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The clinical significance of low-frequent RAS pathway–mutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy. Mol Cancer Ther; 15(5); 1106–12. ©2016 AACR.

Estimation of the prediction quality of mutational status for treatment response. First column (AUC) indicates the Area Under the Curve as a prediction quality measure for all mutation groups. As expected, the AUC increases for lower mutation frequency cut-off. Then, a symmetric matrix for each mutation group with five columns, one column for each cut-off, reports the p-values for all curves comparisons. Highlighted in black the p-values that indicate correlative comparison. Highlighted in red are the p-values close to significant levels that correspond to the comparison between 1% and 1.5% cut-off value. Differences between cut-off of 1% and 1.5% are bigger than differences between the other correlative cut-off comparisons. P-values are close to significance level with a confidence of 90%.

PDF file - 198K

Perspective on This Article from Characterization of New Founder Alu-Mediated Rearrangements in MSH2 Gene Associated with a Lynch Syndrome Phenotype

PDF File - 100K, Pyrosequencing primers used to quantify the methylation status of selected CG sites.

It has been reported that large genomic deletions in the MLH1 and MSH2 genes are a frequent cause of Lynch syndrome in certain populations. Here, a cohort has been screened and two new founder rearrangements have been found in the MSH2 gene. These mutations have been characterized by break point determination, haplotype analysis, and genotype–phenotype correlation. Mutations have been identified in the MLH1, MSH2, and MSH6 genes in 303 subjects from 160 suspected Lynch syndrome unrelated families. All subjects were tested using heteroduplex analysis by capillary array electrophoresis. Multiplex ligation-dependent probe amplification was used to detect rearrangements in mutation-negative index patients and confirmed by reverse transcriptase PCR. The break point of the deletions was further characterized by the array comparative genomic hybridization method. Immunohistochemical staining and microsatellite instability were studied in tumor samples. Hereditary nonpolyposis colorectal cancer–related phenotypes were evaluated. More than 16% (24 of 160) of the families had pathogenic mutations (8 MLH1, 15 MSH2, and 1 MSH6). Twelve of these families (50%) are carriers of a novel mutation. Seven of the 15 positive MSH2 families (47%) are carriers of a rearrangement. The exon 7 deletion and exon 4 to 8 deletion of MSH2 are new founder mutations. The segregation of a common haplotype, a similar phenotype, and anticipation effects were observed in these families. These findings will greatly simplify the diagnosis, counseling, and clinical care in suspected Lynch syndrome families and not just in specific geographic areas, so wide distribution may be explained by migration patterns. Cancer Prev Res; 4(10); 1546–55. ©2011 AACR.

PDF File - 916K, Representative pyrograms in a healthy colonic mucosa sample for the markers analyzed.

DNA methylation biomarkers for noninvasive diagnosis of colorectal cancer (CRC) and precursor lesions have been extensively studied. Different panels have been reported attempting to improve current protocols in clinical practice, although no definite biomarkers have been established.In the present study, we have examined patient biopsies starting from a comprehensive analysis of DNA methylation differences between paired normal and tumor samples in known cancer-related genes aiming to select the best performing candidates informative for CRC diagnosis in stool samples.Five selected markers were considered for subsequent analyses in independent biologic cohorts and in silico data sets. Among the five selected genes, three of them (AGTR1, WNT2 and SLIT2) were validated in stool DNA of affected patients with a detection sensitivity of 78% [95% confidence interval (CI), 56%–89%]. As a reference, DNA methylation of VIM and SEPT9 was evaluated in a subset of stool samples yielding sensitivities of 55% and 20%, respectively. Moreover, our panel may complement histologic and endoscopic diagnosis of inflammatory bowel disease (IBD)-associated neoplasia, as it was also efficient detecting aberrant DNA methylation in non-neoplastic tissue samples from affected patients.This novel panel of specific methylation markers can be useful for early diagnosis of CRC using stool DNA and may help in the follow-up of high-risk patients with IBD. Cancer Prev Res; 6(7); 656–65. ©2013 AACR.

PDF file - 574K, Microarray pre-processing.

