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Despite improvements over the past 20 years, African Americans continue to have the highest incidence and mortality rates of colorectal cancer (CRC) in the United States. While previous studies have found that copy number variations (CNVs) occur at similar frequency in African American and White CRCs, copy-neutral loss of heterozygosity (cnLOH) has not been investigated. In the present study, we used publicly available data from The Cancer Genome Atlas (TCGA) as well as data from an African American CRC cohort, the Chicago Colorectal Cancer Consortium (CCCC), to compare frequencies of CNVs and cnLOH events in CRCs in the two racial groups. Using genotype microarray data, we analyzed large-scale CNV and cnLOH events from 166 microsatellite stable CRCs-31 and 39 African American CRCs from TCGA and the CCCC, respectively, and 96 White CRCs from TCGA. As reported previously, the frequencies of CNVs were similar between African American and White CRCs; however, there was a significantly lower frequency of cnLOH events in African American CRCs compared to White CRCs, even after adjusting for demographic and clinical covariates. Although larger differences for chromosome 18 were observed, a lower frequency of cnLOH events in African American CRCs was observed for nearly all chromosomes. These results suggest that mechanistic differences, including differences in the frequency of cnLOH, could contribute to clinicopathological disparities between African Americans and Whites. Additionally, we observed a previously uncharacterized phenomenon we refer to as small interstitial cnLOH, in which segments of chromosomes from 1 to 5 Mb long were affected by cnLOH.

BACKGROUND: Mismatch repair (MMR) deficiency is the hallmark of tumours from Lynch syndrome (LS), sporadic MLH1 hypermethylated and Lynch-like syndrome (LLS), but there is a lack of understanding of the variability in their mutational profiles based on clinical phenotypes. The aim of this study was to perform a molecular characterisation to identify novel features that can impact tumour behaviour and clinical management. METHODS: We tested 105 MMR-deficient colorectal cancer tumours (25 LS, 35 LLS and 45 sporadic) for global exome microsatellite instability, cancer mutational signatures, mutational spectrum and neoepitope load. RESULTS: Fifty-three percent of tumours showed high contribution of MMR-deficient mutational signatures, high level of global exome microsatellite instability, loss of MLH1/PMS2 protein expression and included sporadic tumours. Thirty-one percent of tumours showed weaker features of MMR deficiency, 62% lost MSH2/MSH6 expression and included 60% of LS and 44% of LLS tumours. Remarkably, 9% of all tumours lacked global exome microsatellite instability. Lastly, HLA-B07:02 could be triggering the neoantigen presentation in tumours that show the strongest contribution of MMR-deficient tumours. CONCLUSIONS: Next-generation sequencing approaches allow for a granular molecular characterisation of MMR-deficient tumours, which can be essential to properly diagnose and treat patients with these tumours in the setting of personalised medicine.

BackgroundMismatch repair (MMR) deficiency is the hallmark of tumors from Lynch syndrome (LS), sporadic MLH1 hypermethylated, and Lynch-like syndrome (LLS), but there is a lack of understanding of the variability in their mutational profiles based on clinical phenotypes. The aim of this study was to perform a molecular characterization to identify novel features that can impact tumor behavior and clinical management.MethodsWe tested 105 MMR-deficient colorectal cancer tumors (25 LS, 35 LLS, and 45 sporadic) for global exome microsatellite instability, cancer mutational signatures, mutational spectrum and neoepitope load.Results78% of tumors showed high contribution of MMR-deficient mutational signatures, high level of global exome microsatellite instability, loss of MLH1/PMS2 protein expression and included sporadic tumors. 22% of tumors showed weaker features of MMR deficiency, 73% lost MSH2/MSH6 expression and included half of LS and LLS tumors. Remarkably, 9% of all tumors lacked global exome microsatellite instability. Lastly, HLA-B07:02 could be triggering the neoantigen presentation in tumors that show the strongest contribution of MMR-deficient tumors.ConclusionsNext-generation sequencing approaches allow for a granular molecular characterization of MMR-deficient tumors, which can be essential to properly diagnose and treat patients with these tumors in the setting of personalized medicine.

Many colorectal cancers (CRCs) that exhibit microsatellite instability (MSI) are not explained by MLH1 promoter methylation or germline mutations in mismatch repair (MMR) genes, which cause Lynch syndrome (LS). Instead, these Lynch-like syndrome (LLS) patients have somatic mutations in MMR genes. However, many of these patients are young and have relatives with cancer, suggesting a hereditary entity. We performed germline sequence analysis in LLS patients and determined their tumor's mutational profiles using FFPE DNA. Six hundred fifty-four consecutive CRC patients were screened for suspected Lynch syndrome using MSI and absence of MLH1 methylation. Suspected LS cases were exome sequenced to identify germline and somatic mutations. Single nucleotide variants were used to characterize mutational signatures. We identified 23 suspected LS cases. Germline sequence analysis of 16 available samples identified 5 cases with LS mutations and 11 cases without LS mutations, LLS. Most LLS tumors had a combination of somatic MMR gene mutation and loss of heterozygosity. LLS patients were relatively young and had excess first-degree relatives with cancer. Four of the 11 LLS patients had rare likely pathogenic variants in genes that maintain genome integrity. Moreover, tumors from this group had a distinct mutational signature compared to tumors from LLS patients lacking germline mutations in these genes. In summary, more than a third of the LLS patients studied had germline mutations in genes that maintain genome integrity and their tumors had a distinct mutational signature. The possibility of hereditary factors in LLS warrants further studies so counseling can be properly informed.

Background: The tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC). Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify regulatory mechanisms underlying APC mutation-negative (APCmut-) CRCs. Methods: We analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut- and APC mutation-positive (APCmut+) microsatellite stable CRCs. We also constructed and compared regulatory networks between APCmut- and APCmut+ CRCs by integrating transcription factor (TF) binding motifs, protein-protein interactions, and gene co-expression using the algorithms PANDA and LIONESS. Single sample networks were used to identify each tumor’s contribution to the differences in transcriptional regulation observed between the APCmut- and APCmut+ groups. Results: APCmut- CRC expression profiles clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut- CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3. Consistent with this, APCmut-CRC regulatory networks showed strong activation of immune pathways relative to APCmut+ CRCs. The second most prominently targeted pathway was RAS signaling, with the TFs TCF15 and RUNX2 regulating key pathway members including RAF1, GEFs, and GAPs. Single sample networks revealed that tumors with the strongest transcriptional regulation of RAS genes tend to lack a KRAS mutation and exhibit a slight skew towards earlier onset. Conclusions: APCmut- CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways. APCmut- tumors exhibit increased regulation of RAS pathway genes compared to APCmut+ tumors. The samples contributing most to this regulatory signature tend to lack a KRAS mutation, suggesting that APCmut- tumors have developed ways to modulate RAS pathway activity through non-mutational mechanisms. Focal emphasis on the RAF1 gene suggests these tumors may be more vulnerable to inhibition of the downstream MAPK pathway. Network analysis implicates TCF15 and RUNX2 as regulators of RAS pathway genes, suggesting a possible WNT-RAS crosstalk occurring in APCmut- CRC that warrants further investigation. Citation Format: Megha Padi, Dante Bellomo, Adam Grant, Rosa M. Xicola, Curtis Thorne, Bodour Salhia, Xavier Llor, Nathan Ellis. Molecular drivers of tumor progression in microsatellite stable APC mutation-negative colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 484.

