 Nature America, Inc. All rights reserved., Vlaamse regering 513943 Compagnia di San Paolo Yorkshire Cancer Research Adessium Foundation European Commission: LSHC-CT-2005 218071 Associazione Italiana per la Ricerca sul Cancro Prinses Beatrix Spierfonds, We thank the individuals who participated in the study and whose contribution made this work possible. We also thank the nurses at deCODE’s recruitment center and the personnel at the deCODE core facilities. We acknowledge the Icelandic Cancer Registry for assistance in the ascertainment of the Icelandic UBC cases. C.Z. and S.B. are funded by a FP7-MC-IAPP Grant agreement no. 218071 (CancerGene). Collection of samples and data in Iceland and The Netherlands was funded in part by the European Commission (POLYGENE: LSHC-CT-2005) and a research investment grant of the Radboud University Nijmegen Medical Centre. Control samples for the Dutch follow-up group were genotyped with generous support from the ‘Prinses Beatrix Fonds’, VSB Fonds, H. Kersten and M. Kersten (Kersten Foundation), The Netherlands ALS Foundation, J.R. van Dijk and the Adessium foundation. The controls from the Dutch Schizophrenia GWA study were genotyped with the support of the US National Institute of Mental Health (R.A.O.). The Leeds Bladder Cancer Study was funded by Cancer Research UK and Yorkshire Cancer Research. The Torino Bladder Cancer Case Control Study was supported by a grant to ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union Sixth Framework Program, Priority 5:‘Food Quality and Safety’ (Contract No. 513943), and by grants of the Compagnia di San Paolo, of the Italian Association for Cancer Research and of the Piedmont Region Progetti de Ricerca Sanitaria Finalizzata, Italy. The Belgian case-control study on bladder cancer risk was supported by a grant of the Flemish government, the government of the Belgian province of Limburg and the Limburg Cancer Fund., 1Department of Epidemiology, Biostatistics and Health Technology Assessment and 2Department of Urology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. 3Comprehensive Cancer Center East, Nijmegen, The Netherlands. 4deCODE Genetics, Reykjavik, Iceland. 5Section of Epidemiology and Biostatistics and 6Section of Experimental Oncology, Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds, UK. 7Christie Hospital National Health Service Foundation Trust, Manchester, UK. 8Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden. 9National Institute of Environmental Health, Budapest, Hungary. 10Environmental Health Centre, Cluj-Napoca, Romania. 11State Health Institute, Banska Bystrica, Slovakia. 12Institute for Scientific Interchange (ISI) Foundation, Torino, Italy. 13Department of Epidemiology and Public Health, Imperial College, London, UK. 14Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy. 15Unit of Cancer Epidemiology, University of Torino, Torino, Italy. 16Centre for Cancer Epidemiology and Prevention (CPO Piemonte), Torino, Italy. 17Department of Experimental and Applied Medicine, Section of Occupational Medicine and Industrial Hygiene, University of Brescia, Brescia, Italy. 18Unit of Genetic Epidemiology, Department of Public Health and Epidemiology, University of Birmingham, Birmingham, UK. 19Department of Complex Genetics, Cluster of Genetics and Cell Biology, Nutrition and Toxicology Research Institute, Maastricht University, Maastricht, The Netherlands. 20Leuven University Centre for Cancer Prevention (LUCK), Leuven, Belgium. 21Department of Medicine, University of Zaragoza, Zaragoza, Spain. 22Division of Urology, San Jorge Hospital, Huesca, Spain. 23Division of Urology, Hospital Clinico, Zaragoza, Spain. 24Department of Urology, University of Zaragoza School of Medicine, Zaragoza, Spain. 25Division of Surgical Pathology, San Jorge Hospital, Huesca, Spain. 26Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany. 27Department of Urology, Paul Gerhardt Foundation, Lutherstadt Wittenberg, Germany. 28Department of Medical Genetics, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht, The Netherlands. 29University of California Los Angeles Center for Neurobehavioral Genetics, Los Angeles, USA. 30Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht, The Netherlands. 31Department of Medical Oncology, 32Department of Urology and 33Department of Pathology, Landspitali–University Hospital, Reykjavik, Iceland. 34Department of Urology, Zonguldak Karaelmas University, Zonguldak, Turkey. 35Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden. 36Department of General Practice, Catholic University of Leuven, Leuven, Belgium. 37Department of General Practice, Maastricht University, Maastricht, The Netherlands. 38Division of Medical Oncology, University of Zaragoza, Zaragoza, Spain. 39Division of Molecular Genetic Epidemiology, German Cancer Research Centre, Heidelberg, Germany. 40Section of Clinical Cancer Epidemiology, Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden. 41Division of Clinical Cancer Epidemiology, Department of Oncology, Institute of Clinical Sciences, The Sahlgrenska Academy, Gothenburg, Sweden. 42Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, UK. 43Faculty of Medicine, University of Iceland, Reykjavik, Iceland. 44These authors contributed equally to this work. Correspondence should be addressed to L.A.K. (b.kiemeney@ebh.umcn.nl) or K.S. (kstefans@decode.is).