Supplementary Table 1 from Genetic Inactivation of ADAMTS15 Metalloprotease in Human Colorectal Cancer

Supplementary Figure 1 from Genetic Inactivation of ADAMTS15 Metalloprotease in Human Colorectal Cancer

Supplementary Table 2 from Genetic Inactivation of ADAMTS15 Metalloprotease in Human Colorectal Cancer

The variants c.306+5G>A and c.1865T>A (p.Leu622His) of the DNA repair gene MLH1 occur frequently in Spanish Lynch syndrome families. To understand their ancestral history and clinical effect, we performed functional assays and a penetrance analysis and studied their genetic and geographic origins. Detailed family histories were taken from 29 carrier families. Functional analysis included in silico and in vitro assays at the RNA and protein levels. Penetrance was calculated using a modified segregation analysis adjusted for ascertainment. Founder effects were evaluated by haplotype analysis. The identified MLH1 c.306+5G>A and c.1865T>A (p.Leu622His) variants are absent in control populations and segregate with the disease. Tumors from carriers of both variants show microsatellite instability and loss of expression of the MLH1 protein. The c.306+5G>A variant is a pathogenic mutation affecting mRNA processing. The c.1865T>A (p.Leu622His) variant causes defects in MLH1 expression and stability. For both mutations, the estimated penetrance is moderate (age-cumulative colorectal cancer risk by age 70 of 20.1% and 14.1% for c.306+5G>A and of 6.8% and 7.3% for c.1865T>A in men and women carriers, respectively) in the lower range of variability estimated for other pathogenic Spanish MLH1 mutations. A common haplotype was associated with each of the identified mutations, confirming their founder origin. The ages of c.306+5G>A and c.1865T>A mutations were estimated to be 53 to 122 and 12 to 22 generations, respectively. Our results confirm the pathogenicity, moderate penetrance, and founder origin of the MLH1 c.306+5G>A and c.1865T>A mutations. These findings have important implications for genetic counseling and molecular diagnosis of Lynch syndrome. Cancer Res; 70(19); 7379–91. ©2010 AACR.

Supplementary Figure 2 from Genetic Inactivation of ADAMTS15 Metalloprotease in Human Colorectal Cancer

Supplementary Table 1 from MLH1 Founder Mutations with Moderate Penetrance in Spanish Lynch Syndrome Families

Supplementary Figure 1 from MLH1 Founder Mutations with Moderate Penetrance in Spanish Lynch Syndrome Families

Supplementary Table 2 from MLH1 Founder Mutations with Moderate Penetrance in Spanish Lynch Syndrome Families

Supplementary Methods from MLH1 Founder Mutations with Moderate Penetrance in Spanish Lynch Syndrome Families

Purpose: The majority of genomic alterations causing intratumoral heterogeneity (ITH) in colorectal cancer are thought to arise during early stages of carcinogenesis as a burst but only after truncal mutations in APC have expanded a single founder clone. We have investigated if the initial source of ITH is consequent to multiple independent lineages derived from different crypts harboring distinct truncal APC and driver KRAS mutations, thus challenging the prevailing monoclonal monocryptal model. Experimental Design: High-depth next-generation sequencing and SNP arrays were performed in whole-lesion extracts of 37 familial adenomatous polyposis colorectal adenomas. Also, ultrasensitive genotyping of hotspot mutations of APC and KRAS was performed using nanofluidic PCRs in matched bulk biopsies (n = 59) and crypts (n = 591) from 18 adenomas and seven carcinomas and adjacent normal tissues. Results: Multiple co-occurring truncal APC and driver KRAS alterations were uncovered in whole-lesion extracts from adenomas and subsequently confirmed to belong to multiple clones. Ultrasensitive genotyping of bulk biopsies and crypts revealed novel undetected APC mutations that were prominent among carcinomas, whereas abundant wild-type APC crypts were detected in adenomas. KRAS mutational heterogeneity within crypts was evident in both adenomas and carcinomas with a higher degree of concordance between biopsy and crypt genotyping in carcinomas. Nonrandom heterogeneity among crypts was also observed. Conclusions: The striking degree of nonrandom intercrypt heterogeneity in truncal and driver gene mutations observed in adenomas and carcinomas is consistent with a polycryptal model derived from multiple independent initiation linages as the source of early ITH in colorectal carcinogenesis. Clin Cancer Res; 23(19); 5936–47. ©2017 AACR.

Increased secretion of EGFR ligands amphiregulin and TGFα by limited KRAS-mutant clones is suggested as a paracrine resistance mechanism to anti-EGFR antibodies in colorectal cancer models. These findings are biologically sound but need to be replicated, including in the clinical setting, to foresee whether they are clinically relevant and therapeutically exploitable. Clin Cancer Res; 20(24); 6227–9. ©2014 AACR.