African Americans (AAs) have higher incidence and mortality rates of colorectal cancer (CRC) compared with other US populations. They present with more right-sided, microsatellite stable disease and are diagnosed at earlier ages compared with non-Hispanic Whites (NHWs). To gain insight into these trends, we conducted exome sequencing (n = 45), copy number (n = 33) and methylation analysis (n = 11) of microsatellite stable AA CRCs. Results were compared with data from The Cancer Genome Atlas (TCGA). Two of the 45 tumors contained POLE mutations. In the remaining 43 tumors, only 27 (63%) contained loss-of-function mutations in APC compared with 80% of TCGA NHW CRCs. APC-mutation-negative CRCs were associated with an earlier onset of CRC (P = 0.01). They were also associated with lower overall mutation burden, fewer copy number variants and a DNA methylation signature that was distinct from the CpG island methylator phenotype characterized in microsatellite unstable disease. Three of the APC-mutation-negative CRCs had loss-of-function mutations in BCL9L. Mutations in driver genes identified by TCGA exome analysis were less frequent in AA CRC cases than TCGA NHWs. Genes that regulate the WNT signaling pathway, including SOX9, GATA6, TET1, GLIS1 and FAT1, were differentially hypermethylated in APC-mutation-negative CRCs, suggesting a novel mechanism for cancer development in these tumors. In summary, we have identified a subtype of CRC that is associated with younger age of diagnosis, lack of APC mutation, microsatellite and chromosome stability, lower mutation burden and distinctive methylation changes.

A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called 'missing heritability'. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (< 50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF > 1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC.

We describe an association between TGFBR1 polymorphism rs868 and mismatch repair proficient hereditary colorectal cancers. This polymorphism results in more binding of TGFBR1 to let-7b-5p miRNA, which would result in lower expression of TGFBR1 and thus higher colorectal cancer risk.The purpose of this study was to identify novel colorectal cancer (CRC)-causing alleles in unexplained familial CRC cases. In order to do so, coding regions in five candidate genes (MGMT, AXIN2, CTNNB1, TGFBR1 and TGFBR2) were sequenced in 11 unrelated microsatellite-stable hereditary non-polyposis CRC (MSS HNPCC) cases. Selected genetic variants were genotyped in a discovery set of 27 MSS HNPCC cases and 85 controls. One genetic variant, rs67687202, in TGFBR1 emerged as significant (P = 0.002), and it was genotyped in a replication set of 87 additional MSS HNPCC-like cases and 338 controls where it was also significantly associated with MSS HNPCC cases (P = 0.041). In the combined genotype data, rs67687202 was associated with a moderate increase in CRC risk (OR = 1.68; 95% CI = 1.13-2.50; P = 0.010). We tested a highly correlated SNP rs868 in 723 non-familial CRC cases compared with 629 controls, and it was not significantly associated with CRC risk (P = 0.370). rs868 is contained in a let-7 miRNA binding site in the 3'UTR of TGFBR1, which might provide a functional basis for the association in MSS HNPCC. In luciferase assays, the risk-associated allele for rs868 was associated with half the luciferase expression in the presence of miRNA let-7b-5p compared with protective allele, suggesting more binding of let-7b-5p and less TGFBR1 expression. Thus, rs868 potentially is a CRC risk-causing allele. Our results support the concept that rs868 is associated with lower TGFBR1 expression thereby increasing CRC risk.

DNA methylation is a fundamental epigenetic mechanism in regulating the expression of genes controlling crucial cell functions in cancer development. Methylation defects (both global hypomethylation and hypermethylation of CpG islands) are implicated in colorectal carcinogenesis. Some nutrients have a clear effect on methylation, suggesting that some dietary-associated differences in the incidence of colorectal cancer could be due to the effect of diet on methylation. The presence of methylation defects has clear diagnostic and prognostic implications. Thus, several tests are being used for colorectal cancer screening based on methylated gene analysis, whether in feces or blood. In addition, the reversibility of methylation processes allows the development of chemotherapies that regulate this process through their antineoplastic activity.

Several studies have suggested that the partially fermentable fibre Plantago ovata husk (PO) may have a protective effect on colorectal cancer (CRC). We studied the potentially pro-apoptotic effect of PO and the implicated mechanisms in CRC cells with different molecular phenotypes (Caco-2, HCT116, LoVo, HT-29, SW480) after PO anaerobic fermentation with colonic bacteria as it occurs in the human colon. The fermentation products of PO induced apoptosis in all primary tumour and metastatic cell lines, independent of p53, adenomatous polyposis coli, β-catenin or cyclo-oxygenase-2 status. Apoptosis was caspase-dependent and both intrinsic and extrinsic pathways were implicated. The intrinsic pathway was activated through a shift in the balance towards a pro-apoptotic environment with an up-regulation of B-cell lymphoma protein 2 homologous antagonist killer (BAK) and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) seen in HCT116 and LoVo cells. This resulted in mitochondrial membrane depolarisation, increased expression of caspase activators second mitochondria-derived activator of caspases (Smac)/Diablo, death effector apoptosis-inducing factor, apoptosome member apoptotic protease activating factor 1 and down-regulation of inhibitors of apoptosis Survivin and X-linked inhibitor of apoptosis in most cells. The extrinsic pathway was activated presumably through the up-regulation of death receptor (DR5). Some important differences were seen between primary tumour and metastatic CRC cells. Thus, metastatic PO-treated LoVo cells had a remarkable up-regulation of TNF-α ligand along with death-inducing signalling complex components receptor interacting protein and TNF-α receptor 1-associated death domain protein. The extrinsic pathway modulator FCICE-inhibitory protein (FLIP), an inhibitor of both spontaneous death ligand-independent and death receptor-mediated apoptosis, was significantly down-regulated after PO treatment in all primary tumour cells, but not in metastatic LoVo. These findings suggest that PO could potentially be a useful chemotherapy adjuvant.