The occurrence of oncogenic alleles can display striking tissue specificity. For example KRAS mutations are very frequent in pancreatic cancers but relatively rare in melanomas. The opposite is true for BRAF mutations. Somatic mutations in the gene encoding for the phosphatidylinositol 3-kinase (PI3KCA) catalytic subunit, PIK3CA, occur at high frequency in many solid cancers. We have examined whether PI3K oncogenic mutations (exons 9 and 20) might exhibit gender and/or tissue specificity. By examining large cohorts of breast and colorectal cancers affecting both men and women we found that the pattern of PIK3CA mutations is distinctive. In colorectal cancers, PIK3CA (but not KRAS, APC, or TP53) mutations display a gender bias occurring at higher frequencies in women. We also found that male breast cancers display PIK3CA mutations at an overall frequency similar to that observed in female breast tumors. In male breast cancers, however, PIK3CA mutations are found mainly in exon 20. We conclude that PI3KCA mutations affecting exons 9 and 20 display gender- and tissue-specific patterns, thus suggesting that the different amino acid changes could exert distinct functional effects on the oncogenic properties of this enzyme. Furthermore, we propose that sexual dimorphisms and tissue specific factors might directly or indirectly influence the occurrence of PI3KCA cancer alleles. Hum Mutat 29(2), 284–288, 2008. © 2007 Wiley-Liss, Inc.

// Marco Agostini 1, 2, 3 , Klaus-Peter Janssen 4 , ll-Jin Kim 5 , Edoardo D’Angelo 1, 2 , Silvia Pizzini 6 , Andrea Zangrando 2, 7 , Carlo Zanon 8 , Chiara Pastrello 9 , Isacco Maretto 1 , Maura Digito 1 , Chiara Bedin 1, 3 , Igor Jurisica 9 , Flavio Rizzolio 10, 11 , Antonio Giordano 11 , Stefania Bortoluzzi 12 , Donato Nitti 1, * , Salvatore Pucciarelli 1, * 1 Department of Surgical, Oncological and Gastroenterological Sciences, Section of Surgery, University of Padova, Padua, Italy 2 Istituto di Ricerca Pediatrica-Citta della Speranza, Padua, Italy 3 The Methodist Hospital Research Institute, Houston, USA 4 Department of Surgery, UCSF, San Francisco, CA, USA 5 Department of Surgery, UCSF, San Francisco, CA 94143, California, CA 6 Department of Biology, University of Padua, Padua, Italy 7 Department of Woman and Child Health, University of Padua, Padua, Italy 8 Neuroblastoma Laboratory, Istituto di Ricerca Pediatrica-Citta della Speranza, Padua, Italy 9 Ontario Cancer Institute, the Campbell Family Institute for Cancer Research, and Techna Institute, University Health Network, Toronto, ON, Canada 10 Department of Translational Research, National Cancer Institute – CRO-IRCSS, Aviano, Italy 11 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA 12 Department of Molecular Medicine, University of Padua, Padua, Italy * These authors have contributed equally to this work Correspondence to: Marco Agostini, e-mail: m.agostini@unipd.it Keywords: rectal cancer, integrated approach, biological network, prognostic, predictive Received: April 28, 2015 Accepted: August 20, 2015 Published: September 02, 2015 ABSTRACT Introduction: Colorectal cancer is the third most common cancer in the world, a small fraction of which is represented by locally advanced rectal cancer (LARC). If not medically contraindicated, preoperative chemoradiotherapy, represent the standard of care for LARC patients. Unfortunately, patients shows a wide range of response rates in which approximately 20% has a complete pathological response, whereas in 20 to 40% the response is poor or absent. Results: The following specific gene signature, able to discriminate responders’ patients from non-responders, were founded: AKR1C3, CXCL11, CXCL10, IDO1, CXCL9, MMP12 and HLA-DRA. These genes are mainly involved in immune system pathways and interact with drugs traditionally used in the adjuvant treatment of rectal cancer. Discussion: The present study suggests that new ideas for therapy could be found not only limited to studying genes differentially expressed between the two groups of patients but deepening the mechanisms, associated to response, in which they are involved. Methods: Gene expression studies performed by: Agostini et al ., Rimkus et al . and Kim et al . have been merged through a meta-analysis of the raw data. Gene expression data-sets have been processed using A-MADMAN. Common differentially expressed gene (DEG) were identified through SAM analysis. To further characterize the identified DEG we deeply investigated its biological role using an integrative computational biology approach.

