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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.

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.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of kidney cancer, yet molecular biomarkers have not been used for the prognosis of ccRCC to aide clinical decision making. This study aimed to identify genes associated with ccRCC aggressiveness and overall survival (OS). Samples of ccRCC tumor tissue were obtained from 33 patients who underwent nephrectomy. Gene expression was determined using whole-transcriptome sequencing. The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) RNA-seq data was used to test association with OS. 290 genes were differentially expressed between tumors with high and low stage, size, grade, and necrosis (SSIGN) score (≥7 vs. ≤3) with P ADJ

Cancer disparities in Native Americans (NAs) and Hispanic Americans (HAs) vary significantly in terms of cancer incidence and mortality rates across geographic regions. This review reports that kidney and renal pelvis cancers are unevenly affecting HAs and NAs compared to European Americans of non-Hispanic origin, and that currently there is significant need for improved data and reporting to be able to advance toward genomic-based precision medicine for the assessment of such cancers in these medically underserved populations. More specifically, in states along the US-Mexico border, HAs and NAs have higher kidney cancer incidence rates as well as a higher prevalence of kidney cancer risk factors, including obesity and chronic kidney disease. They are also more likely to receive suboptimal care compared to European Americans. Furthermore, they are underrepresented in epidemiologic, clinical, and molecular genomic studies of kidney cancer. Therefore, we maintain that progress in precision medicine for kidney cancer care requires an understanding of various factors among HAs and NAs, including the real kidney cancer burden, variations in clinical care, issues related to access to care, and specific clinical and molecular characteristics.

Background: Hispanic Americans (HAs) in Arizona have a heavy burden of renal cell carcinoma (RCC) presenting at a younger age at diagnosis with higher incidence and mortality rates than European Americans (EAs). However, HAs are underrepresented in RCC research, and tumor molecular characteristics in this population are unknown. We report preliminary findings from a study of molecular differences between HAs and EAs exploring the molecular basis of early-onset clear cell RCC (ccRCC). Methods: Clinical records of patients who underwent surgical treatment for RCC between 2011 and 2020 at the University of Arizona Department of Urology was reviewed. A total of 539 patients were identified, and paraffin embedded surgical specimens of a subset of patients with ccRCC were selected for this study. Three exons of VHL gene, the most commonly altered gene in ccRCC, were screened for somatic mutations in a total of 117 patients. Thirty-three patients were included for TempO-Seq analysis to correlate molecular subtype with demographic and clinicopathologic characteristics. Results: Among 117 patients, 55 somatic mutations in coding regions of VHL were found in 42 patients (35.9%). Nine patients had more than 2 somatic mutations. There were 37 substitutions, 14 deletions, and 4 insertions. There was one patient with a mutation of an intron variant that was a splice acceptor. Mutations in the coding regions were more common in EAs (40.9%) than HAs (31%), but the difference was not statistically significant (P=0.32). For moderate or high impact mutations, EAs had mutations at a significantly higher frequency than HAs (39.4% vs. 20.5%, P=0.04). Patients with high impact mutations tend to be diagnosed before the age of 50 (37.5%) compared to patients without high impact mutations (23.9%). We were able to assign 32 out of 33 patients into molecular subtypes (ccA and ccB). Molecular subtype could not be assigned to one HA patient with high grade and advanced stage ccRCC. Compared to patients with ccB subtype, patients who had ccA subtype were younger (mean age of 52.2 vs. 61.4) and tend to be obese (55.6% vs. 28.6% body mass index ≥30). Molecular subtype, ccA, was more common in HAs than EAs (64.3% vs. 41.2%), but this difference was not statistically significant. Conclusion: Somatic mutations within the VHL gene often altered in early during ccRCC pathogenesis were less common in HAs, but ccA associated with early age of diagnosis and obesity was more common in HAs. Impact: HAs and EAs have different molecular basis of early-onset ccRCC which may impact clinical managements. Citation Format: Yuliang Chen, Karleen M. Meiklejohn, Nathan Ellis, Erika R. Bracamonte, Benjamin R. Lee, Ken Batai. Clear cell renal cell carcinoma molecular differences between Hispanic Americans and European Americans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2191.

African Americans (AAs) have higher incidence and mortality rates of colorectal cancer (CRC) compared to other US populations. AAs present with more right-sided, microsatellite stable disease, and they are diagnosed at earlier ages compared to non-Hispanic whites (NHWs). Data from the Chicago Colorectal Cancer Consortium (CCCC) suggest that the right-sided and early-onset CRCs are distinct diseases, because early-onset CRC arises more often in the distal colon (1). In addition, analysis of stage at presentation data from the SEER database suggests that early-onset CRC is on average a more rapidly developing disease that is less likely to be prevented by colonoscopy (2). To better understand 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 to data from The Cancer Genome Atlas (TCGA). In the 43 non-hypermutable tumors, only 27 (63%) contained loss-of-function mutations in APC as compared to 80% of TCGA NHW CRCs. Importantly, APC mutation-negative CRCs were associated with an earlier age of onset of CRC (p=0.01). In the TCGA, APC mutation-negative CRCs were also associated with an earlier age of onset of CRC (p=10−5). In CCCC CRCs, APC mutation-negative CRCs were also associated with previous cancer, lower overall mutation burden, and fewer copy number variants. We conducted an analysis of DNA methylation patterns and found an epigenetic signature that was distinct from the CpG island methylator phenotype characterized in microsatellite unstable disease, referred to as CIMP. Included in the list of genes that were differentially hypermethylated in APC mutation-negative CRCs were genes that regulate the WNT signaling pathway, such as SOX9, GATA6, TET1, GLIS1, and FAT1. Using the most variable differentially methylated regions from the CCCC data, we found that these regions similarly clustered in APC mutation-negative CRCs from the TCGA. These data strongly suggest that a novel epigenetic mechanism accounts for cancer development in early-onset CRCs. Contrary to the mechanism that predominates in later-onset CRC, the early-onset mechanism does not depend on mutation in the APC gene but is associated with differential methylation of WNT pathway regulating genes instead. Our data support the claim that early-onset CRC is driven by a distinct subtype of CRC that is associated with lack of APC mutation, microsatellite and chromosome stability, lower mutation burden, and distinctive DNA methylation changes. CRC driven by epigenetic changes is consistent with the epidemiologic data suggesting that early-onset CRC develops as a more rapidly advancing disease. A deeper understanding is needed of the pathways affected by the epigenetic changes and the exposures that drive those changes in order to develop therapeutic approaches to early-onset CRC. References 1. Xicola et al. Clin Cancer Res 2014;20:4962-70. 2. Augustus et al. PLOS One 2018 13:e0200462. Citation Format: Rosa Xicola, Zarko Manojlovic, Gaius Augustus, Sonia Kupfer, Rajyasree Emmadi, Victoria Alagiozian-Angelova, Tim Triche, Salhia Bodour, John Carpten, Xavier Llor, Nathan Ellis. Mechanistic differences in early-onset colorectal cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr IA26.

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.

Firefighters are exposed to carcinogens and have elevated cancer rates. We hypothesized that occupational exposures in firefighters would lead to DNA methylation changes associated with activation of cancer pathways and increased cancer risk. To address this hypothesis, we collected peripheral blood samples from 45 incumbent and 41 new recruit non-smoking male firefighters and analyzed the samples for DNA methylation using an Illumina Methylation EPIC 850k chip. Adjusting for age and ethnicity, we performed: 1) genome-wide differential methylation analysis; 2) genome-wide prediction for firefighter status (incumbent or new recruit) and years of service; and 3) Ingenuity Pathway Analysis (IPA). Four CpGs, including three in the YIPF6, MPST, and PCED1B genes, demonstrated above 1.5-fold statistically significant differential methylation after Bonferroni correction. Genome-wide methylation predicted with high accuracy incumbent and new recruit status as well as years of service among incumbent firefighters. Using IPA, the top pathways with more than 5 gene members annotated from differentially methylated probes included Sirtuin signaling pathway, p53 signaling, and 5' AMP-activated protein kinase (AMPK) signaling. These DNA methylation findings suggest potential cellular mechanisms associated with increased cancer risk in firefighters.