Purpose: Several studies have described molecular differences between microsatellite stable hereditary nonpolyposis colorectal cancer (MSS-HNPCC) and microsatellite unstable Lynch syndrome tumors (MSI-HNPCC). These differences highlight the possibility that other instability forms could explain cancer susceptibility in this group of families. The base excision repair (BER) pathway is the major DNA repair pathway for oxidative DNA damage. A defect in this pathway can result in DNA transversion mutations and a subsequent increased cancer risk. Mutations in MUTYH have been associated with increased colorectal cancer (CRC) risk while no association has been described for OGG1 or NUDT1. Experimental Design: We performed mutational screening of the three genes involved in defense against oxidative DNA damage in a set of 42 MSS-HNPCC families. Results: Eight rare variants and 5 frequent variants were found in MSS-HNPCC patients. All variants were previously described by other authors except variant c.285C>T in OGG1. Segregation studies were done and in silico programs were used to estimate the level of amino acid conservation, protein damage prediction, and possible splicing alterations. Variants OGG1 c.137G>A; MUTYH c.1187G>A were detected in Amsterdam I families and cosegregate with cancer. Analysis of OGG1 c.137G>A transcripts showed an inactivation of the splicing donor of exon 1. Conclusions: Two rare variants (OGG1 c.137G>A; MUTYH c.1187G>A) and one common polymorphism (NUDT1 c.426C>T) were associated with CRC risk. We show that the BER pathway can play a significant role in a number of MSS-HNPCC colorectal cancers. More studies could be of interest in order to gain further understanding of yet unexplained CRC susceptibility cases. Clin Cancer Res; 17(7); 1701–12. ©2011 AACR.

Several studies have suggested that the n-3 fatty acids Docosahexaenoic (DHA) and Eicosapentaenoic (EPA) have an important protective effect on colorectal cancer, and this could be at least partly due to their proapoptotic activity. It is unclear, however, how this phenomenon is triggered and what mechanisms are implicated. Here, we show that both DHA and EPA have an important proapoptotic effect on colorectal cancer cells with different molecular phenotypes but not in noncancerous cells. Apoptosis is caspase dependent, and both intrinsic and extrinsic pathways are implicated. The dimerization of Bax and Bak, the depolarization of the mitochondrial membrane, and the subsequent release of cytochrome c and Smac/Diablo to the cytosol evidence the activation of the intrinsic pathway. The implication of the extrinsic pathway is shown by the activation of caspase-8, along with the down-regulation of FLIP. The timing of caspase-8 activation, and the oligomerization of Bid with Bax, suggest a cross-talk with the intrinsic pathway. None of the death receptors that commonly initiate the extrinsic pathway: FAS, TNF-R1, and TRAIL-R2 are found to be responsible for triggering the apoptosis cascade induced by DHA and EPA. Neither PPARγ nor cyclooxygenase-2, two likely candidates to regulate this process, play a significant role. Our findings suggest that the down-regulation of two key regulatory elements of the extrinsic and intrinsic pathways, FLIP and XIAP, respectively, is determinant in the induction of apoptosis by DHA and EPA. These fatty acids could potentially be useful adjuvant anticancer agents in combination with other chemotherapeutic elements.

Background & Aims: Mismatch repair (MMR) deficiencies are the hallmark of tumors arising in Lynch syndrome, however, in approximately 15% of sporadic colorectal cancers (CRC) these deficiencies most often are associated with somatic methylation of the MMR gene MLH1. Recently, an oncogenic mutation in the BRAF gene has been involved in sporadic CRC showing MMR deficiencies as a result of MLH1 promoter methylation. The aim of this study was to evaluate the contribution of BRAF V600E mutation analysis in the identification of MSH2/MLH1 gene mutation carriers in newly diagnosed CRC patients. Methods:BRAF V600E mutation was analyzed in CRC patients with MMR deficiencies (microsatellite instability and/or lack of MLH1/MSH2 protein expression) in the EPICOLON population-based study. The effectiveness and efficiency of different strategies were evaluated with respect to the presence of MSH2/MLH1 germline mutations. Results: MMR deficiencies were detected in 119 of the 1222 CRC patients with tumors showing either microsatellite instability (n = 111) or loss of protein expression (n = 81). BRAF mutation was detected in 22 (18.5%) of the patients. None of the patients with unambiguous germline mutation had BRAF mutation. Regardless of the strategy used to identify MSH2/MLH1 gene carriers, the introduction of BRAF analysis in these patients slightly improves their effectiveness. The introduction of BRAF mutation analysis as a step before germline genetic testing in patients with MMR deficiencies achieved a significant reduction in costs per mutation detected. Conclusions: Detection of BRAF V600E mutation could simplify and improve the cost effectiveness of genetic testing for hereditary nonpolyposis colorectal cancer, especially in patients whose family history is incomplete or unknown.

ARLTS1 was recently identified in chromosome 13q14 as a tumor suppressor gene of the ADP-ribosylation factor family with pro-apoptotic characteristics. Additionally, one of its genetic variants (W149X) was hypothesized to be a polymorphism associated with familial cancer. We performed a large case-control association study within the EPICOLON project aimed at evaluating the sporadic and familial colorectal cancer (CRC) risk associated with ARLTS1 genetic variants. Whereas P131L and W149X did not seem to affect CRC risk, C148R did show, for the first time in CRC, statistically significant differences between cases and controls [odds ratio (OR) = 1.45, 95% confidence interval (95% CI) = 1.13-1.86, P = 0.003], sporadic cases and controls (OR = 1.59, 95% CI = 1.13-2.23, P = 0.007) and familial cases and controls (OR = 1.55, 95% CI = 1.10-2.19, P = 0.01) in agreement with a hypothetical moderate increase of the cancer risk linked to the C148R ARLTS1 variant, both in sporadic and familial CRC cases.

Background Colorectal tumors caused by failure of the DNA mismatch repair system commonly show microsatellite instability. Our goals were to compare the performance of two panels of markers (a panel previously recommended by the National Cancer Institute [NCI] and a pentaplex of mononucleotide repeats) and to devise the simplest diagnostic strategy for identification of patients with colorectal cancer characterized by defects in mismatch repair. Methods We recruited 1058 patients who were newly diagnosed with colorectal cancer. DNA from fresh-frozen and paraffin-embedded tumors was tested for microsatellite instability, using the NCI-recommended panel of microsatellite markers and the pentaplex panel of mononucleotide repeats , respectively, as templates for polymerase chain reactions (PCRs). Microsatellite instability in fresh-frozen tumors was also assessed using the pentaplex panel of mononucleotides in a crossover analysis. The expression of mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) in the tumors was determined immunohistochemically. The sensitivity and specificity with which the marker panels identified tumors with deficiencies in the expression of mismatch repair proteins were calculated. All statistical tests were two-sided. Results The sensitivity and positive predictive value of the NCI panel were 76.5% (95% confidence interval [CI] = 61% to 92%) and 65.0% (95% CI = 49% to 81%), respectively; corresponding values for the mononucleotide pentaplex panel were 95.8% (95% CI = 89% to 103%) and 88.5% (95% CI = 79% to 98%), respectively. A panel consisting of the mononucleotide repeat markers BAT26 and NR24 alone had the same predictive value as the pentaplex panel of mononucleotide repeats. Conclusions The pentaplex panel of mononucleotide repeats performs better than the NCI panel for the detection of mismatch repair – deficient tumors. Simultaneous assessment of the instability of BAT26 and NR24 is as effective as use of the pentaplex panel for diagnosing mismatch repair deficiency. J Natl Cancer Inst 2007;99: 244 – 52