DNA methylation biomarkers for noninvasive diagnosis of colorectal cancer (CRC) and precursor lesions have been extensively studied. Different panels have been reported attempting to improve current protocols in clinical practice, although no definite biomarkers have been established. In the present study, we have examined patient biopsies starting from a comprehensive analysis of DNA methylation differences between paired normal and tumor samples in known cancer-related genes aiming to select the best performing candidates informative for CRC diagnosis in stool samples. Five selected markers were considered for subsequent analyses in independent biologic cohorts and in silico data sets. Among the five selected genes, three of them (AGTR1, WNT2 and SLIT2) were validated in stool DNA of affected patients with a detection sensitivity of 78% [95% confidence interval (CI), 56%–89%]. As a reference, DNA methylation of VIM and SEPT9 was evaluated in a subset of stool samples yielding sensitivities of 55% and 20%, respectively. Moreover, our panel may complement histologic and endoscopic diagnosis of inflammatory bowel disease (IBD)-associated neoplasia, as it was also efficient detecting aberrant DNA methylation in non-neoplastic tissue samples from affected patients. This novel panel of specific methylation markers can be useful for early diagnosis of CRC using stool DNA and may help in the follow-up of high-risk patients with IBD. Cancer Prev Res; 6(7); 656–65. ©2013 AACR.

Purpose: Epithelial ovarian cancer (EOC) is the fifth leading cause of death in women diagnosed with gynecologic malignancies. The low survival rate is because of its advanced-stage diagnosis and either intrinsic or acquired resistance to standard platinum-based chemotherapy. So, the development of effective innovative therapeutic strategies to overcome cisplatin resistance remains a high priority. Experimental Design: To investigate new treatments in in vivo models reproducing EOCs tumor growth, we generated a preclinical model of ovarian cancer after orthotopic implantation of a primary serous tumor in nude mice. Further, matched model of acquired cisplatin-resistant tumor version was successfully derived in mice. Effectiveness of lurbinectedin (PM01183) treatment, a novel marine-derived DNA minor groove covalent binder, was assessed in both preclinical models as a single and a combined-cisplatin agent. Results: Orthotopically perpetuated tumor grafts mimic the histopathological characteristics of primary patients' tumors and they also recapitulate in mice characteristic features of tumor response to cisplatin treatments. We showed that single lurbinectedin or cisplatin-combined therapies were effective in treating cisplatin-sensitive and cisplatin-resistant preclinical ovarian tumor models. Furthermore, the strongest in vivo synergistic effect was observed for combined treatments, especially in cisplatin-resistant tumors. Lurbinectedin tumor growth inhibition was associated with reduced proliferation, increased rate of aberrant mitosis, and subsequent induced apoptosis. Conclusions: Taken together, preclinical orthotopic ovarian tumor grafts are useful tools for drug development, providing hard evidence that lurbinectedin might be a useful therapy in the treatment of EOC by overcoming cisplatin resistance. Clin Cancer Res; 18(19); 5399–411. ©2012 AACR.

The family of receptor tyrosine kinases EPH and their Ephrin ligands regulate cell proliferation, migration, and attachment. An important role in colorectal carcinogenesis is emerging for some of its members. In this study, we evaluate the role of EPHB4 in colorectal cancer and its value as a prognostic marker. EPHB4 levels were assessed by immunohistochemical staining of tissue microarrays of 137 colorectal tumors and aberrant hypermethylation of the EPHB4 promoter was investigated using methylation-specific PCR. We found that EPHB4 expression is frequently reduced or lost in colorectal tumors. Patients with low EPHB4 tumor levels had significantly shorter survival than patients in the high EPHB4 group (median survival, 1.8 and >9 years, respectively; P < 0.01, log-rank test), and this finding was validated using an independent set of 125 tumor samples. In addition, we show that EPHB4 promoter hypermethylation is a common mechanism of EPHB4 inactivation. Moreover, reintroduction of EPHB4 resulted in a significant reduction in the clonogenic potential of EPHB4-deficient cells, whereas abrogation of EPHB4 in cells with high levels of this receptor lead to a significant increase in clonogenicity. In summary, we identified EPHB4 as a useful prognostic marker for colorectal cancer. In addition, we provide mechanistic evidence showing that promoter methylation regulates EPHB4 transcription and functional evidence that EPHB4 can regulate the long-term clonogenic potential of colorectal tumor cells, revealing EPHB4 as a potential new tumor suppressor gene in colorectal cancer. (Cancer Res 2006; 66(18): 8943-8)