Colorectal cancer (CRC) is one of the most significant causes of cancer morbidity and mortality in the United States. CRC incidence rates increase with age with greater than 90 % of cases occurring in persons over the age of 50. Twin and family studies have shown that genetic risk factors influence CRC incidence in up to 35 % of cases, but within that 35 % the level of risk conferred by genetic factors that predispose to CRC varies enormously. The correlations between family history and CRC susceptibility are propelled by a range of genetic mechanisms, from high-risk, disease-causing mutations that ablate gene function to low-risk genetic variants that perturb gene function in subtler ways. In addition, genetic risk factors can interact with each other and with nongenetic factors to increase or decrease CRC risk. However, the impact of interaction on risk is poorly understood.

Background Screening in the average risk population for colorectal cancer (CRC) is expected to reduce the incidence of distant (i.e., metastatic) CRCs at least as much as less advanced CRCs. Indeed, since 2000, during which time colonoscopy became widely used as a screening tool, the overall incidence of CRC has been reduced by 29%. Objective The purpose of the current study was to determine whether the reduction of incidence rates is the same for all stages of disease. Methods We evaluated incidence data from the Surveillance, Epidemiology, and End Results (SEER) program from 2000-2014 for Localized, Regional, and Distant disease. Joinpoint models were compared to assess parallelism of trends. Data were stratified by race, age, tumor location, and sex to determine whether these subgroupings could explain overall trends. Results Inconsistent with the expectations of a successful screening program, the reduction in incidence rates of distant CRCs from 2000-2014 has been slower than the reductions in incidence rates of both regional and localized CRCs. This trend is evident even when the data are stratified by age at diagnosis, sex, race, or tumor location. Conclusions The slower decrease in the incidence rate of distant disease is not consistent with a screening effect, that is, CRC screening may not be effective in preventing many distant CRCs. As a consequence, distant CRCs represent an increasing fraction of all CRCs, accounting for 21% of all CRCs in 2014. The analysis indicates that inadequate screening does not explain the slower decrease in incidence of distant CRCs. Consequently, we suggest that a subtype of CRC exists that advances rapidly, evading detection because screening intervals are too long to prevent it. Microsatellite unstable tumors represent a known subtype that advances more rapidly, and we suggest that another rapidly advancing subtype very likely exists that is microsatellite stable.

Background: Recent reports have shown that chromosomal copy number gains and losses associated with the development of colorectal cancer (CRC) occur at similar frequencies in Whites and African Americans (AAs), but no studies have compared copy-neutral loss of heterozygosity (cnLOH) between these two populations. cnLOH can arise by at least two different mechanisms. One involves recombination between homologous chromosomes, but cnLOH most commonly arises from mis-segregation of chromosomes via a mitotic form of uniparental disomy. Preliminary analysis of copy number data from Chicago AA CRC cases by our lab suggested that cnLOH occurred less frequently in AA CRCs. The present study aimed to use publicly available data from The Cancer Genome Atlas (TCGA) to test whether cnLOH was less frequent in AA CRC cases in the TCGA. Methods: Raw Affymetrix GenomeWide SNP Array 6.0 files were obtained from TCGA Data Portal and processed using R package Rawcopy and PennCNV to determine segments of loss, gain, and cnLOH. Samples from AAs and Whites were processed separately, using normals from their respective populations as reference. cnLOH events involving less than 20 markers were removed, and the proportion of each chromosome arm affected by cnLOH was calculated for each tumor. Chromosome arms with over 50% cnLOH were counted as cnLOH events. Linear regression models were used to determine associations between the number of chromosome arm events and clinical variables. AA and White cases were identified per TCGA clinical data. Additionally, logistic regression models were used to determine associations between affected and unaffected tumors, defining affected as a tumor with > 2 cnLOH events. The value of 2 was chosen because it was the median value of cnLOH events per tumor among all samples. Chromosome arm-specific differences were tested by Fisher exact tests, using a Bonferroni correction to adjust for multiple testing. Results: Data from 56 AA and 210 White CRCs were analyzed. Race was associated with a decreased number of cnLOH events per tumor, with AA CRCs on average having significantly fewer chromosome arms affected than White CRCs (p = 0.01). This difference remained significant after correction for age at diagnosis, sex, stage, and BMI. Additionally, AA CRCs were half as likely to have developed more than two cnLOH events compared to White CRCs. In AA CRCs, the frequency of cnLOH events was lower on 32 of the 39 chromosome arms compared to frequency of cnLOH events in White CRCs. Chromosomes 17p and 5q were the arms most frequently subject to cnLOH, comprising 13% of all cnLOH events in White tumors and 15% of all cnLOH events in AA tumors. 19.6% and 17.9% of AA CRC cases had cnLOH events on 17p and 5p, respectively, whereas 24.8% and 24.3% of White CRC cases had events on 17p and 5p. These differences were not significant. cnLOH of chromosome 6p was nominally significantly different (3% of AA cases vs 17% of White cases, p = 0.009); however, that difference did not remain significant after adjustment for multiple testing. No other differences were significant. No differences were found in chromosome arm gains or losses. Conclusions: The results in our present study show that cnLOH affects AA CRCs less than White CRCs. cnLOH is not a rare event in CRC. Only 64 of 266 CRCs examined from TCGA (~24%) had zero chromosome arms affected. This mechanism of genomic instability is more frequent for certain chromosome arms, specifically chromosomes 5q and 17p, where the known tumor suppressors APC and TP53 are located. In AA CRCs, the majority of chromosome arms showed lower levels of cnLOH, suggesting that the difference in the frequency of cnLOH events is the result of a reduced susceptibility to cnLOH and not of the selective action of different cancer driver genes. Taken together, these results suggest that there is a racial difference in mechanisms that drive genomic instability as it affects and drives CRC. Citation Format: Gaius J. Augustus, Nathan A. Ellis. Copy-neutral loss of heterozygosity is decreased in African American colorectal cancers [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B52.

Background: Replication forks that are persistently blocked cannot restart, even after DNA damage is repaired. These forks are termed collapsed forks. In order to fully duplicate the genome, collapsed forks must be rescued by activation of nearby dormant origins of replication. Homologous recombination (HR) is essential in collapsed-fork rescue. Cells deficient in the SUMO E3 ligase NSMCE2 exhibit mitotic defects, are sensitive to DNA damaging agents, and have defects in HR. Thus, NSMCE2 regulates HR during replication stress, but the molecular mechanisms are poorly understood. In yeasts, hypomorphic NSMCE2 (MMS21) alleles accumulate HR intermediates during replication stress. We therefore hypothesized that NSMCE2 regulates RAD51 function during collapsed fork rescue. Results: It was previously shown that sumoylation of the BLM helicase results in recruitment of RAD51 to stalled forks. Consistent with data in yeast, we found that BLM sumolyation is dependent on NSMCE2. However, contrary to our expectation, we found that the amount of RAD51 protein that accumulated at collapsed forks was over two times greater in NSMCE2-deficient cells than in normal cells. In contrast, the levels of BLM, RPA, single-stranded DNA, and γH2AX at stalled forks is reduced by half. Consistent with the low levels of γH2AX, the double-strand breaks and sister chromatid exchanges that accumulate during collapsed fork rescue were also greatly diminished in NSMCE2-deficient cells. Thus, despite the over-accumulation of RAD51 to sites of collapsed replication forks, cells are unable to perform HR efficiently, indicating that RAD51 is unable to complete its function there. In NSMCE2-deficient cells, the hyper-accumulated RAD51 at collapsed forks persists into mitosis where excess under-replicated DNA causes mitotic DNA damage. Conclusions: The hyper-accumulation of RAD51 at stalled forks we observed in NSMCE2-deficient cells suggests that NSMCE2 is required for the remodeling of collapsed forks that normally leads to the rescue of collapsed forks, namely, the unloading of RAD51, DNA breakage, repair by HR, and completion of DNA synthesis. We suggest that the excess accumulation of RAD51 that is observed in a substantial number of cancers is not sufficient to demonstrate that the cells are HR proficient. The identification of NSMCE2 as a controller of HR-mediated fork rescue also highlights NSMCE2's potential as a new therapeutic target for combinatorial therapy of HR-dependent cancers. Citation Format: Kelvin W. Pond, Christelle DeRenty, Mary K. Yagle, Nathan Ellis. NSMCE2 enables rescue of collapsed replication forks to prevent mitotic DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1356.