Disruption of the gene encoding Protein Tyrosine Kinase 6 ( Ptk6 ) delayed differentiation and increased growth in the mouse intestine. However, Ptk6 -null mice were also resistant to azoxymethane-induced colon tumorigenesis. To further explore functions of PTK6 in colon cancer, expression of epithelial and mesenchymal markers, as well as proliferation, migration, and xenograft tumor growth, was examined in human colon tumor cell lines with knockdown or overexpression of PTK6. PTK6 protein, transcript, and activation were also examined in a human colon tumor tissue array, using immunohistochemistry and qRT-PCR. Knockdown of PTK6 led to the epithelial–mesenchymal transition (EMT) in SW480 and HCT116 cells, whereas overexpression of PTK6 in SW620 cells restored an epithelial phenotype in a kinase-independent manner. PTK6 knockdown also increased xenograft tumor growth of SW480 cells, suggesting tumor suppressor functions. In clinical specimens, PTK6 expression was highest in normal differentiated epithelial cells and reduced in tumors. In contrast, overexpression of constitutively active PTK6 promoted STAT3 and ERK5 activation in colon cancer cells, and endogenous PTK6 promoted cell survival and oncogenic signaling in response to DNA-damaging treatments. These data indicate that PTK6 has complex, context-specific functions in colon cancer; PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner, whereas activation of PTK6 promotes oncogenic signaling. Implications: Understanding context-specific functions of PTK6 is important, because although it promotes cell survival and oncogenic signaling after DNA damage, expression of PTK6 in established tumors may maintain the epithelial phenotype, preventing tumor progression. Mol Cancer Res; 14(6); 563–73. ©2016 AACR . This article is featured in Highlights of This Issue, [p. 505][1] [1]: /lookup/volpage/14/505?iss=6

Purpose: African Americans (AA) have the highest incidence of colorectal cancer compared with other U.S. populations and more proximal colorectal cancers. The objective is to elucidate the basis of these cancer disparities. Experimental design: Of note, 566 AA and 328 non-Hispanic White (NHW) colorectal cancers were ascertained in five Chicago hospitals. Clinical and exposure data were collected. Microsatellite instability (MSI) and BRAF (V600E) and KRAS mutations were tested. Statistical significance of categorical variables was tested by the Fisher exact test or logistic regression and age by the Mann–Whitney U test. Results: Over a 10-year period, the median age at diagnosis significantly decreased for both AAs (68–61; P < 0.01) and NHWs (64.5– 62; P = 0.04); more AA patients were diagnosed before age 50 than NHWs (22% vs. 15%; P = 0.01). AAs had more proximal colorectal cancer than NHWs (49.5% vs. 33.7%; P < 0.01), but overall frequencies of MSI, BRAF and KRAS mutations were not different nor were they different by location in the colon. Proximal colorectal cancers often presented with lymphocytic infiltrate (P < 0.01) and were diagnosed at older ages (P = 0.02). Smoking, drinking, and obesity were less common in this group, but results were not statistically significant. Conclusions: Patients with colorectal cancer have gotten progressively younger. The excess of colorectal cancer in AAs predominantly consists of more proximal, microsatellite stable tumors, commonly presenting lymphocytic infiltrate and less often associated with toxic exposures or a higher BMI. Younger AAs had more distal colorectal cancers than older ones. These data suggest two different mechanisms driving younger age and proximal location of colorectal cancers in AAs. Clin Cancer Res; 20(18); 4962–70. ©2014 AACR.

Purpose Disparities in both colorectal cancer (CRC) incidence and survival impact African Americans (AAs) more than other US ethnic groups. Because vitamin D is thought to protect against CRC and AAs have lower serum vitamin D levels, genetic variants that modulate the levels of active hormone in the tissues could explain some of the cancer health disparity. Consequently, we hypothesized that genetic variants in vitamin D-related genes are associated with CRC risk. Methods To test this hypothesis, we studied 39 potentially functional single-nucleotide polymorphisms (SNPs) in eight genes (CYP2R1, CYP3A4, CYP24A1, CYP27A1, CYP27B1, GC, DHCR7, and VDR) in 961 AA CRC cases and 838 healthy AA controls from Chicago and North Carolina. We tested whether SNPs are associated with CRC incidence using logistic regression models to calculate p values, odds ratios, and 95 % confidence intervals. In the logistic regression, we used a log-additive genetic model and used age, gender, and percent West African ancestry, which we estimated with the program STRUCTURE, as covariates in the models. Results A nominally significant association was detected between CRC and the SNP rs12794714 in the vitamin D 25-hydroxylase gene CYP2R1 (p = 0.019), a SNP that has previously been associated with serum vitamin D levels. Two SNPs, rs16847024 in the GC gene and rs6022990 in the CYP24A1 gene, were nominally associated with left-sided CRC (p = 0.015 and p = 0.018, respectively). Conclusions Our results strongly suggest that genetic variation in vitamin D-related genes could affect CRC susceptibility in AAs. Electronic supplementary material The online version of this article (doi:10.1007/s10552-014-0361-y) contains supplementary material, which is available to authorized users.

Colorectal cancer (CRC) is the second leading cause of cancer-related death among men and women in Western countries. Once a tumour develops, a differentiated prognosis could be determined by lifestyle habits or inherited and somatic genetic factors. Finding such prognostic factors will be helpful in order to identify cases with a shorter survival or at a higher risk of recurrence that may benefit from more intensive treatment and follow-up surveillance. Sixteen CRC genetic susceptibility variants were directly genotyped in a cohort of 1235 CRC patients recruited by the EPICOLON Spanish consortium. Univariate Cox and multivariate regression analyses were performed taking as primary outcomes overall survival (OS), disease-free survival and recurrence-free interval. Genetic variants rs9929218 at 16q22.1 and rs10795668 at 10p14 may have an effect on OS. The G allele of rs9929218 was linked with a better OS [GG genotype, genotypic model: hazard ratio (HR) = 0.65, 95% confidence interval (CI) 0.45-0.93, P = 0.0179; GG/GA genotypes, dominant model: HR = 0.66, 95% CI 0.47-0.94, P = 0.0202]. Likewise, the G allele of rs10795668 was associated with better clinical outcome (GG genotype, genotypic model: HR = 0.73, 95% CI 0.53-1.01, P = 0.0570; GA genotype, genotypic model: HR = 0.66, 95% CI 0.47-0.92, P = 0.0137; GG/GA genotypes, dominant model: HR = 0.68, 95% CI 0.50-0.94, P = 0.0194). In conclusion, CRC susceptibility variants rs9929218 and rs10795668 may exert some influence in modulating patient's survival and they deserve to be further tested in additional CRC cohorts in order to confirm their potential as prognosis or predictive biomarkers.