Objectives: We have undertaken a comprehensive study of common polymorphisms in genes of DNA repair, exploring both the risk of developing colorectal cancer and the prognosis of patients. Methods: Subjects from a case-control study (377 cases and 329 controls) designed to assess gene-environment interactions were genotyped by use of an oligonucleotide microarray and the arrayed primer extension technique. Twenty-eight single nucleotide polymorphisms in 15 DNA repair genes were included. The candidate genes belong to different DNA repair pathways: base excision repair (OGG1, LIG3, APEX, POLB, XRCC1, PCNA, and MUTYH), nucleotide excision repair (ERCC1, ERCC2, ERCC4, and ERCC5), double-strand breaks repair (XRCC2, XRCC3, and XRCC9), and reversion repair (MGMT) genes. Results: Polymorphism OGG1 S326C was associated with an increased risk of colorectal cancer [odds ratio (OR), 2.3; 95% confidence interval (95% CI), 1.1-5.0], the risk being higher in younger individuals. A haplotype of ERCC1 was associated with increased risk (OR, 2.3; 95% CI, 1.0-5.3). POLB P242R was also associated with decreased risk (OR, 0.23; 95% CI, 0.05-0.99), although the number of variant allele carriers was low. In the univariate analysis, adjusted for age, sex, and Dukes' stage, three polymorphisms were significantly associated with better prognosis: XRCC1 R399Q [hazard ratio (HR), 0.38; 95% CI, 0.17-0.85], XRCC3 T141M (HR, 0.66; 95% CI, 0.45-0.97), and MGMT L84F (HR, 0.14; 95% CI, 0.02-0.99). ERCC1 19007T>C was associated with worse prognosis (HR, 1.51; 95% CI, 1.01-2.27). In a multivariate analysis, only XRCC1 R399Q and ERCC1 19007T>C remained significant. These associations were stronger among patients receiving adjuvant chemotherapy. Conclusions: Although the overall effect of DNA repair genes in colorectal cancer etiology seems limited, their influence in the response to chemotherapy and prognosis may be more relevant. This knowledge may help to clarify the utility of specific adjuvant treatments according to the individual genetic background.

Among K-ras mutations, codon 12 mutations have been identified as those conferring a more aggressive phenotype. This aggressiveness is primarily associated with slow proliferation but greatly increased resistance to apoptosis. Using transfected NIH3T3 fibroblasts with a mutated K-ras minigene either at codon 12 (K12) or at codon 13 (K13), and taking advantage of [1,2-13C2]glucose tracer labeling, we show that codon 12 mutant K-ras (K12)-transformed cells exhibit greatly increased glycolysis with only a slight increase in activity along pathways that produce nucleic acid and lipid synthesis precursors in the oxidative branch of the pentose phosphate pathway and via pyruvate dehydrogenase flux. K13 mutants display a modest increase in anaerobic glycolysis associated with a large increase in oxidative pentose phosphate pathway activity and pyruvate dehydrogenase flux. The distinctive differences in metabolic profiles of K12 and K13 codon mutated cells indicate that a strong correlation exists between the flow of glucose carbons towards either increased anaerobic glycolysis, and resistance to apoptosis (K12), or increased macromolecule synthesis, rapid proliferation, and increased sensitivity to apoptosis.

Purpose: To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage. Experimental Design: Tumor pieces (2 × 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21Cip1, p27Kip1, p16INK4a, pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression. Results: Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27kip1 overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete α-fetoprotein, and has TP53 missense mutations in codons 192 and 242. Conclusions: The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.