Regions on chromosome 8q24 harbor susceptibility alleles for multiple cancers including colorectal (region 3) and prostate cancer (regions 1–4). The objectives of the present study were (i) to test whether single-nucleotide polymorphisms (SNPs) in region 4 are associated with colorectal cancer (CRC) in European or African Americans; (ii) to test whether 8q24 SNPs previously shown to be associated with colorectal and prostate cancer also show association in our multiethnic series and (iii) to test for association between 100 ancestry informative markers (AIMs) and CRC in both the African American and European American cohorts. In total, we genotyped nine markers on 8q24 and 100 unlinked AIMs in 569 CRC cases and 439 controls (490 European Americans and 518 African Americans) obtained retrospectively from a hospital-based sample. We found rs7008482 in 8q24 region 4 to be significantly associated with CRC in European Americans (P = 0.03). Also in region 4, we found that a second SNP, rs16900305, trended toward association with CRC in African Americans. The rs6983267 in region 3, previously implicated in CRC risk, trended toward association with disease in European Americans but not in African Americans. Finally, none of the 100 AIMs tested for association reached statistical significance after correction for multiple hypothesis testing. In summary, these results are evidence that 8q24 region 4 contains novel CRC-associated alleles in European and African Americans.

// Hassan Ashktorab 1 , Sadhna Ahuja 2 , Lakshmi Kannan 1 , Xavier Llor 3 , Nathan A. Ellis 4 , Rosa M. Xicola 3 , Adeyinka O. Laiyemo 1 , John M. Carethers 5 , Hassan Brim 2 , Mehdi Nouraie 1 1 Department of Medicine and Cancer Center, Howard University College of Medicine, Washington DC, USA 2 Department of Pathology, Howard University College of Medicine, Washington DC, USA 3 Department of Medicine and Cancer Center, Yale University, New Haven, CT, USA 4 Cancer Biology Research Program, The University of Arizona Cancer Center, Tucson, AZ, USA 5 Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA Correspondence to: Hassan Ashktorab, email: hashktorab@howard.edu Keywords: MSI, colorectal cancer, African Americans, Hispanics, Caucasians Received: December 14, 2015 Accepted: March 28, 2016 Published: April 23, 2016 ABSTRACT PURPOSE: African Americans (AA) are at a higher risk of colorectal cancer (CRC) and some studies report a higher frequency of microsatellite instability (MSI) in this population while others report lower frequency compared to Caucasians. AIM: To determine and evaluate the association of race and clinical factors with MSI frequency through meta- analysis. METHODS: Twenty-two studies out of 15,105 (1997-2015) were evaluated after a search in different literature databases, using keywords “colorectal cancer, microsatellite instability, African Americans, Caucasians and Hispanics”. 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 frequency. RESULTS: The overall MSI frequency among CRCs was 17% (95%CI: 15%-19%, I²=91%). In studies with available race data, The MSI rate among AAs, Hispanics and Caucasians were 12%, 12% and 14% respectively and was not significantly different. Sub-group analysis of studies with racial information indicates MSI OR of 0.78 for AAs compared to Caucasians. CONCLUSION: CRCs demonstrate an overall MSI frequency of 17%. MSI frequency differences between AAs and Caucasians were not pronounced, suggesting that other factors contribute to the racial disparity. The methodological approaches and biological sources of the variation seen in MSI frequency between different studies need to be further investigated.

Nearly one in eight US women will develop breast cancer in their lifetime. Most breast cancer is not associated with a hereditary syndrome, occurs in postmenopausal women, and is estrogen and progesterone receptor-positive. Estrogen exposure is an epidemiologic risk factor for breast cancer and estrogen is a potent mammary mitogen. We studied single nucleotide polymorphisms (SNPs) in estrogen receptors in 615 healthy subjects and 1011 individuals with histologically confirmed breast cancer, all from New York City. We analyzed 13 SNPs in the progesterone receptor gene (PGR), 17 SNPs in estrogen receptor 1 gene (ESR1), and 8 SNPs in the estrogen receptor 2 gene (ESR2). We observed three common haplotypes in ESR1 that were associated with a decreased risk for breast cancer [odds ratio (OR), ∼ O.4; 95% confidence interval (CI), 0.2–0.8; P < 0.01]. Another haplotype was associated with an increased risk of breast cancer (OR, 2.1; 95% CI, 1.2–3.8; P < 0.05). A unique risk haplotype was present in ∼7% of older Ashkenazi Jewish study subjects (OR, 1.7; 95% CI, 1.2–2.4; P < 0.003). We narrowed the ESR1 risk haplotypes to the promoter region and first exon. We define several other haplotypes in Ashkenazi Jews in both ESR1 and ESR2 that may elevate susceptibility to breast cancer. In contrast, we found no association between any PGR variant or haplotype and breast cancer. Genetic epidemiology study replication and functional assays of the haplotypes should permit a better understanding of the role of steroid receptor genetic variants and breast cancer risk.

With the large numbers of single nucleotide polymorphisms (SNPs) available and new technologies that permit high throughput genotyping, we have investigated the possibility of the localization of disease genes with genome-wide panels of SNP markers and taking advantage of the linkage-disequilibrium (LD) between the disease gene and closely linked markers. For this purpose, we selected cases from the Ashkenazi Jewish population, in which the mutant alleles are expected to be identical by descent from a common founder and the regions of LD encompassing these mutant alleles are large. As a validation of this approach for localization, we performed two trials: one in autosomal recessive Bloom syndrome, in which a unique mutation of the BLM gene is present at elevated frequencies in cases, and the other in autosomal dominant hereditary nonpolyposis colorectal cancer (HNPCC), in which a unique mutation of MSH2 is present at elevated frequencies. In the Bloom syndrome trial, we genotyped 3,258 SNPs in 10 Jewish Bloom syndrome cases and 31 non-Bloom syndrome Jewish persons as a comparison group. In the HNPCC trial, we genotyped 8,549 SNPS in 13 Jewish HNPCC cases whose colon cancers exhibited microsatellite instability and in 63 healthy Jews as a comparison group. To identify significant associations, we performed (a) Fisher’s exact test comparing genotypes at each locus in cases versus controls and (b) a haplotype analysis by estimating the frequency of haplotypes with the expectation-maximization algorithm and comparing haplotype frequencies in cases versus controls by logistic regression and a maximum likelihood ratio method. In the Bloom syndrome trial, by Fisher’s exact test, statistically significant association was detected at a single locus, TSC0754862, which is a locus 1.7 million bp from BLM. Two-locus, three-locus, and four-locus haplotypes that included TSC0754862 and flanked BLM were also statistically more frequent in cases versus controls. In the HNPCC trial, although a significant P value was not obtained by the single SNP genotype analysis, significant associations were detected for several multilocus haplotypes in an 11-million-bp region that contained the MSH2 gene. This work demonstrates the power of the LD mapping approach in an isolated population and its general applicability to the identification of novel cancer-causing genes.