Preoperative chemoradiotherapy is widely used to improve local control of disease, sphincter preservation and to improve survival in patients with locally advanced rectal cancer. Patients enrolled in the present study underwent preoperative chemoradiotherapy, followed by surgical excision. Response to chemoradiotherapy was evaluated according to Mandard’s Tumor Regression Grade (TRG). TRG 3, 4 and 5 were considered as partial or no response while TRG 1 and 2 as complete response. From pretherapeutic biopsies of 84 locally advanced rectal carcinomas available for the analysis, only 42 of them showed 70% cancer cellularity at least. By determining gene expression profiles, responders and non-responders showed significantly different expression levels for 19 genes (P < 0.001). We fitted a logistic model selected with a stepwise procedure optimizing the Akaike Information Criterion (AIC) and then validated by means of leave one out cross validation (LOOCV, accuracy D 95%). Four genes were retained in the achieved model: ZNF160, XRCC3, HFM1 and ASXL2. Real time PCR confirmed that XRCC3 is overexpressed in responders group and HFM1 and ASXL2 showed a positive trend. In vitro test on colon cancer resistant/susceptible to chemoradioterapy cells, finally prove that XRCC3 deregulation is extensively involved in the chemoresistance mechanisms. Protein-protein interactions (PPI) analysis involving the predictive classifier revealed a network of 45 interacting nodes (proteins) with TRAF6 gene playing a keystone role in the network. The present study confirmed the possibility that gene expression profiling combined with integrative computational biology is useful to predict complete responses to preoperative chemoradiotherapy in patients with advanced rectal cancer.

Only a small proportion of cancers result from familial cancer syndromes with Mendelian inheritance. Nonfamilial, 'sporadic' cancers, which represent most cancer cases, also have a significant hereditary component1,2, but the genes involved have low penetrance and are extremely difficult to detect2,3. Therefore, mapping and cloning of quantitative trait loci (QTLs) for cancer susceptibility in animals could help identify homologous genes in humans. Several cancer-susceptibility QTLs have been mapped in mice and rats4,5, but none have been cloned so far. Here we report the positional cloning of the mouse gene Scc1 (Susceptibility to colon cancer 1)6 and the identification of Ptprj, encoding a receptor-type protein tyrosine phosphatase, as the underlying gene. In human colon, lung and breast cancers, we show frequent deletion of PTPRJ, allelic imbalance in loss of heterozygosity (LOH) and missense mutations. Our data suggest that PTPRJ is relevant to the development of several different human cancers.