Clonality in carcinomas is thought to arise from the accumulation of mutations and changes in selective pressure during carcinogenesis and further stages of tumor development. However, the identification of clonality through different molecular and systems biology tools has challenged the current understanding of carcinogenesis and progression. Familial Adenomatous Polyposis (FAP) is a genetic condition secondary to germline mutations in APC and characterized by the development of hundreds of polyps in the colorectum. Therefore, FAP is an ideal model to study the presence of clonality during early stages of colorectal carcinogenesis in humans. Second hits in APC are a valuable and intuitive method to establish the presence of clonality in FAP, as additional somatic APC mutations will reflect the presence of additional clones. Here, we present a targeted high-depth sequencing analysis of APC and KRAS in conjunction with high density SNP array data in 37 colorectal adenomas and matched adjacent uninvolved mucosa samples. Ampliseq sequencing was performed using the next-generation sequencing platform IT Personal Genome Machine (PGM; Life Technologies) using the Ion PGM 200 Sequencing Kit on an Ion 318 Chip Kit (Life Technologies, average of 70% bases at 500x). Ion Torrent Variant Caller v4.2 was run in the somatic low stringency proton mode to detect variants in APC and KRAS against hg19. Then, normal variants were subtracted from matched adenoma variants to create a list of somatic candidates for each adenoma and normal pair. SNP array analysis was performed using the HumanOmniExpressExome-8 v1.2 BeadChip (Illumina, San Diego, CA). To validate the presence of double somatic hits in APC we performed a colony genotyping assay from one adenoma sample. First, we identified APC somatic mutations or loss of heterogeneity (LOH) in 5q (second hits) in 81% (30/37) and KRAS somatic mutations in 35% (16/37) of adenomas analyzed. Six adenomas (16%) presented double somatic APC mutations with different allelic frequencies with three of them showing a significant difference in allelic frequencies among the somatic APC hits (>30%); in addition, one adenoma presented two KRAS mutations. The APC clonality was validated in one adenoma using colony analysis. The region that contains the two somatic mutations (c.4348C>T and c.4267_4280del) and the germline mutation (c.2894delA) was amplified by Long-Range PCR and cloned into the pGEM-T plasmid. DNA from one hundred colonies was extracted and genotyped using Taqman assay probes. All samples were genotyped in triplicate and positive and negative controls for all mutations were included in every single plate. A total of 25% of the colonies harbored a wild-type allele, 36% the known germline APC mutation, 15% the APC somatic c.4348C>T mutation and 24% the APC somatic c.4267_4280del mutation. In conclusion, our analysis showed that there are multiple mutated APC subclones driving initiation of colorectal carcinogenesis, thus supporting experimentally a finding of multi-clonality in premalignant colonic lesions. These findings support a model of colorectal carcinogenesis that is based on polycryptal polyclonal adenomas and calls for revisiting the “Big-Bang” model based on the presence of multiple initiator clones. Further analyses aimed to understand the functional impact and the hierarchical relations among the different identified subclones are warranted. Citation Format: Ester Borras, Kyle Chang, Anthony San Lucas, Gita Masand, Axel Delgado Amador, Jerry Fowler, Gareth E. Davies, Erik A. Ehli, Y. Nancy You, Patrick M. Lynch, Ernest T. Hawk, Gabriel Capella, Paul A. Scheet, Eduardo Vilar. High-depth sequencing reveals the presence of multi-clonality in colorectal premalignancy. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A02.

In the article by Sanz-Pamplona and colleagues ([1][1]), functionally relevant mutations in AMER1 were identified in approximately 10% of stage II colorectal tumors, reinforcing its role as a driver gene in colorectal cancer, as occurs with APC , KRAS , or TP53 . AMER1 codifies a membrane protein

Clonality of tumors arises from accumulation of mutations and changes in selective pressure during carcinogenesis and further stages of tumor development. The identification of clonality through different molecular and systems biology tools has challenged the current understanding of the carcinogenesis and progression process, as well as drug resistance, and clinical outcomes. With the advent of next-generation sequencing, numerous bioinformatics methods are being developed to study clonality of tumors at a genomic scale. However, these studies have mostly been focusing on late stage carcinomas and little is known about the clonality in pre-malignant lesions. Here we present an analysis of clonality on colonic polyps from patients diagnosed with Familial Adenomatous Polyposis (FAP). FAP is an autosomal dominant disease secondary to germline mutations in APC and characterized by the development of hundreds to thousands of polyps in the gastrointestinal tract at a young age. We performed whole-exome sequencing on 25 polyps and matched blood samples using an Illumina HiSeq2000 instrument. We obtained somatic mutations calls using MuTect and copy number variations from VarScan2. Then, we used two systems biology tools (ABSOLUTE and EXPANDS) to assess the levels of tumor purity and ploidy, and to determine the presence of clonal and subclonal populations. We found that EXPANDS predicted an average tumor purity of polyps (54%) lower than that of 20 TCGA stage I colorectal carcinomas (77%); and an average number of clones in polyps (2.72) lower than that of TCGA stage I colorectal carcinomas (7.04). ABSOLUTE predicted an average tumor purity in polyps (40%) lower than that in TCGA stage I colorectal carcinomas (68%) as well, although there were differences between the purity estimates between both tools. Second hits in APC are a valuable and intuitive method to establish the presence of clonality in FAP polyps as any additional APC mutation has to be acquired in the context of progression of carcinogenesis as a second hit. We identified 12 APC somatic mutations from 12 polyps and 2 KRAS somatic mutations from 2 polyps that were predicted to be in clonal populations by EXPANDS. Furthermore, we performed targeted high-depth IonTorrent Ampliseq in a second set of 37 polyps (average of 70% bases at 500x) and matched normal mucosa from 14 FAP patients. We identified evidence of multiple somatic APC mutations at different allelic frequencies in 7 polyp samples with 3 of them showing significant differences in allelic frequencies between different somatic APC hits (>30%); in addition, 2 KRAS mutations were observed in a polyp. In conclusion, our analysis show that there are multiple mutated APC subclones driving progression and evidence of clonality in pre-malignant colonic lesions. Further analysis will be conducted to understand the functional impact and the relation among the different identified subclones. Citation Format: Kyle Chang, Ester Borras, Anthony San Lucas, Jerry Fowler, Ernest T. Hawk, Gabriel Capella, Paul Scheet, Eduardo Vilar. Genomic analysis reveals evidence of clonality in premalignant colonic polyps. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2015-1087