Purpose: The Breast Cancer Linkage Consortium and other family-based ascertainments have suggested that male carriers of BRCA mutations are at increased risk of prostate cancer. Several series looking at the frequency of BRCA mutations in unselected patients with prostate cancer have not confirmed this finding. To clarify this issue, we conducted a large case-control study. Experimental Design: Blood specimens from 251 unselected Ashkenazi men with prostate cancer were screened for the presence of one of the three common Ashkenazi founder mutations in BRCA1 and BRCA2. The incidence of founder mutations was compared with the incidence of founder mutations in 1472 male Ashkenazi volunteers without prostate cancer using logistic regression analysis after adjusting for age. Results: Thirteen (5.2%) cases had a deleterious mutation in BRCA1 or BRCA2 compared with 28 (1.9%) controls. After adjusting for age, the presence of a BRCA1 or BRCA2 mutation was associated with the development of prostate cancer (odds ratio, 3.41; 95% confidence interval, 1.64–7.06; P = 0.001). When results were stratified by gene, BRCA2 mutation carriers demonstrated an increased risk of prostate cancer (odds ratio, 4.78; 95% confidence interval, 1.87–12.25; P = 0.001), whereas the risk in BRCA1 mutation carriers was not significantly increased. Conclusions: BRCA2 mutations are more likely to be found in unselected individuals with prostate cancer than age-matched controls. These results support the hypothesis that deleterious mutations in BRCA2 are associated with an increased risk of prostate cancer.

Hereditary non-polyposis colon cancer (HNPCC) is an autosomal dominant disorder featuring familial clustering of colorectal and/or endometrial cancer, and other malignancies. Except for a rare case report, Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) have not been considered part of HNPCC. Recent murine models for HNPCC have shown an increased incidence of B- and T-cell lymphoma, as well as tumors of the gastrointestinal tract and other organ systems, involving defects in genes resulting in faulty mismatch repair (MMR) of DNA. These MMR genes include MLH1, MSH2, MSH3, MSH6, PMS1 and PMS2. We sought to analyze the occurrence of NHL and HD in families with clusters of colorectal cancers (CRC). Probands from 21 kindreds were classified as HNPCC (3), HNPCC-like (5), and HNPCC-variant (13); seen and followed by Clinical Genetics at Memorial Hospital the kindreds were assessed for the occurrence of NHL or HD. Of the 21 pedigrees, a total of 37 patients were identified who were diagnosed with leukemia, lymphoma, or HD. Fourteen of the 37 patients with a diagnosis of NHL or HD were further classified and showed varying histologies ranging from chronic lymphocytic leukemia/small lymphocytic lymphoma (2), mycosis fungoides (1), follicular lymphoma (1), extranodal marginal zone lymphoma of MALT type (2), diffuse large B-cell lymphoma (4), nodular sclerosis HD (3), and mixed cellularity HD (1). Microsatellite instability studies were performed on 6 cases but none showed evidence of replication error repair defects. Immunohistochemical stains performed on paraffin sections from these 6 representative cases showed differential protein expression of MLH1, MSH2, MSH6, and PMS2 when compared to normal reactive tissues from the same patient but showed no significant differences when compared to controls of non-familial, sporadic lymphomas. These results suggest that lymphomas arising in the setting of familial CRC do not bear the molecular hallmarks of HNPCC. Further studies are needed to explain the differential patterns of expression of RER-associated proteins in lymphomas, as well as the association of lymphomas and possibly renal cell cancers in a subset of kindreds in which CRC clustering is evident.

Introduction: The purpose of this study is to determine the role of the E3 SUMO ligase NSMCE2 in BLM regulation and homologous recombination (HR). Background: DNA damage generated during replication is a major source of mutations and failure to repair this damage can cause genomic instability. HR is a high-fidelity DNA repair pathway activated primarily by replication-associated DNA damage. Numerous HR proteins are regulated by sumoylation, but the mechanisms that control sumoylation and their roles in HR are poorly understood. Cells deficient in the SUMO E3 ligase NSMCE2 are sensitive to DNA damaging agents and have defects in HR. Our preliminary data indicated that BLM sumoylation is dependent on NSMCE2. Because BLM sumolyation is required to recruit RAD51 to stalled forks, we hypothesized that NSMCE2-deficient cells are deficient in HR due to a defect in BLM-dependent RAD51 recruitment. Results: To test this hypothesis, we transfected HeLa cells with siRNAs specific to NSMCE2 and tested whether cells could recruit RAD51 to replication forks stalled with hydroxyurea (HU). Contrary to our hypothesis, we found that the amount of RAD51 protein that accumulated at stalled forks was greater in HU-treated NSMCE2-deficient cells compared to HU-treated control cells. To our surprise, the amount of single-stranded DNA binding protein RPA was diminished by half in HU-treated NSMCE2-deficient cells and DNA damage signaling was similarly diminished as evidenced by the lower levels of γ-H2AX. Consistent with the low levels of γ-H2AX, the double-strand breaks (DSB) that normally form after extended treatment with HU were also greatly diminished in NSMCE2-deficient cells. Consistent with previous reports, we found that the levels of HU-induced sister chromatid exchange were also low. These data indicated that despite the over-accumulation of RAD51 to sites of stalled replication forks, cells are unable to perform HR repair efficiently, indicating that RAD51 is unable to complete its function there. Conclusions: The hyper-accumulation of RAD51 at stalled forks we observed in NSMCE2-deficient cells suggests the sumoylation of one or more substrates by NSMCE2 is required for the remodeling of stalled forks that normally leads to unloading of RAD51 at the fork, strand breakage, and repair by HR. We suggest that the RAD51 accumulation that is observed in a substantial fraction of cancers may not be sufficient to demonstrate that the cells are HR proficient. Based on our data, RPA staining combined with RAD51 may be able to distinguish HR-proficient and deficient cells. Citation Format: Kelvin W. Pond, Christelle DeRenty, Mary Yagle, Nathan Ellis. Regulation of homologous recombination by the SUMO ligase NSMCE2 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A06.