Background Glioblastoma is a highly malignant brain tumor for which no cure is available. To identify new therapeutic targets, we performed a mutation analysis of kinase genes in glioblastoma. Methods Database mining and a literature search identified 76 kinases that have been found to be mutated at least twice in multiple cancer types before. Among those we selected 34 kinase genes for mutation analysis. We also included IDH1, IDH2, PTEN, TP53 and NRAS, genes that are known to be mutated at considerable frequencies in glioblastoma. In total, 174 exons of 39 genes in 113 glioblastoma samples from 109 patients and 16 high-grade glioma (HGG) cell lines were sequenced. Results Our mutation analysis led to the identification of 148 non-synonymous somatic mutations, of which 25 have not been reported before in glioblastoma. Somatic mutations were found in TP53, PTEN, IDH1, PIK3CA, EGFR, BRAF, EPHA3, NRAS, TGFBR2, FLT3 and RPS6KC1. Mapping the mutated genes into known signaling pathways revealed that the large majority of them plays a central role in the PI3K-AKT pathway. Conclusions The knowledge that at least 50% of glioblastoma tumors display mutational activation of the PI3K-AKT pathway should offer new opportunities for the rational development of therapeutic approaches for glioblastomas. However, due to the development of resistance mechanisms, kinase inhibition studies targeting the PI3K-AKT pathway for relapsing glioblastoma have mostly failed thus far. Other therapies should be investigated, targeting early events in gliomagenesis that involve both kinases and non-kinases. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-718) contains supplementary material, which is available to authorized users.

Item does not contain fulltext BACKGROUND: Genetic and epigenetic profiling of glioblastomas has provided a comprehensive list of altered cancer genes of which only O(6)-methylguanine-methyltransferase (MGMT) methylation is used thus far as a predictive marker in a clinical setting. We investigated the prognostic significance of genetic and epigenetic alterations in glioblastoma patients. METHODS: We screened 98 human glioblastoma samples for genetic and epigenetic alterations in 10 genes and chromosomal loci by PCR and multiplex ligation-dependent probe amplification (MLPA). We tested the association between these genetic and epigenetic alterations and glioblastoma patient survival. Subsequently, we developed a 2-gene survival predictor. RESULTS: Multivariate analyses revealed that mutations in isocitrate dehydrogenase 1 (IDH1), promoter methylation of MGMT, irradiation dosage, and Karnofsky Performance Status (KFS) were independent prognostic factors. A 2-gene predictor for glioblastoma survival was generated. Based on the genetic and epigenetic status of IDH1 and MGMT, glioblastoma patients were stratified into 3 clinically different genotypes: glioblastoma patients with IDH1mt/MGMTmet had the longest survival, followed by patients with IDH1mt/MGMTunmet or IDH1wt/MGMTmet, and patients with IDH1wt/MGMTunmet had the shortest survival. This 2-gene predictor was an independent prognostic factor and performed significantly better in predicting survival than either IDH1 mutations or MGMT methylation alone. The predictor was validated in 3 external datasets. DISCUSSION: The combination of IDH1 mutations and MGMT methylation outperforms either IDH1 mutations or MGMT methylation alone in predicting survival of glioblastoma patients. This information will help to increase our understanding of glioblastoma biology, and it may be helpful for baseline comparisons in future clinical trials.

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification. Significance: Amplification of the MET proto-oncogene is responsible for de novo and acquired resistance to anti-EGFR therapy in a subset of colorectal cancers. As multiple anti-MET therapeutic strategies are available, these findings offer immediate novel opportunities to design clinical studies. Cancer Discov; 3(6); 658–73. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 591