Genome-wide association studies have successfully identified 20 colorectal cancer susceptibility loci. Amongst these, four of the signals are defined by tagging single nucleotide polymorphisms (SNPs) on regions 14q22.2 (rs4444235 and rs1957636) and 20p12.3 (rs961253 and rs4813802). These markers are located close to two of the genes involved in bone morphogenetic protein (BMP) signaling (BMP4 and BMP2, respectively). By investigating these four SNPs in an initial cohort of Spanish origin, we found substantial evidence that minor allele frequencies (MAFs) may be different in northern and southern European populations. Therefore, we genotyped three additional southern European cohorts comprising a total of 2028 cases and 4273 controls. The meta-analysis results show that only one of the association signals (rs961253) is effectively replicated in the southern European populations, despite adequate power to detect all four. The other three SNPs (rs4444235, rs1957636 and rs4813802) presented discordant results in MAFs and linkage disequilibrium patterns between northern and southern European cohorts. We hypothesize that this lack of replication could be the result of differential tagging of the functional variant in both sets of populations. Were this true, it would have complex consequences in both our ability to understand the nature of the real causative variants, as well as for further study designs.

BACKGROUND: African Americans(AA) are at a higher risk of colorectal cancer (CRC) and some studies report a higher frequency of microsatellite instability (MSI) in their tumors while others report lower frequency compared to Caucasians. AIM: To determine and evaluate the association of race and clinical factors with MSI rate through meta- analysis. METHODS: Twenty-two studies out of 15105 (1997-2015) were evaluated after a search in different literature databases, using keywords “colorectal cancer, microsatellite instability”. We used random effect meta-analysis to calculate the MSI frequency in all studies as well as in African American and Caucasian samples. Meta-regression analysis was used to assess the univariate effect of race, gender, age, tumor location and stage on MSI rate. RESULTS: The overall MSI frequency among CRCs was 17% (95%CI: 15%-19%, I2 = 91%). The MSI rate among racial groups were 12%, 12% and 14% in AAs, Hispanics and Caucasians respectively and was not significantly different. Sub-group analysis of studies with racial information indicates MSI OR (95% CI) of 0.78 (0.58-1.06) for AAs compared to Caucasians. CONCLUSION: CRCs demonstrate an MSI frequency of 17%. MSI frequency differences between AAs and Caucasians were not pronounced, suggesting other factors contribute to the racial disparity. There is a large variation in MSI rate between different studies. Methodology approaches and biological sources of this variation should be investigated. Citation Format: Hassan Ashktorab, Sadhna Ahuja, Lakshmi Kannan, Xavier Llor, Nathan Ellis, Rosa M. Xicola, Adeyinka O. Laiyemo, John M. Carethers, Hassan Brim, Mehdi Nouraie. A meta-analysis of MSI frequency and race in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5013.

Colorectal cancer (CRC) is the second most common cancer in Western countries. Hereditary forms only correspond to 5% of CRC burden. Recently, genome-wide association studies have identified common low-penetrant CRC genetic susceptibility loci. Early-onset CRC (CRC50 years old) is especially suggestive of hereditary predisposition although 85-90% of heritability still remains unidentified. CRC50 patients (n = 191) were compared with a late-onset CRC group (CRC65 years old) (n = 1264). CRC susceptibility variants at 8q23.3 (rs16892766), 8q24.21 (rs6983267), 10p14 (rs10795668), 11q23.1 (rs3802842), 15q13.3 (rs4779584), 18q21 (rs4939827), 14q22.2 (rs4444235), 16q22.1 (rs9929218), 19q13.1 (rs10411210) and 20p12.3 (rs961253) were genotyped in all DNA samples. A genotype-phenotype correlation with clinical and pathological characteristics in both groups was performed. Risk allele carriers for rs3802842 [Odds ratio (OR) = 1.5, 95% confidence interval (CI) 1.1-2.05, P = 0.0096, dominant model) and rs4779584 (OR = 1.39, 95% CI 1.02-1.9, P = 0.0396, dominant model) were more frequent in the CRC50 group, whereas homozygotes for rs10795668 risk allele were also more frequent in the early-onset CRC (P = 0.02, codominant model). Regarding early-onset cases, 14q22 (rs4444235), 11q23 (rs3802842) and 20p12 (rs961253) variants were more associated with family history of CRC or tumors of the Lynch syndrome spectrum excluding CRC. In our entire cohort, sum of risk alleles was significantly higher in patients with a CRC family history (OR = 1.40, 95% CI 1.06-1.85, P = 0.01). In conclusion, variants at 10p14 (rs10795668), 11q23.1 (rs3802842) and 15q13.3 (rs4779584) may have a predominant role in predisposition to early-onset CRC. Association of CRC susceptibility variants with some patient's familiar and personal features could be relevant for screening and surveillance strategies in this high-risk group and it should be explored in further studies.

The aim of this study is to evaluate if mismatch repair (MMR) defective colorectal cancer has a different response to adjuvant 5-fluorouracil (5-FU) chemotherapy in a cohort of patients prospectively followed during 5 years.The cohort included 754 surgically treated patients with colorectal cancer. MMR status was diagnosed by MLH1 and MSH2 immunohistochemistry and microsatellite instability analysis. Median follow-up was 49.2 months (range 1-73). At inclusion, 505 patients were diagnosed as TNM II or III stage, analysis of the efficacy of adjuvant chemotherapy was made on this population. Adjuvant chemotherapy was applied to 248 patients (98.2% 5-FU based).MMR deficiency was found in 76 patients (10.1%). No differences were found in overall survival (log-rank p=0.3) or disease-free survival (log-rank p=0.3) regarding MMR status. Adjuvant chemotherapy improves survival in patients in the II or III stage, but this improvement is only evident in patients with MMR-competent tumours (log-rank p=0.00001). Survival of patients with MMR-defective tumours does not improve with adjuvant chemotherapy (log-rank p=0.7). A multivariate analysis showed an independent effect of the interaction between MMR status and adjuvant chemotherapy (Hazard ratio 2.04; 95% confidence interval: 1.42-2.93).In a cohort of colorectal cancer patients, those with MMR-deficient tumours seem not to benefit from 5-FU-based chemotherapy.

Background: Protein Tyrosine Kinase 6 (PTK6, also called BRK) is a non-receptor tyrosine kinase expressed in differentiated epithelial cells of the skin, gastrointestinal tract, and prostate. Disruption of the Ptk6 gene led to impaired intestinal differentiation and increased intestinal proliferation in mice. However, PTK6 also has a tumor-promoting role in the colon, as disruption of the Ptk6 gene impaired carcinogen-induced STAT3 activation and tumorigenesis in mice. PTK6 also promotes survival of human colon cancer cell lines following DNA damaging treatments including γ-irradiation and chemotherapeutic drugs via STAT3 activation. Results: We examined and manipulated colon cancer cell lines to understand these two seemingly contradictory roles for PTK6 in the colon. The human colon adenocarcinoma cell lines SW480 and SW620 represent a primary colon tumor and a metastasis from the same patient, respectively. PTK6 is highly expressed in SW480 cells and significantly reduced in SW620 cells. Active PTK6, phosphorylated on tyrosine residue 342, is not detectable in either cell line. Stable knockdown of PTK6 in SW480 cells results in an epithelial-to-mesenchymal transition (EMT), evidenced by decreased E-cadherin expression and increased ZEB-1 and Vimentin; as well as increased migration, wound-healing, and anchorage-independent growth of cells in culture. Additionally, knockdown of PTK6 led to increased in vivo tumorigenesis of xenograft cells in nude mice. Ectopic expression of wild-type PTK6 in SW620 cells drives the reverse process (MET), demonstrating a rescue of the EMT phenotype. When a constitutively active PTK6 construct is expressed in SW620 cells, it promotes activation of cytoplasmic oncogenic targets and downstream effectors, STAT3, FAK, BCAR1 and ERK5. Analysis of patient tissues demonstrates a downregulation of mRNA and protein levels, as well as a change in protein localization in tumor versus normal tissues; while PTK6 is detectable in the nuclei of differentiating normal cells it is excluded from nuclei in colon tumors. Conclusions: Our studies indicate that PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner and a kinase switch activates PTK6 to promote oncogenic signaling. PTK6 protein localization may function to regulate its specific role. Further examination of the complex role of this kinase could allow for a more nuanced understanding of colon cancer initiation and progression for the development of novel treatments. Citation Format: Priya S. Mathur, Jessica J. Gierut, Rosa M. Xicola, Xavier Llor, Angela L. Tyner. Opposing roles for protein tyrosine kinase 6 (PTK6) in colon cancer. [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 LB-059. doi:10.1158/1538-7445.AM2015-LB-059