Objectives. Colorectal cancer (CRC) incidence is higher in post-industrial cultures, and the incidences varies among different populations. In the US, there is a higher incidence of CRC in African Americans (AAs) compared to non-Hispanic whites (NHWs). Recent evidence links consumption of a diet high in animal protein and fat as an environmental risk factor for the development of CRC. The intestinal microbiota is postulated to modulate the effects of diet in promoting or preventing CRC development. Hydrogen sulfide, which is produced by normal members of the colonic microenvironment (sulfidogenic bacteria), triggers pro-inflammatory and hyper-proliferative pathways, and it is genotoxic. We hypothesized that the production of hydrogen sulfide by sulfidogenic bacteria is a key environmental carcinogen contributing to CRC risk. Design. The abundance of sulfidogenic bacteria via quantitative PCR (qPCR) was compared in non-involved colonic mucosa of 97 AA and 56 NHW CRC patients and in 100 AA and 76 NHW healthy controls. In addition, we performed 16S rDNA sequencing in 61 AA cases and 94 AA controls. Additionally, we tested correlations among race, dietary intake, disease status, and sulfidogenic bacterial abundance. Results. Overall, the functional gene for hydrogen sulfide production in sulfate-reducing bacteria, dissimilatory sulfate reductase (dsrA), was more abundant in AAs than in NHWs, in both cases and controls. In addition, AA CRC cases exhibited a significantly higher abundance of Bilophila wadsworthia-specific dsrA. Linear discriminant analysis of 16S rDNA sequencing results revealed several taxa that differed between AA cases and controls, including the known butyrate producer Faecalibacterium that was more abundant in AA controls, and the sulfidogenic Pyramidobacter that was more abundant in AA CRC cases. Importantly, we found that dietary intake of protein and fat was higher in AAs compared to NHWs, and these dietary components correlated with a higher abundance of sulfidogenic bacteria. Conclusion. There were significant differences in sulfidogenic bacterial abundance between AAs and NHWs, in both cases and controls, and implicate sulfidogenic bacteria as an important diet-driven environmental exposure that contributes to the increased risk of CRC in AAs. Replication studies are needed to test that effectiveness of using B. wadsworthia as a biomarker for increased CRC risk. Citation Format: Cemal Yazici, Patricia G. Wolf, Tzu-Wen Liu, Karin Vermillion, Timothy Carroll, Ece Mutlu, Lisa Tussing-Humphreys, Carol Braunschweig, Rosa M. Xicola, Barbara Jung, Xavier Llor, Nathan A. Ellis, H. Rex Gaskins. Sulfidogenic bacteria are an important diet-driven exposure promoting colorectal cancer in African Americans. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B31.

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.

Paolo Peterlongo, Nandita Mitra, Shaokun Chuai, Tomas Kirchhoff, Crystal Palmer, Helen Huang, Khedoudja Nafa, Kenneth Offit and Nathan A. Ellis* Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

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.

Polymerase stalling results in uncoupling of DNA polymerase and the replicative helicase, which generates single-stranded DNA (ssDNA). After stalling, RAD51 accumulates at stalled replication forks to stabilize the fork and to repair by homologous recombination (HR) double-strand breaks (DSBs) that accumulate there. We showed recently that SUMO modification of the BLM helicase is required in order for RAD51 to accumulate at stalled forks. In order to investigate how BLM SUMOylation controls RAD51 accumulation, we characterized the function of HR proteins and ssDNA-binding protein RPA in cells that stably expressed either normal BLM (BLM+) or SUMO-mutant BLM (SM-BLM). In HU-treated SM-BLM cells, mediators BRCA2 and RAD52, which normally substitute RAD51 for RPA on ssDNA, failed to accumulate normally at stalled forks; instead, excess RPA accumulated. SM-BLM cells also exhibited higher levels of HU-induced chromatin-bound RPA than BLM+ cells did. The excess RPA did not result from excessive intrinsic BLM helicase activity, because in vitro SUMOylated BLM unwound similar amounts of replication-fork substrate as unSUMOylated BLM. Nor did BLM SUMOylation inhibit binding of RPA to BLM in vitro; however, in immunoprecipitation experiments, more BLM-RPA complex formed in HU-treated SM-BLM cells, indicating that BLM SUMOylation controls the amount of BLM-RPA complex normally formed at stalled forks. Together, these results showed that BLM SUMOylation regulates the amount of ssDNA that accumulates during polymerase stalling. We conclude that BLM SUMOylation functions as a licensing mechanism that permits and regulates HR at damaged replication forks.

Mutations in BRCA1 and BRCA2 that predispose to breast and ovarian cancer are detected in approximately 2.5% of the Ashkenazi Jewish population. To explore whether carriers of Ashkenazi founder mutations in BRCA1 or BRCA2 have an increased risk for colorectal cancer, we screened 586 unselected Ashkenazi Jewish case patients with colorectal cancer for the three common founder mutations in BRCA1 and BRCA2. We identified six carriers (1.02%) among these case patients. After adjusting for age at diagnosis and sex by use of logistic regression analysis, we compared the incidence of carriers in this group of 586 case patients with that of 5012 Ashkenazi Jewish control subjects without a known history of colorectal cancer. The presence of a founder BRCA mutation was not associated with the risk of colorectal cancer (relative risk = 0.50, 95% confidence interval = 0.22 to 1.14). We thus recommend that counseling for colorectal cancer screening and prevention in individuals with BRCA mutations be based on the personal and family history of colorectal cancer or associated syndromic malignancies.

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.

Background: African Americans (AAs) have the highest incidence and mortality rates of colorectal cancer (CRC) cases in the United States, with higher levels of early-onset and right-sided CRC, yet there are little data on carcinogenesis in AA CRC. Copy number variations (CNVs) affect large portions of the cancer genome, and they provide information about mechanisms of cancer initiation and progression. Given the unique clinical-pathological features of AA CRC, we hypothesized that different mechanisms operate in colorectal carcinogenesis in AAs. To test this hypothesis, we evaluated CNVs in AA CRC as compared to publically available data on CRC from the TCGA. Methods: Tumor DNA samples from 46 AA CRC cases were available through the Chicago Colorectal Cancer Consortium. Determinations of CNVs were performed using the Affymetrix Cytoscan HD platform, which provides data from 2.67 million copy number probes and 750,000 genotype probes for analysis of loss of heterozygosity (LOH) with and without copy number change. Whole chromosome-arm gains and losses were defined as the gain or loss of >50% of probes on an arm. Gains and losses were tabulated and compared to data from the TCGA. Statistical analyses of arm losses and gains were performed with Fisher exact tests, adjusting for multiple testing using a Bonferroni correction. Statistical analysis of total chromosome number alterations was performed with the chi-square test. Results: Overall, significantly fewer chromosome-arm gains and losses occurred in AA CRC cases than in TCGA CRC cases with 10% of gains and losses in AA and 34% of gains and losses from TCGA (p < 0.0001). More specifically, we found chromosome-arm gains occurred significantly less frequently for 8p (28% to 0%), 13q (56% to 26%), and 20 p and q (58% to 17% and 72% to 39%, respectively). Similarly, we found chromosome-arm losses occurred significantly less frequently for 1p (19% to 0%), 5q (17% to 0%), 8p (50% to 20%), 14q (30% to 0%), 17p (56% to 24%), 18p and q (61% to 26% and 66% to 28%, respectively), 20p (32% to 7%), and 21q (22% to 2%). All adjusted p values were less than 0.05 for these comparisons. No chromosome-arm gains or losses were significantly more frequent in AA CRC cases than in TCGA cases. The difference in chromosome-arm gain and loss was not the result of segmental gain or loss. However, analysis of genotype data revealed that copy-number neutral LOH in AA CRC cases substantially compensates for the lower frequency of chromosome-arm losses and focal amplifications for the lower frequency of gains. For example, in AAs, for chromosomal region 17p where TP53 localizes, 24% of cases exhibited 17p loss and 26% exhibited copy-number neutral LOH, for a total of 50% TP53 loss. For chromosomal region 13q where CDK8 localizes, 26% of cases exhibited 13q gain and 28% exhibited focal gains around CDK8, for a total of 54% CDK8 gain. The total frequency of tumor-suppressor gene losses and oncogene gains was similar in AA CRC and TCGA cases. Conclusion: These data show that, although the major carcinogenesis genes (as identified by LOH and gene amplification) appear to be similar in AA and TCGA CRC cases, the mechanisms of genomic instability that generate these genetic changes are different. Chromosome-arm losses are more frequent in TCGA CRC cases whereas AA CRC is driven by homologous recombination and segregation resulting in copy-number neutral LOH. Chromosome-arm gains are more frequent in TCGA CRC cases whereas AA CRC is driven by focal amplification, which could result from unequal sister chromatid exchange. Thus, these CNV differences could reflect variation in the regulation of fundamental genomic integrity mechanisms in AAs. More studies are needed to confirm these results and elucidate the mechanisms of carcinogenesis in AA CRC. This abstract is also presented as Poster A62. Citation Format: Gaius J. Augustus, Rosa Xicola, Arthur R. Brothman, Xavier Llor, Nathan A. Ellis. Homologous recombination drives African American colorectal carcinogenesis. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr PR02.