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field Genome-wide association studies (GWAS) have identified 3 genomic regions, at 15q24-25.1, 5p15.33, and 6p21.33, which associate with the risk of lung cancer. Large meta-analyses of GWA data have failed to find additional associations of genome-wide significance. In this study, we sought to confirm 7 variants with suggestive association to lung cancer (P < 10(-5)) in a recently published meta-analysis. In a GWA dataset of 1,447 lung cancer cases and 36,256 controls in Iceland, 3 correlated variants on 15q15.2 (rs504417, rs11853991, and rs748404) showed a significant association with lung cancer, whereas rs4254535 on 2p14, rs1530057 on 3p24.1, rs6438347 on 3q13.31, and rs1926203 on 10q23.31 did not. The most significant variant, rs748404, was genotyped in an additional 1,299 lung cancer cases and 4,102 controls from the Netherlands, Spain, and the United States and the results combined with published GWAS data. In this analysis, the T allele of rs748404 reached genome-wide significance (OR = 1.15, P = 1.1 × 10(-9)). Another variant at the same locus, rs12050604, showed association with lung cancer (OR = 1.09, 3.6 × 10(-6)) and remained significant after adjustment for rs748404 and vice versa. rs748404 is located 140 kb centromeric of the TP53BP1 gene that has been implicated in lung cancer risk. Two fully correlated, nonsynonymous coding variants in TP53BP1, rs2602141 (Q1136K) and rs560191 (E353D) showed association with lung cancer in our sample set; however, this association did not remain significant after adjustment for rs748404. Our data show that 1 or more lung cancer risk variants of genome-wide significance and distinct from the coding variants in TP53BP1 are located at 15q15.2. info:eu-repo/grantAgreement/EC/FP7/218071

Gain-of-function (GOF) mutations of Janus kinase 2 (JAK2) are frequently seen in myeloproliferative disorders (MPDs). Meanwhile, JAK3 activating substitutions have been found in a few megakaryocytic cell lines and in primary myeloid leukemia (AMKL). Here, we sought to discover novel leukemogenetic mutations in de novo acute myeloid leukemia of non-Down syndrome (N-DS) by DNA sequencing. A total of 191 normal Caucasian individuals were studied to define single nucleotide polymorphisms (SNPs) within the JH2 and JH6 domains. Although known activating substitutions were observed in rare cases of acute myeloid leukemia (AML) (V722I [2/134] or P132T [1/119]), all samples were wild-type (WT) for the oncogenic A572V (119/119). Interestingly, a novel homozygous mutation (P132A) was discovered in a patient with acute megakaryoblastic leukemia and in vivo studies demonstrated that its ectopic expression was oncogenic in a mouse xenotransplant model. This study defines a novel JAK3 mutation among patients with N-DS AML and demonstrates that normal individuals can also display germline JAK3 substitutions, previously proven to have oncogenic properties, in vitro and in vivo. The discovery of these substitutions in normal donors encourages future studies to define new risk factors among patients with MPDs.

Plexins are transmembrane high-affinity receptors for semaphorins, regulating cell guidance, motility, and invasion. Functional evidences implicate semaphorin signals in cancer progression and metastasis. Yet, it is largely unknown whether plexin genes are genetically altered in human tumors. We performed a comprehensive gene copy analysis and mutational profiling of all nine members of the plexin gene family (plexinome), in melanomas and pancreatic ductal adenocarcinomas (PDACs), which are characterized by high metastatic potential and poor prognosis. Gene copy analysis detected amplification of PLXNA4 in melanomas, whereas copy number losses of multiple plexin genes were seen in PDACs. Somatic mutations were detected in PLXNA4, PLXNB3, and PLXNC1; providing the first evidence that these plexins are mutated in human cancer. Functional assays in cellular models revealed that some of these missense mutations result in loss of plexin function. For instance, c.1613G>A, p.R538H mutation in the extracellular domain of PLXNB3 prevented binding of the ligand Sema5A. Moreover, although PLXNA4 signaling can inhibit tumor cell migration, the mutated c.5206C>T, p.H1736Y allele had lost this activity. Our study is the first systematic analysis of the "plexinome" in human tumors, and indicates that multiple mutated plexins may be involved in cancer progression