Background. There are disparities in both colorectal cancer (CRC) incidence and survival between African Americans (AAs) and Non-Hispanic Whites (NHWs). Genetic susceptibility factors are believed to contribute to CRC development but have been inadequately studied in AAs. Our previous work showed both shared and independent CRC-associated risk alleles are present in some gene regions identified in genome-wide association studies (GWAS) in NHWs. In the present work, using a functional bioinformatics approach, we searched for risk alleles in these regions in AAs. Methods. 10 chromosomal regions (8q23.3, 8q24.21, 10p14, 11q23.1, 14q22.2, 15q13.3, 16q22.1, 18q21.1, 19q13.11, 20p12) from COGENT consortium meta-analysis were selected for DNA sequencing. Three additional high-interest regions were included. The region surrounding each associated SNP was analyzed using r2 plots, based on European-ancestry genotype data from HapMap Phase 3. DNAs from 63 individuals (25 AA CRC cases and 38 AA controls) were sequenced using Agilent SureSelect enrichment arrays and Illumina HiSeq platform technology. Variants from the targeted sequencing were combined with 1000 genomes variant data for a total of 16,193 variants. Potentially functional variants were identified based on the following criteria: the variant is (i) in a coding region, (ii) an expression quantitative trait locus (eQTL) or a DNAseI eQTL (DsQTL), (iii) in a miRNA binding site, and (iv) in a transcription factor binding site. Golden Helix software was used to process the data. Using the Sequenom Massarray platform, we genotyped variants in discovery and replication sets for a total of 1177 AA CRC cases and 1264 AA controls. We calculated odds ratios (OR) and 95% confidence intervals using logistic regression, assuming an additive genetic model and adjusting for age, sex, and African ancestry. Results. We identified 81 variants in coding sequences, 82 eQTLs, 5 dsQTLs, 38 variants in putative microRNA binding sites, 33 variants in possible transcription factor binding sites, and 6 variants that are in two or more of the preceding categories, for a total of 245 variants. After assay development and quality control, 163 SNPs were available for genetic analysis. Nominal associations (p Citation Format: Fabio Pibiri, Ricky Rechenmacher, Rosa Xicola, Rick Kittles, Robert Sandler, Temitope Keku, Xavier Llor, John Carpten, Sonia Kupfer, Nathan Ellis. Identification of functional risk alleles in colorectal cancer-associated regions in African Americans. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2191. doi:10.1158/1538-7445.AM2014-2191

Introduction: Approximately 10% to 15% of all sporadic colorectal cancers have microsatellite instability (MSI). MSI has not been well studied in Hispanics, an ethnic minority at high risk for colorectal cancer (CRC). Patients with MSI tumors present with better prognosis, higher 5-year survival and limited response to 5-FU based chemotherapy, compared to microsatellite stable (MSS) tumors. The aim of this study was to define the clinic-pathologic phenotype of Hispanics with sporadic MSI CRC and to evaluate the association of dietary factors and tumor MSI-status. Methods: The case-case study evaluated the MSI status of sporadic CRC diagnosed during the period January 1, 2002 to October 31, 2012. MSI analysis was performed using six markers (BAT26, BAT25, NR21, NR22, NR24 and NR27). Tumors were classified as MSI-high if two or more of the six microsatellite repeats were mutated and MSS if none of the replicates was mutated. Frequency analysis, chi-square, Fisher's exact test and Wilcoxon rank-sum test were used for statistical analysis methods. Unconditional logistic regression was employed to estimate the odds ratio (OR) between exposures and MSI using STATA 10.0. Results: A total of 85 participants (mean age 57.5 ± 12.5 yrs, 51.8% males) were examined. 80 patients had MSS tumors, while 5 (6%) tumors had MSI. The colorectal neoplasia specimens analyzed were mostly adenocarcinomas (96.5%) located in the distal colon (72.0%) with TNM stage III (34.4%). Tumors with MSI did not significantly differ from MSS tumors with regards to gender (p=0.36), age (p=0.66), education (p>0.99), family history of CRC (p=0.58) or obesity (p>0.99). MSI tumors were more likely located in the proximal colon compared to MSS tumors (OR=12.21; 95% CI 1.4-306.6). There were no statistical significant associations between MSI and reported consumption of dark green leaf vegetables (2-4 times/week) (OR=0.77; 95% CI 0.11-6.88), fruits (≥ 7 times/week) (OR=3.54; 95% CI 0.24-108.52) and red meats (once per day) (OR=1.08; 95% CI: 0.04-9.55). Conclusions: We report a lower prevalence of MSI in Hispanics with sporadic CRC than other Western populations. As previously reported in other non-Hispanic populations, MSI tumors were mostly located in the proximal colon. There were no statistical significant associations between sociodemographic, clinical and nutritional characteristics and MSI. Ongoing efforts to continue to examine additional Hispanic patients are underway to determine if dietary patterns in Hispanics are associated with CRC MSI status. Citation Format: Yaritza Diaz-Algorri, Sofia Margarita Lopez-Diaz, Maria del Mar Gonzalez-Pons, Katerina Freyre, Maritere Olascoaga, Sylvia B. Saldaña-Villafañe, Mercedes Y. Lacourt-Ventura, Sharon C. Fonseca-Williams, Raul D. Bernabe-Dones, Xavier LLord, Rosa Xicola, Katherine Tucker, Marcia R. Cruz-Correa. Clinicopathological characterization and nutritional assessment of Hispanics with sporadic microsatellite unstable colorectal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 109. doi:10.1158/1538-7445.AM2013-109