Low vitamin D levels are associated with an increased incidence of colorectal cancer (CRC) and higher mortality from the disease. In the US, African Americans (AAs) have the highest CRC incidence and mortality and the lowest levels of vitamin D. Single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene have been previously associated with CRC, but few studies have included AAs. We studied 795 AA CRC cases and 985 AA controls from Chicago and North Carolina as well as 1324 Caucasian cases and 990 Caucasian controls from Chicago and Spain. We genotyped 54 tagSNPs in VDR (46586959 to 46521297 Mb) and tested for association adjusting for West African ancestry, age, gender, and multiple testing. Untyped markers were imputed using MACH1.0. We analyzed associations by gender and anatomic location in the whole study group as well as by vitamin D intake in the North Carolina AA group. In the joint analysis, none of the SNPs tested was significantly associated with CRC. For four previously tested restriction fragment length polymorphisms, only one (referred to as ApaI), tagged by the SNP rs79628898, had a nominally significant p-value in AAs; none of these polymorphisms were associated with CRC in Caucasians. In the North Carolina AAs, for whom we had vitamin D intake data, we found a significant association between an intronic SNP rs11574041 and vitamin D intake, which is evidence for a VDR gene-environment interaction in AAs. In summary, using a systematic tagSNP approach, we have not found evidence for significant associations between VDR and CRC in AAs or Caucasians.

BACKGROUND: Defective DNA repair has a causal role in hereditary colorectal cancer (CRC). Defects in the base excision repair gene MUTYH are responsible for MUTYH-associated polyposis and CRC predisposition as an autosomal recessive trait. Numerous reports have suggested MUTYH mono-allelic variants to be low penetrance risk alleles. We report a large collaborative meta-analysis to assess and refine CRC risk estimates associated with bi-allelic and mono-allelic MUTYH variants and investigate age and sex influence on risk.METHODS: MUTYH genotype data were included from 20 565 cases and 15 524 controls. Three logistic regression models were tested: a crude model; adjusted for age and sex; adjusted for age, sex and study.RESULTS: All three models produced very similar results. MUTYH bi-allelic carriers demonstrated a 28-fold increase in risk (95% confidence interval (CI): 6.95-115). Significant bi-allelic effects were also observed for G396D and Y179C/G396D compound heterozygotes and a marginal mono-allelic effect for variant Y179C (odds ratio (OR) = 1.34; 95% CI: 1.00-1.80). A pooled meta-analysis of all published and unpublished datasets submitted showed bi-allelic effects for MUTYH, G396D and Y179C (OR = 10.8, 95% CI: 5.02-23.2; OR = 6.47, 95% CI: 2.33-18.0; OR = 3.35, 95% CI: 1.14-9.89) and marginal mono-allelic effect for variants MUTYH (OR = 1.16, 95% CI: 1.00-1.34) and Y179C alone (OR = 1.34, 95% CI: 1.01-1.77).CONCLUSIONS: Overall, this large study refines estimates of disease risk associated with mono-allelic and bi-allelic MUTYH carriers. British Journal of Cancer (2010) 103, 1875-1884. doi:10.1038/sj.bjc.6605966 www.bjcancer.com Published online 9 November 2010 (C) 2010 Cancer Research UK

We tested for germline variants showing association to colon cancer metastasis using a genome-wide association study that compared Ashkenazi Jewish individuals with stage IV metastatic colon cancers versus those with stage I or II non-metastatic colon cancers. In a two-stage study design, we demonstrated significant association to developing metastatic disease for rs60745952, that in Ashkenazi discovery and validation cohorts, respectively, showed an odds ratio (OR) = 2.3 (P = 2.73E-06) and OR = 1.89 (P = 8.05E-04) (exceeding validation threshold of 0.0044). Significant association to metastatic colon cancer was further confirmed by a meta-analysis of rs60745952 in these datasets plus an additional Ashkenazi validation cohort (OR = 1.92; 95% CI: 1.28-2.87), and by a permutation test that demonstrated a significantly longer haplotype surrounding rs60745952 in the stage IV samples. rs60745952, located in an intergenic region on chromosome 4q31.1, and not previously associated with cancer, is, thus, a germline genetic marker for susceptibility to developing colon cancer metastases among Ashkenazi Jews.

This study summarizes our initial experience with prospective, single-amplicon (mutation-specific) A636P testing in Ashkenazi Jewish patients at risk for Hereditary Nonpolyposis Colorectal Cancer (HNPCC).We previously described a founder mutation, MSH2*1906GC (A636P) that causes HNPCC in 8/1345 (0.59%) of Ashkenazim with colorectal cancer. The mutation was more common in Ashkenazim diagnosed ator=40 years (7%).Twenty-seven Ashkenazi probands at risk for HNPCC were ascertained. Single-amplicon A636P testing was performed on 21 by polymerase chain reaction of exon 12 of MSH2, followed by direct DNA sequencing. Mutational analysis of the entire open reading frame of MLH1 and MSH2 was performed on 7 by PCR of each exon, followed by heteroduplex analysis using denaturing high-performance liquid chromatography and direct sequencing of exons with variant chromatographs. One patient received both studies,The A636P mutation was detected in 3/21 (14%) prospectively evaluated patients using single amplicon testing. In 6 patients, the entire open reading frame of MLH1 and MSH2 was analyzed, and 1 additional A636P carrier and 2 carriers of previously unrecognized mutations were identified. The A636P mutation was present in 2 patients who met Amsterdam criteria and in 2 patients who did not.Although rare in the general population, A636P mutations are found at increased frequency in Ashkenazim with a personal or family history of colorectal or other HNPCC-associated cancers. This inexpensive and rapid approach may be useful preoperatively in helping determine the extent of colon resection for a subset of patients.

Background: African Americans (AAs) have the highest incidence of colorectal cancer (CRC) compared to other US populations and more proximal CRCs. The objective is to elucidate the basis of these cancer disparities. Methods: 566 AA and 328 Non-Hispanic White (NHW) CRCs were ascertained in five Chicago hospitals. Clinical and exposure data were collected. Microsatellite instability and BRAF (V600E) and KRAS mutations were tested. Statistical significance of categorical variables was tested by Fisher's exact test or logistic regression and age by Mann-Whitney U test. Results: Over a ten-year period, the median age at diagnosis significantly decreased for both AAs (68 to 61; P Conclusions: Patients with CRC have gotten progressively younger. The excess of CRC 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 CRCs than older ones. These data suggest two different mechanisms driving younger age and proximal location of CRCs in AAs. Citation Format: Rosa Xicola, The Chicago Colorectal Cancer Consortium, Sonia Kupfer, Carol Braunschweig, Nathan Ellis, Xavier Llor. Excess of proximal microsatellite-stable colorectal cancer in African Americans from a multiethnic study. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A75.