Background: Since fibroblasts are the predominant stromal cell type in colorectal carcinomas (CRC), it is reasonable to assume that fibroblast-tumor cell crosstalk play an important role in the production and maintenance of the malignant phenotype. Thus, it is very relevant to abort the supply of such factors responsible for this crosstalk in order to hamper tumor growth. In this work, we studied the crosstalk between Normal Colonic Fibroblasts (NCF) and tumor cells, its relation with de novo chemoresistance and fibroblast activation, providing hints for the treatment of CRC. Methods: After 5 days of Transwell coculture between fibroblasts and tumor cells we obtained mRNA of each cell type to obtain differentially expressed genes compared to monocultures (Affymetrix GeneChip Human Gene 1.0 ST Array). From genes with fold change>2, we selected those codifying for secreted or membrane proteins in both tumor cells and fibroblast. Protein-protein interaction (PPI) analysis was carried out by means of BIANA software searching for fibroblast secreted proteins with receptors in tumor cells plasma membrane and viceversa. We also studied the main pathways involved in such crosstalk in control conditions or pharmacologically disrupted. In addition we performed functional assays (proliferation, migration and citotoxicity against Oxaliplatin and 5FU). We also performed a cytokine array (Raybiotech) to characterise products mediating such crosstalk. Results: After integrating our transcriptomic/PPI data with information obtained by means of GSEA we stated our working hypothesis as: cytokines released by NCF stimulate tumor cells to secrete IL1β and TGFβ1. These two soluble factors are responsible for the NCF activation. Activated fibroblasts in turn began to produce large amounts of IL1β/TGFβ1 targets, mainly chemokines, cytokines, EGFR-ligands and PAI-1, signalling basically through IL6R, CCR2 (JAK-STAT pathway), CCR6, EGFR and CXCR3 (PI3K-AKT pathway), pathways involved in chemoresistance in CRC. Hampering the crosstalk by means of a blocking IL1β antibody and TGFBR1 inhibitor sensitizes tumor cells to Oxaliplatin and 5FU and induces a change in the cytokines mediating the crosstalk. Cell signalling reveals an activation of non-canonical TGFβ1 evidenced by TAK1 phosphorilation and NFkβ activation. Conclusions: Disrupting the crosstalk between fibroblasts and tumor cells with TGFBR1 inhibitor and a blocking IL1β antibody induces a change in the cytokine repertoire of both cells that activates NFkβ pathway in fibroblasts and a sustained activation of AKT in tumor cells. We can avoid such activation inhibiting non-canonical TGFβ pathway with a TAK1 inhibitor. The combined use of canonical and non-canonical TGFβ inhibitors could an interesting approach to overcome microenvironment-mediated drug resistance. Citation Format: NATALIA GUILLEN, REBECA SANZ-PAMPLONA, SAMUEL GONÇALVES, RAMON SALAZAR, GABRIEL CAPELLA, DAVID G. MOLLEVI. Hampering the crosstalk between fibroblasts and tumor cells reveals the need for blocking both canonical and non-canonical TGFβ pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2396. doi:10.1158/1538-7445.AM2015-2396