Background: Colorectal cancer (CRC) is among all common malignancies one with the highest percentage of familial clustering. Thus almost 20% of cases develop in families with at least another affected member. Individuals in these families have a higher risk of developing CRC. Known mutations responsible for several CRC syndromes only explain a small proportion of familial cases but the underlying basis of most still remains unknown. Over half of CRCs that are highly suspicious for an autosomal dominant hereditary pattern of malignancy, as expressed by the Amsterdam criteria, are not explained by known genetic mutations. These cases are known as microsatellite stable hereditary non-polyposis CRC (MSS-HNPCC) or colorectal cancer type X. Aims: To identify new genetic variants that can potentially modulate CRC risk and contribute to hereditability. Methods: We sequenced the coding sequence of MGMT, AXIN2, CTNNB1, TGFβRI and TGFβRII genes and we genotyped 10 common low risk variants, in 30 MSS-HNPCC patients. Potentially relevant variants were genotyped in 308 sporadic cases and 425 cancer-free controls. Logistic regression was used to estimate cancer risk (OR (95%CI)) associated with the genetic variants identified using SPSS (IBM v20.0). Results: A haplotype containing 3 variants (rs67687202, rs868 and rs334354) in high linkage disequilibrium (LD) was found to be associated in a very strong manner with MSS-HNPCC patients and to a lesser degree with sporadic CRC. Thus both MSS-HNPCC and sporadic CRC showed a significantly lower minor allele frequency (MAF) than controls (MAF 0.07, MAF 0.23 vs MAF 0.29) for rs67687202. The AA genotype of rs868 associates with a strong risk for CRC in MSS-HNPCC patients (OR=7.9 (2.7-23.1) P Citation Format: Rosa M. Munoz Xicola, Brian Doyle, Jamie Rawson, Pilar Garre, Anna Abuli, Esther Lee, Sathyaraj Murugappan, Xavier Bessa, Luis Bujanda, Francesc Balaguer, Sergi Castellvi-Bel, Juan Clofent, Cristina Alenda, Rodrigo Jover, Clara Ruiz-Ponte, Sapna Singal, Montserrat Andreu, Angel Carracedo, Antoni Castells, Nathan Ellis, Trinidad Caldes, Xavier LLor. Implication of the 3′UTR region of TGFβR1 with MSS HNPCC and sporadic colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1334. doi:10.1158/1538-7445.AM2013-1334

Background: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in both sexes in the United States. There is a large disparity in both CRC incidence and survival between African Americans (AAs) and all other US racial groups. Differences in serum vitamin D levels could contribute to this disparity because vitamin D is thought to protect against CRC and AAs have lower serum vitamin D levels than whites. We hypothesized that genetic variants in vitamin D-related genes are associated with CRC risk in AAs. Methods: To evaluate association with CRC risk, we genotyped 39 putatively functional single nucleotide polymorphisms (SNPs) in vitamin D-related genes (CYP27A1, GC, CYP3A4, CYP2R1, DHCR7/NADSYN1, VDR, CYP27B1 and CYP24A1, in 961 AA cases (292 right colon, 340 left colon, 113 rectal) and 838 AA controls from the North Carolina Colorectal Cancer Study and the Chicago Colorectal Cancer Consortium. We calculated odds ratios (OR) using logistic regression, assuming an additive genetic model and controlling for age, gender, and West African ancestry. We further evaluated whether the genetic polymorphisms conferred differential risk for right-sided CRC (R-CRC) and left-sided CRC (L-CRC, including rectal). Results: Nominal associations (p Conclusion: Our results indicate that several SNPs in the vitamin D pathway contribute to CRC susceptibility in AAs. The minor allele of rs73913755 was present in at least one dose in 48% of AA controls and only 30% of cases, conferring increased protection against L-CRC. We hypothesize that the minor allele of rs73913755 causes a reduction in the expression levels of CYP24A1. Because rs73913755 is African-specific, showing high Fst value in comparison to ancestrally European populations, its presence could explain in part the lower proportion of L-CRC in AAs compared to whites. Future studies include analysis of vitamin D pathway gene expression levels relative to genotype and analysis of possible gene-vitamin D level interactions in AA CRC. Citation Format: Fabio Pibiri, Nathan Ellis, Rick Kittles, Xavier LLor, Rosa Xicola, Sonia Kupfer, Robert S. Sandler, Temitope Keku. Genetic variation in the vitamin D pathway and risk for colorectal cancer in African Americans. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1330. doi:10.1158/1538-7445.AM2013-1330

Colorectal cancer (CRC) affects disproportionally African Americans (AAs) who have a 20% higher incidence and a 40% higher mortality than Caucasians. Very few studies have specifically addressed CRC in AAs. In order to uncover the factors that underlie this disparity we set up the CCCC, a large, robust and well-characterized database and biorepository of CRC patients from the Chicago metropolitan area that is highly enriched with AAs. The project enrolls newly diagnosed CRCs, polyps at different stages, and cancer/polyp-free controls in 5 large hospitals. Extensive clinical, family history, demographic, dietary, toxic exposure data is collected along with tumor and uninvolved mucosa as well as plasma, serum, and paraffin-embedded samples. Tumors are molecularly characterized and germline DNA is used to assess genetic factors implicated in CRC development. The primary goal of the project is to study how genetic and environmental factors as well as their interaction contribute to CRC development and which factors make AAs more prone to develop this cancer. We will also assess specific factors that may contribute to the worse prognosis in AAs. Results: A total of 205 CRCs, (58% AAs), 86 patients with high-risk adenomas, 75 mild-risk adenomas, and 173 controls have been recruited so far. The mean age at diagnosis was 60.2 for AAs and 61.8 for whites (P=0.08), in both cases significantly lower than the mean age reported for the US CRCs in general (68-70). There was a significantly higher number of AA patients younger than age 50 at diagnosis (19.5% vs. 7.1% whites; P=0.043). In AA patients tumors were more often on the right side of the colon (43.3% vs. 22.7%; P=0.02) and more were undifferentiated (19.1% vs. 3.5%; P=0.03). Both groups had a similar number of colonoscopies before cancer diagnosis (60.2% for AAs vs. 68.3% for whites; P=0.11). While there was no significant difference among AA cases and controls regarding high tobacco use (>20 pack/years)(16.9% cases vs. 17.1% controls), more whites with CRC were heavy smokers (45.7% cases vs. 29.7 controls). Less AA patients reported family history of CRC than whites (16.4% vs. 35.2%; P0.02) or family history of polyps (21.7% vs. 44%). Conclusions: In our urban cohort both AA and white patients present CRC significantly earlier than expected. Very important differences are seen between AAs and whites that may have significant implications when considering CRC screening approaches, such as the high percentage of AA patients diagnosed before age 50 or the much higher number of right-sided tumors. Heavy tobacco use seems to associate with CRC in whites but not in AAs. Altogether points towards important biological differences that need to be further assessed. As we are ascertaining toxic and dietary exposure and molecularly characterizing all tumors, eventually we should be able to explain the biological basis of the significant disparity in CRC 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 3597. doi:1538-7445.AM2012-3597