Background: Genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with diseases of the colon including colorectal cancer (CRC) and inflammatory bowel diseases (IBD). However, the functional role of many of these SNPs is largely unknown. Expression quantitative trait loci (eQTL) mapping is a tool to identify target genes of disease-associated SNPs. We have comprehensively assessed eQTLs in the human colon and tested for enrichment of GWAS-associated variants of colon diseases. We also characterized the genetic and functional properties of colonic eQTLs. Methods: Distal colonic biopsies were obtained from 48 healthy African American males and total RNA was extracted. Genomic DNA was obtained from the same patients and genotyped using the Axiom Pan-African array. In total, 8.4 million genotyped and imputed SNPs were included. RNA expression levels were estimated using the Illumina HT-12v4 Expression Beadchip. eQTLs were identified using Matrix eQTL. False discovery rate calculations were performed according to the Benjamini and Hochberg method. Significance thresholds for other associations were determined using permutation-based methods. Results: 1941 significant cis-eQTLs, corresponding to 122 independent signals, were identified at a false discovery rate of 0.01. There was substantial overlap between cis-eQTLs found in liver and ileum and less overlap in lymphoblastoid cells, brain, and skin fibroblasts. Overall, there was enrichment for GWAS variants associated with CRC and IBD, as well as 2 metabolic traits–Type 2 diabetes and body mass index–among colon cis-eQTLs. The CRC-associated eQTL rs3802842 was significantly associated with the expression of C11orf93 (COLCA2). The CRC-associated SNP rs1862748 is associated with 2 cis-eQTLs that are associated with expression differences in the NFATC3 gene, which regulates beta-catenin activation in response to WNT signaling. We also identified UBA7, INPP5E, ERAP2, SFMBT1, NXPE1, REXO2 as target genes for IBD-associated variants. There was significant enrichment of colonic cis-eQTLs near the transcription start sites (TSSs), for active histone marks and for SNPs with high population differentiation. Conclusion: Through this comprehensive study of eQTLs in the human colon, we have identified novel and known target genes for IBD- and CRC-associated genetic variants. The cis-eQTLs are useful in identification of the causal functional variation underlying IBD and CRC associations. The eQTL catalog is now expanded to include the human colon and the functional characteristics of colonic eQTLs. Note: This abstract was not presented at the meeting. Citation Format: Sonia S. Kupfer, Imge Hulur, Eric Gamazon, Andrew Skol, Xavier Llor, Kenan Onel, Nathan A. Ellis. Enrichment of colorectal cancer and inflammatory bowel disease risk variants in colon expression quantitative trait loci in African Americans. [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 4582. doi:10.1158/1538-7445.AM2015-4582

Regulation of dormant origin firing under conditions of replication stress is poorly understood. The Bloom syndrome DNA helicase BLM functions in maintaining replication fork stability, with sumoylation regulating this function, and BLM deficiency is associated with increased dormant origin firing. We found here that BLM sumoylation levels increase in response to replication stalling by hydroxyurea (HU) and to proteasome inhibition; depletion of the SUMO-targeted ubiquitin ligase RNF4 also causes increased levels of BLM sumoylation. RNF4 directly interacts with BLM and can ubiquitylate sumoylated BLM in vitro. These data indicate that sumoylated BLM is an RNF4 substrate. RNF4 is recruited to DNA repair foci in response to replication stalling. RNF4 depletion causes the accumulation of increased numbers of HU-induced BLM foci, whereas the numbers of RPA, RAD51, and gamma-H2AX foci are unaffected by fork stalling in RNF4-depleted cells. Sister chromatid exchanges are normal in RNF4-depleted cells. These data indicate that RNF4 can regulate BLM retention at stalled forks without affecting repair by homologous recombination. RNF4 depletion reduces cell proliferation and survival of untreated cells, and it confers hypersensitivity to replication stalling by HU. Studies of cell-cycle progression using bromodeoxyuridine incorporation and flow cytometry show that RNF4 depletion causes a delay of re-entry into S phase in HU-treated cells and the replication delay is partially rescued by BLM mutation. The delay is not caused by modification of checkpoint kinase activities. Analysis of replication dynamics using the DNA fiber assay show that RNF4 depletion causes an increase of permanently stalled replication forks and a decrease in activation of dormant origins following recovery from HU-induced replication stress. Co-depletion of RNF4 and BLM partially rescues these defects. Replication dynamics is unaffected by RNF4 depletion in untreated cells. These data indicate that RNF4 regulates BLM's functions in replication fork stability and dormant origin firing, without affecting BLM's roles in homologous recombination. We propose that sumoylation of BLM at stalled replication forks leads to RNF4-mediated ubiquitylation and subsequent proteasome-dependent degradation of BLM, which relieves BLM's active inhibition of dormant origin firing. Citation Format: Nathan A. Ellis, Wei-Chih Yang, Mary Yagle, Jianmei Zhu, Jing Huang, Michael Seidman, Michael J. Matunis. The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing. [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 3036. doi:10.1158/1538-7445.AM2015-3036

Purpose TGFBR1☆6A is a tumor susceptibility gene that increases breast, colon, and ovarian cancer risk. Fourteen percent of the general population carries TGFBR1☆6A, and TGFBR1☆6A homozygotes have a greater than 100% increased colon cancer risk compared with noncarriers. Low-penetrance genes such as TGFBR1☆6A may account for a sizable proportion of familial colorectal cancer occurrences. To test this hypothesis, we determined whether TGFBR1☆6A contributes to a proportion of mismatch repair (MMR) gene mutation–negative hereditary nonpolyposis colorectal cancer (HNPCC) patients. Patients and Methods A case-case study was performed of 208 index patients with HNPCC meeting the Amsterdam criteria. Patients were examined for mutations and genomic rearrangements in the MLH1, MSH2, and MSH6 genes and genotyped for TGFBR1☆6A. Tumor microsatellite instability status was available for 95 patients. Results A total of 144 patients (69.2%) carried a deleterious mutation and were classified as positive for MMR gene mutation; 64 patients (30.8%) had no evidence of mutations and were classified as MMR negative. TGFBR1☆6A allelic frequency was significantly higher among MMR-negative patients (0.195) than among MMR-positive patients (0.104; P = .011). The proportion of TGFBR1☆6A homozygotes was nine-fold higher among MMR-negative (6.3%) than among MMR-positive patients (0.7%; P = .032). The highest TGFBR1☆6A allelic frequency was found among MMR-negative patients with tumors exhibiting no microsatellite instability (0.211), and the lowest frequency was found among MMR-positive patients with tumors exhibiting microsatellite instability (0.121); the difference was not statistically significant (P = .17). Conclusion TGFBR1☆6A may be causally responsible for a proportion of HNPCC occurrences.

Li-Fraumeni syndrome (LFS) is a dominantly inherited cancer predisposition syndrome characterized by a wide spectrum of neoplasms occurring at young age. Germline mutations in the TP53 tumor suppressor gene have been identified in approximately 71 of LFS patients and 22 of Li-Fraumeni-like (LFL) patients. Mutations within the cell cycle checkpoint gene CHEK2 have also been reported in some patients with LFS, LFL, and phenotypically suggestive of LFS (PS-LFS) not carrying a TP53 mutation. In this study, we show that 7 of the 23 patients with LFS/LFL tested positive for deleterious mutations in p53. Fifteen of the remaining sixteen were not found to carry the CHEK2* 1100delCmutation. These results indicate that CHEK2*1100delC is not a common cause of LFS, LFL, or PS-LFS in North American kindreds not carrying a TP53 mutation. Of note, two patients were found to carry p53* R72P, which is of unknown clinical significance. Lack of segregation of this allele in one of these kindreds provides strong evidence that the R72P allele is not disease-causing. While mutations in p53 account for a proportion of patients with LFS/LFL, future studies are needed to determine if other genes are responsible for LFS/LFL families not carrying germline p53 mutations.