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field We report a prostate cancer genome-wide association follow-on study. We discovered four variants associated with susceptibility to prostate cancer in several European populations: rs10934853[A] (OR = 1.12, P = 2.9 x 10(-10)) on 3q21.3; two moderately correlated (r2 = 0.07) variants, rs16902094[G] (OR = 1.21, P = 6.2 x 10(-15)) and rs445114[T] (OR = 1.14, P = 4.7 x 10(-10)), on 8q24.21; and rs8102476[C] (OR = 1.12, P = 1.6 x 10(-11)) on 19q13.2. We also refined a previous association signal on 11q13 with the SNP rs11228565[A] (OR = 1.23, P = 6.7 x 10(-12)). In a multivariate analysis using 22 prostate cancer risk variants typed in the Icelandic population, we estimated that carriers in the top 1.3% of the risk distribution are at a 2.5 times greater risk of developing the disease than members of the general population. info:eu-repo/grantAgreement/EC/FP7/218071 European Union 202059 V Foundation US Department of Veterans Affairs Academy of Finland Sigrid Juselius Foundation Pirkanmaa Hospital District, Tampere University Hospital

The serine- threonine kinase AKT1 is a central player in the oncogenic pathway controlled by PI3K. Recently, a somatic mutation in AKT1 (E17K) has been detected in breast, colorectal, lung and ovarian cancers. The E17K change results in constitutive AKT1 activation and induces leukaemia in mice. We determined the occurrence of the E17K variant in a panel of 764 tumour samples. These included breast, lung, ovarian, colorectal and pancreatic carcinomas as well as melanomas and glioblastomas. Despite the fact that these tumours are known to bear alterations in genes involved in the PI3K signalling pathway, AKT1(E17K) was detected only in breast (16/273), colorectal (1/88) and lung(1/155) cancers. Within the neoplasms of breast origin, the AKT1(E17K) variant was mutually exclusive with respect to the PIK3CA(E454K) (or H1047R) alleles and was present only in ductal and lobular histotypes. Our results, showing that AKT1 mutations seem to occur in a tissue-specific fashion have basic and clinical implications. First, the activity of mutated AKT1 in oncogenic PI3K signalling could be strictly dependent on the cell and tissue milieu. Second, therapeutic efforts aimed at selective targeting the AKT1(E17K) variant could be effective mainly in specific cancer types

A recent systematic sequence analysis of well-annotated human protein coding genes or consensus coding sequences led to the identification of 189 genes displaying somatic mutations in breast and colorectal cancers. Based on their mutation prevalence, a subset of these genes was identified as cancer candidate (CAN) genes as they could be potentially involved in cancer. We evaluated the mutational profiles of 19 CAN genes in the highly aggressive tumors: glioblastoma, melanoma, and pancreatic carcinoma. Among other changes, we found novel somatic mutations in EPHA3, MLL3, TECTA, FBXW7, and OBSCN, affecting amino acids not previously found to be mutated in human cancers. Interestingly, we also found a germline nucleotide variant of OBSCN that was previously reported as a somatic mutation. Our results identify specific genetic lesions in glioblastoma, melanoma, and pancreatic cancers and indicate that CAN genes and their mutational profiles are tumor specific. Some of the mutated genes, such as the tyrosine kinase EPHA3, are clearly amenable to pharmacologic intervention and could represent novel therapeutic targets for these incurable cancers. We also speculate that similar to other oncogenes and tumor suppressor genes, mutations affecting OBSCN could be involved in cancer predisposition. [Cancer Res 2007;67(8):3545–50]

Monoclonal antibodies (mAbs) against the extracellular domain of the epidermal growth factor receptor (EGFR) have been introduced for the treatment of metastatic colorectal cancer (mCRC). We have reported recently that increased copy number of the EGFR can predict response to anti-EGFR mAbs and that patients might be selected for treatment based on EGFR copy number. Here, we show that mutations activating the RAS/RAF signaling pathway are also predictive and prognostic indicators in mCRC patients, being inversely correlated with response to anti-EGFR mAbs. In cellular models of CRCs, activation of the RAS signaling pathway by introduction of an activated K-RAS allele (Gly12Val) impairs the therapeutic effect of anti-EGFR mAbs. In cancer cells carrying constitutively active RAS, the pharmacologic inhibition of the mitogen-activated protein kinase (MAPK) signaling cascade improves anti-EGFR treatment based on mAbs. These results have implications for the identification of patients who are likely to respond to anti-EGFR treatment. They also provide the rationale for combination therapies, targeted simultaneously to the EGFR and RAS/RAF/MAPK signaling pathways in CRC patients. [Cancer Res 2007;67(6):2643–8]