Background & Aims Approximately half of the families that fulfill Amsterdam criteria for Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC) do not have evidence of the germline mismatch repair gene mutations that define this syndrome and result in microsatellite instability (MSI). The carcinogenic pathways and the best diagnostic approaches to detect microsatellite stable (MSS) HNPCC tumors are unclear. We investigated the contribution of epigenetic alterations to the development of MSS HNPCC tumors. Methods Colorectal cancers were divided into 4 groups: (1) microsatellite stable, Amsterdam-positive (MSS HNPCC) (N = 22); (2) Lynch syndrome cancers (identified mismatch repair mutations) (N = 21); (3) sporadic MSS (N = 92); and (4) sporadic MSI (N = 46). Methylation status was evaluated for CACNAG1 , SOCS1 , RUNX3 , NEUROG1 , MLH1 , and long interspersed nucleotide element-1 ( LINE-1 ). KRAS and BRAF mutation status was analyzed. Results MSS HNPCC tumors displayed a significantly lower degree of LINE-1 methylation, a marker for global methylation, than any other group. Although most MSS HNPCC tumors had some degree of CpG island methylation, none presented a high index of methylation. MSS HNPCC tumors had KRAS mutations exclusively in codon 12, but none harbored V600E BRAF mutations. Conclusions Tumors from Amsterdam-positive patients without mismatch repair deficiency (MSS HNPCC) have certain molecular features, including global hypomethylation, that distinguish them from all other colorectal cancers. These characteristics could have an important impact on tumor behavior or treatment response. Studies are underway to further assess the cause and effects of these features.

Comparison Between Routine Immunohistochemistry for Mismatch Repair Proteins Versus Revised Bethesda Guidelines in the Diagnosis of Lynch Syndrome in a Non-Selected Population of Colorectal Cancer Patients Lucia Perez-Carbonell, Clara Ruiz-Ponte, Xavier Bessa, Jose-Luis Soto, Adela Castillejo, Victor Barbera, Alejandro Brea, Laura Sempere, Cristina Sanchez-Fortun, Sergi CastellviBel, Francesc Balaguer, Rosa M Xicola, Xavier Llor, Anna Abuli, Montserrat Andreu, Cristina Alenda, Artemio Paya, Angel Carracedo, Antoni Castells, Rodrigo Jover

Introduction: The intestinal epithelium undergoes a constant and fast renewal that is maintained in check thanks to a tight equilibrium between cell death and proliferation. An alteration of this equilibrium in the colonic epithelium can result in colorectal cancer development. Nutrients capable of either inducing programmed cell death or decreasing the proliferative rate of this epithelium could presumably be useful as either chemo preventive or even adjuvant therapeutic elements in colorectal cancer. Fish oil, through its main fatty acids, Docosahexaenoic (DHA) and Eicosahexaenoic (EPA) has shown some promising effects in colorectal cancer both, epidemiologically and experimentally. We have previously shown that DHA and EPA induce apoptosis in colorectal cancer cells. We also showed that both, the intrinsic and extrinsic pathways are involved though the down-regulation of two key elements: FLIP and XIAP. Aims: The aims of this study were: to determine if the n-3 fatty acids DHA and EPA can regulate colorectal cancer cell proliferation; to determine if this regulation is dependent on the type of molecular alterations present in these cells and; to determine if any phase of the cell cycle is affected in this potentially anti neoplastic effect. Methods: Non-confluent HCT116, LoVo, SW480, Caco-2 and HT-29 human colorectal cancer cells were grown in their regular media and, 24 h before the experiments, they were synchronized by feeding them with sera-free media supplemented with DHA, EPA or vehicle at final concentrations of 30 to 50 uM according to the type of cell studied. Cell proliferation was assessed by MTT analysis at 24 and 48h after fatty acid supplementation. Cell cycle phases were analyzed by flow citometry through propidium iodide staining. Results: DHA and EPA supplementation resulted in a decrease in the proliferation rate of most colorectal cancer cells, independently of APC, p53 or mismatch repair status. This coincided with a significant decrease in the DNA synthesis of these cells. Neither DHA nor EPA was able to stop proliferation of the metastatic LoVo cells. DNA synthesis was preserved in the latter cells as opposed to the non-metastatic cells. Conclusions: DHA and EPA can exert an anti neoplastic effect in colorectal cancer not only through a significant apoptosis induction but also through a significant decrease in cell proliferation and in DNA synthesis. These effects are independent of the responsible underlying molecular pathways. Metastatic colorectal cancer cells may have developed mechanisms that make these cells resistant to the anti proliferative effects of DHA and EPA.

Background Colorectal cancer (CRC) is the second leading cause of cancer death in developed countries. Familial aggregation in CRC is also important outside syndromic forms and, in this case, a polygenic model with several common low-penetrance alleles contributing to CRC genetic predisposition could be hypothesized. Mucins and GALNTs (N-acetylgalactosaminyltransferase) are interesting candidates for CRC genetic susceptibility and have not been previously evaluated. We present results for ten genetic variants linked to CRC risk in previous studies (previously identified category) and 18 selected variants from the mucin gene family in a case-control association study from the Spanish EPICOLON consortium. Methods CRC cases and matched controls were from EPICOLON, a prospective, multicenter, nationwide Spanish initiative, comprised of two independent stages. Stage 1 corresponded to 515 CRC cases and 515 controls, whereas stage 2 consisted of 901 CRC cases and 909 controls. Also, an independent cohort of 549 CRC cases and 599 controls outside EPICOLON was available for additional replication. Genotyping was performed for ten previously identified SNPs in ADH1C, APC, CCDN1, IL6, IL8, IRS1, MTHFR, PPARG, VDR and ARL11, and 18 selected variants in the mucin gene family. Results None of the 28 SNPs analyzed in our study was found to be associated with CRC risk. Although four SNPs were significant with a P-value < 0.05 in EPICOLON stage 1 [rs698 in ADH1C (OR = 1.63, 95% CI = 1.06-2.50, P-value = 0.02, recessive), rs1800795 in IL6 (OR = 1.62, 95% CI = 1.10-2.37, P-value = 0.01, recessive), rs3803185 in ARL11 (OR = 1.58, 95% CI = 1.17-2.15, P-value = 0.007, codominant), and rs2102302 in GALNTL2 (OR = 1.20, 95% CI = 1.00-1.44, P-value = 0.04, log-additive 0, 1, 2 alleles], only rs3803185 achieved statistical significance in EPICOLON stage 2 (OR = 1.34, 95% CI = 1.06-1.69, P-value = 0.01, recessive). In the joint analysis for both stages, results were only significant for rs3803185 (OR = 1.12, 95% CI = 1.00-1.25, P-value = 0.04, log-additive 0, 1, 2 alleles) and borderline significant for rs698 and rs2102302. The rs3803185 variant was not significantly associated with CRC risk in an external cohort (MCC-Spain), but it still showed some borderline significance in the pooled analysis of both cohorts (OR = 1.08, 95% CI = 0.98-1.18, P-value = 0.09, log-additive 0, 1, 2 alleles). Conclusions ARL11, ADH1C, GALNTL2 and IL6 genetic variants may have an effect on CRC risk. Further validation and meta-analyses should be undertaken in larger CRC studies.