Adenomatous polyps are the premalignant lesions that precede the development of colorectal cancers (CRC). The normal mucosa-adenoma-carcinoma sequence was originally described more than 2 decades ago; however, no comprehensive annotation of the genomic landscape of polyps has been reported to date. Familial Adenomatous Polyposis (FAP) is a mendelian disorder caused by germline mutations in the APC gene, has an autosomal dominant pattern of inheritance and is characterized by the development of adenomas in the entire intestine, thus conferring patients with a life-time risk for CRC development of 100%. Patients diagnosed with hereditary polyposis syndromes such as FAP represents the perfect model to acquire tissue samples from polyps, at-risk normal mucosa and germline allowing this type of high-throughput studies. We have performed whole exome sequencing in paired polyps and germline blood samples of 12 individuals with FAP (25 polyps and 12 blood samples) using the Illumina HiSeq 2000 platform. Using standard bioinformatics pipelines, we have identified a total of 1064 functional mutations (somatic mutations and indels) that we have classified using a 5-tier system based on functional predictions generated by several in silico tools. We have used hapLOH to ascertain for genomic imbalances from exome data and identified 26 events. Validation was performed using Sanger sequencing in a set of the identified events confirming 90% of the somatic mutations and 50% of the indels. Allelic imbalances in 5q were partially confirmed using microsatellite analysis. APC, KRAS, FBXW7, TCFL2 and ARID1A were among the 16 genes found to be recurrently mutated. Pathway enrichment analysis of the mutated gene list revealed deregulation of the Wnt, MAPK kinase and ERBB pathways, thus indicating potential avenues for chemoprevention drug development. Moreover, we assessed and compared the genomic characteristics such as the rate of transitions/transversion, mutational rate [mutations/Megabase (Mb)] and genomic imbalances with stage I CRC from the The Cancer Genome Atlas in order to understand better the carcinogenic process in the colonic epithelium at the genomic level. The signature profile of transition/transversions displayed by polyps showed abundance of C>T (followed by T>C changes) and was very similar to stage I non-hypermutator CRCs. In contrast, the mutational rate was lower in polyps (1.75 vs 3.95 mutations/Mb) and the pattern of genomic imbalances shared some events with stage I CRCs (loss of 5q and events in chromosome 7) but there were notable events seen only in the stage I CRCs most likely involved with further steps in the carcinogenesis process (loss of 18q and 17p). In conclusion, genomic assessment of premalignant colonic polyps revealed mutational patterns that are similar to stage I CRC and pointed to deregulated pathways that are potential candidates for biomarker and chemoprevention drug development. Citation Format: Ester Borras, Anthony San Lucas, Kyle Chang, Gita Bhatia, Hong Wu, Jerry Fowler, Y. Nancy You, Patrick M. Lynch, Melissa W. Taggart, Ernest T. Hawk, Gabriel Capella, Paul Scheet, Eduardo Vilar. Characterizing the genomic landscape of premalignant colorectal polyps using next-generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4806. doi:10.1158/1538-7445.AM2015-4806

One of the emerging Next Generation Sequencing (NGS) applications is amplicon resequencing, which can be applied to screen for mutations in defined genes with diagnostic value. However, in this scenario, methods and protocols are still poorly developed. To address this need, we have established a study workflow that integrates experimental work and bioinformatics analyses. Our Unit of Genetic Testing for Hereditary Cancer is using kits for Multiplex Amplification of Specific Targets for Resequencing (MASTR) from the Multiplicom Company in order to generate highly homogeneous gene specific libraries for point mutation detection. Currently we have completed a proof of concept for the Breast Cancer Susceptibility kit (BRCA1 and BRCA2). We analysed a training set of 267 variants in 28 samples, and a validation set of 137 variants in 14 samples. Results from the GS Junior were combined with those resulting from the analysis of the homopolymer sequences in each gene. Sanger confirmation was performed in all the identified DNA variants. Low coverage ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4445. doi:1538-7445.AM2012-4445