11005 Background: The anti EGFR monoclonal antibodies cetuximab and panitumumab are used to treat metastatic colorectal cancer patients but their clinical efficacy is limited by the development of acquired resistance. We recently reported that secondary KRAS mutations are responsible for acquired resistance in approximately 50% of the patients who initially respond to cetuximab or panitumumab (Misale et al., Nature 2012; Diaz et al., Nature 2012). Here we studied the molecular bases of relapse in CRC patients who do not develop KRAS mutations during the course of anti-EGFR therapy. Methods: Next generation sequencing was applied to tumor biopsies to identify genetic alterations associated with relapse to cetuximab and panitumumab in mCRC patients. Detection and quantitation of genetic alterations in circulating tumor DNA was used to monitor the occurrence of KRAS mutations and MET amplification in blood samples. Results: Molecular analyses of tumor biopsies from patients who did not develop KRAS mutations during anti-EGFR therapy revealed high level of amplification of the MET proto-oncogene in 3/5 cases. Quantitative PCR, FISH and IHC analysis confirmed high level of MET amplification in the post-therapy samples but not in the matched pre-treatment tissues. We developed a PCR based assay to detect the presence of the MET amplicon in circulating, cell-free, DNA. We found that MET amplification could be detected in the blood as early as 3 months after initiation of anti EGFR therapy. To functionally evaluate the role of MET amplification on resistance to anti EGFR antibody therapies we exploited patient-derived CRC xenografts (‘xenopatients). We found that (2/2) xenopatients established from MET amplified tumors were completely refractory to cetuximab but showed sensitivity to the Met inhibitor crizotinib. Conclusions: Amplification of the MET proto-oncogene is responsible for acquired acquired resistance to anti-EGFR antibody therapy in a subset of CRCs. The emergence of MET amplification in circulating, cell-free, DNA may be used to select patients most likely to benefit from anti MET therapies.

A main limitation of therapies that selectively target kinase signaling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of EGFR, is effective in a subset of KRAS wild type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here, we show for the first time that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance but resistant cells remained sensitive to combinatorial inhibition of EGFR and MEK (Figure 1). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60 % (6/10) of the cases (Table 1). KRAS mutant alleles were detectable in the blood of cetuximab treated patients as early as 10 months prior to radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

4582 Background: Biliary tract carcinomas express EGFR and are potential candidates to EGFR target therapies. We recently described somatic mutations of EGFR that can enhance MAPK or Akt activation (Clin Cancer Res, 2006). Some of them are identical to those previously reported to confer sensitivity to some tyrosine kinase inhibitors (TKIs) like erlotinib or gefitinib in lung cancer. Here we report a molecular analysis of EGFR transducers potentially involved in TKI response. Methods: In 49 samples of biliary tract carcinoma we performed mutational analysis of exons from 18 to 21 of EGFR, exons 9 and 20 of phosphatidylinositol 3’-kinase (PI3K), exon 2 of K-Ras, exon 15 of B-Raf and exons from 5 to 8 of PTEN. Nuclear PTEN expression was analyzed by immunohistochemistry and the expression in cancer cells was compared to that of normal cholangiocites. Results: Mutations of EGFR have been detected in 7 out of 49 samples (14.3%). One of them was a new stop-codon mutation. Five hotspot mutations of PI3K (codon 545, 546, 1048 and 1059) were found in 4 cases (8.2%); 3 cases (6.1%) had single mutations in K-Ras and 4 (8.2%) had the V599E mutation in B-Raf. In some samples, mutations of multiple trasducers were present simultaneously. PI3K mutations were significantly more frequent in EGFR mutated samples compared to wild type (28% vs. 4.7% respectively, p No significant financial relationships to disclose.