Background. A genome-wide study performed in a Japanese population identified a strong association between SNP rs2294008 (Met1Thr) in the Prostate Stem Cell Antigen gene (PSCA) and diffuse-type gastric cancer (GC). This association has been validated in different Asian populations but no study has yet been conducted in Caucasians. Objective. To analyze the association between PSCA variants and gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Methods. As part of a wider study in which we used the Illumina GoldenGate technology to genotype 1287 SNPs in 249 candidate genomic regions, we genotyped six tagSNPs covering the PSCA gene region in 411 incident gastric adenocarcinoma cases and 1530 matched controls from a nested case-control study in the European EPIC cohort. Among GC cases, 28% were in the cardia or in the gastroesophageal junction, 48% were noncardia and 24% were unspecified. Regarding the histological type, 36% were intestinal, 35% were diffuse and 29% were unspecified. After quality control analysis and data filtering, association was analyzed by unconditional logistic regression, adjusting for age, sex and country. Results. The T allele of rs2294008 in PSCA (frequency of 0.44 in controls and 0.53 in cases) was found to be a significant risk factor for GC (per allele OR=1.42, 95%CI:1.23-1.6; uncorrected p-value=6.5×10-6; Bonferroni adjusted p-value=0.0126). The A allele of rs12155758 also appeared to increase GC risk, while the C allele of rs9297976, in LD with rs2294008, was inversely associated with GC. These 3 SNPs jointly cover 92% of the common variation (MAF≥0.05) within 10 kb around PSCA in Caucasians. Haplotype analysis did not provide additional information. No statistically significant differences were observed in the effect of rs2294008 between diffuse (OR=1.54, 95%CI:1.20-1.96; p-value=0.0005) and intestinal-types (OR=1.52, 95%CI:1.20-1.93; p-value=0.0005), or between cardia (OR=1.19, 95%CI:0.91-1.56; p-value=0.20) and noncardia (OR=1.47, 95%CI:1.19-1.81; p-value=0.0003) sub-sites, although the associations were stronger in noncardia GC. Conclusions. Variation in the promoter region of PSCA, specifically the functional rs2294008 variant, is associated with GC risk in Caucasians. Supported by “LaCaixa” (BM06-130-0); Spanish Ministry of Health (PI070130 and PI081420); European Commission FP5 (ref.QLG1-CT-2001-01049). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3746. doi:10.1158/1538-7445.AM2011-3746

Colorectal cancer is the second cause of cancer related death in the western world and although the genetic and molecular mechanisms involved in initiation and progression of these tumors is among the best characterized, there are significant gaps in our understanding of this disease. The family of receptor tyrosine kinases EPH and their Ephrin ligands regulate cell proliferation, migration and attachment. An important role in colorectal carcinogenesis is emerging for some of its members. In this study we used animal models and analysis of large tumor collections to investigate the possible role of EPHB4 as a new tumor suppressor gene in colorectal cancer. Modulation of EPHB4 levels in colon cancer cell lines resulted in significant growth differences in vitro and in a xenograft model, with low levels of EPHB4 associated with faster growth. In addition, using a genetic model of intestinal tumorigenesis where Apc mutations lead to initiation of the tumorigenic process (Apcmin mice), we show that inactivation of a single allele of EphB4 results in: a) higher proliferation both in the normal epithelium and intestinal tumors, b) significantly larger tumors in the small intestine and c) a 10-fold increase in the number of tumors in the large intestine. This was associated with a 25% reduction in the lifespan of Apcmin mice (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5756.

Current chemotherapy for colorectal cancer is still based in combined 5-fluorouracil (5-FU) and oxaliplatin administration with or without other new drugs. However, an important percentage of patients show resistance to this treatment. Tumor initiating cells (TICs) or cancer stem cells, are able to reconstitute a tumor of similar characteristics to the primary tumor after transplantation into immunodeficient mice. TICs show the ability to self-renew and give rise to non-TIC progeny, supporting the tumor growth. Recent studies suggest that TICs may be intrinsically resistant to therapy, leading to tumor relapse after chemotherapy. To gain insight into the dynamics of TICs during tumor progression and in response to chemotherapy, we have orthotopically implanted and perpetuated in nude mice 14 human colorectal carcinomas (CRC) that produce local and distal dissemination. We have characterized the chemotherapy response in 7 of these perpetuated xenografts. Four of these tumors showed a significant reduction of growth after 5-FU and oxaliplatin combined treatment (FUOX treatment) compared to the correspondent non-treated control tumors. To evaluate the role of TICs in chemotherapy response, we analyzed survival of non-TICs and TICs -identified by CD133/CD44/EpCAM cell surface markers- after chemotherapy. Our results show that, although in 2 out of 4 of the tumors showing efficient response to chemotherapy, TICs were more resistant to FUOX than the rest of tumor cells, in the other 2 tumors TICs and non-TICs were similarly sensitive to treatment. These results indicate that the resistance to chemotherapy is not an intrinsic characteristic of TICs and offer the possibility to characterize sensitive and resistant TICs to FUOX to identify genes and pathways directly involved in chemoresistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3337.