Purpose: Somatic mutations that result in the activation of the growth factor receptor KIT are commonly found in gastrointestinal stromal tumors (GISTs). Six families have been reported in which a germ-line mutation in KIT is associated with an autosomal dominant predisposition to the development of GISTs. Hyperpigmentation, urticaria pigmentosa, and dysphagia have been described in some, but not all, families. Preliminary correlations between the site of mutation and the clinical phenotype have been proposed, but the strength of these associations is not defined. Design: A large kindred with multiple GISTs, hyperpigmentation, and dysphagia was identified after the index case presented with multiple GISTs. A germ-line mutation in KIT (W557R) was identified in an affected cousin, after which a large family meeting was held and testing offered. Clinical data were obtained by interview and, whenever possible, medical record documentation. Results: To date, 19 individuals have been tested, and the mutation has been shown to cosegregate with the syndrome. The phenotypic expression, however, is variable. GISTs, often presenting as upper gastrointestinal bleeding, and hyperpigmentation are common, but not diagnosed in all documented or obligate carriers. Dysphagia is a less prevalent complaint. The diagnosis of GISTs appears to be made at a younger age in more recent generations. Metastatic disease is uncommon. Conclusions: A germ-line mutation in KIT resulting in an amino acid substitution in the juxtamembrane region is associated with a syndrome of GIST, hyperpigmentation, and dysphagia, although the prominence of each component varies.

Germline mutations in MSH6 can cause HNPCC, which is associated with a tumor phenotype featuring MSI. However, tumors arising in persons with disease-causing mutations of MSH6 may or may not exhibit MSI. We used D-HPLC to screen for germline mutations in the promoter region, the coding region and the 3'-UTR of MSH6. Eighty-four families, enrolled on the basis of Amsterdam I and II criteria (HNPCC families) and less stringent criteria (HNPCC-like families), were tested for MMR gene mutations; 27 families had a disease-causing mutation in MLH1 or MSH2, and the remaining 57 families were tested for mutations in MSH6. Two protein-truncating mutations were identified in each of 2 families fulfilling the Amsterdam I criteria, being present in persons affected with early-onset colorectal cancers exhibiting MSI. Immunohistochemical analysis showed that expression of both MSH2 and MSH6 proteins was lost in the cancer cells of the 2 mutation carriers but only MSH6 protein expression was lost in 2 adenomatous polyps. A third possibly disease-causing mutation was found in a person affected with a tumor that did not exhibit MSI. In addition, we found 4 new polymorphisms and determined that neither of the 2 studied by association analysis conferred susceptibility to colorectal or endometrial cancer. Altogether, our results indicate that disease-causing germline mutations of MSH6 are rare in HNPCC and HNPCC-like families.

Although it is widely stated that 5-10% of all breast cancers arise as the result of an inherited predisposition, the prevalence of mutations in BRCA1 or BRCA2 in unselected ascertainments of women with breast cancer is somewhat lower. In two population based series from the United States, presumably deleterious BRCA1 mutations were identified in only 14 female breast cancer patients out of a combined total of 884 women (1.6%).1,2 In two European series, BRCA1 or BRCA2 mutations were identified in 19/1035 (1.6%) Finnish breast cancer patients3 and 24/1220 (2.0%) breast cancer patients from the UK.4 Because of the low prevalence of detectable mutations, screening of unselected breast cancer patients has not been recommended. Mutation analysis is more often suggested for specific groups of breast cancer patients in whom mutations are more likely to be detected. The decision whether or not to offer genetic testing usually revolves around the presence or absence of a significant family history of breast or ovarian cancer, although age at diagnosis, bilaterality, and ethnicity may be important considerations. A negative family history, however, clearly does not exclude the presence of a germline mutation in BRCA1 or BRCA2 . In a population based series from the United Kingdom, none of the 13 mutation carriers diagnosed with breast cancer before the age of 36 was reported to have had a family history of breast or ovarian cancer in first degree relatives.5 In contrast, in a series from the United States, most mutation carriers in a group of women with early onset breast cancer had a history of breast cancer in either first or second degree relatives.6 However, even in this series, one of the mutation carriers had no family history of breast cancer. The lack of a family history among mutation carriers …

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

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

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, with the minor allele of rs73913755 conferring increased protection against L-CRC. We hypothesize that the minor allele of rs73913755 causes a reduction in the expression levels of CYP24A1. We further hypothesize that there is a gradient of expression levels of CYP24A1 in the colon with higher levels on the left side than on the right side. The protective effect of rs73913755 would then be explained by increased levels of calcidiol in the tissue in combination with a threshold effect. Because rs73913755 is African-specific, 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. This abstract is also presented as Poster B72. Citation Format: Fabio Pibiri, Rick A. Kittles, Robert S. Sandler, Temitope O. Keku, Sonia S. Kupfer, Rosa Xicola, Xavier Llor, Nathan A. Ellis. Genetic variation in the vitamin D pathway and risk for colorectal cancer in African Americans. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PR02.

Background: Genome-wide association studies (GWAS) of colorectal cancer (CRC) have identified risk variants in candidate genes involved in TGF-ß signaling including BMP4, GREM1, CDH1, RHPN2 and SMAD7. We previously replicated the association with a 3′-UTR SNP in GREM1 (rs10318) in African Americans but did not find evidence of associations in the other genes. This finding could be explained by different genomic structure in African Americans. Therefore, we sought to fine-map these genes to identify CRC risk variants in African Americans who are at highest risk of CRC in the US. Methods: DNA from cases and controls was obtained from two US institutions. In total, we included DNA from 795 AA cases and 985 AA controls. TagSNPs with minor allele frequencies >5% and pairwise r2 >0.8 were identified in five genes using YRI Hapmap data. A total of 81 SNPs were genotyped in cases and controls using Sequenom MassARRAY. We calculated odds ratios and 95% confidence intervals using logistic regression controlling for ancestry, age, gender and multiple testing in PLINK. Results: Association analysis was completed for four genes: BMP4, GREM1, CDH1 and RHPN2. The top tagSNP associations for each gene are shown in the Table. None of the SNPs were significant after permutation testing. In GREM1, the strongest SNP associations were found at the 3′ end of the gene. In BMP4, we identified two SNPs (rs1957852 and rs7146040) that are in LD with rs4444235 previously identified in a European GWAS. In CDH1, rs12931189 is located in the same LD block as the strongest association signal (rs7199991) in a recent fine-mapping study in Europeans. Finally, we have no evidence for associations in RHPN2 in African Americans. Conclusion: These results provide preliminary evidence of associations in African American colorectal cancer patients in BMP4, GREM1 and CDH1 identified by GWAS in Europeans. Ongoing work is testing associations in SMAD7 and further fine-mapping by imputation and resequencing in these regions. 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 1661. doi:1538-7445.AM2012-1661

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: Colorectal cancer (CRC) is the third leading cause of cancer-related death in both sexes in the United States. While the overall CRC incidence rates have declined over the past 10 years, the disparity between African Americans (AAs) and non-Hispanic whites has increased and the incidence in AAs remains higher than in other populations. Very few studies have specifically addressed CRC in AAs, and important studies on CRC almost never include significant numbers of AAs to draw any firm conclusions. Objective: To establish a large, robust and well-characterized database and bio-repository collection of CRC from AA patients and controls strengthening a collaborative working group, the Chicago Colorectal Cancer Consortium (CCCC), which spans 5 of the main hospitals of the Chicago area. We collect clinical, demographic and dietary data; and a biological repository of specimens consisting of tumor and non-tumor genomic DNA, RNA, plasma, serum, and paraffin-embedded samples. Specific Aims: The primary goal of the project is to study how genetic and environmental factors and their interaction result in the development of CRC. We will also study prognosis determinants in the AA population. Results: Recruitment and data collection: So far we have recruited 56 AA and 17 white-non Hispanic adenocarcinoma colorectal patients. We have also recruited 84 AAs and 32 white patients with adenomatous polyps, and 44 AAs and 30 white controls (individuals without polyps or previous history of polyps). From all individuals we have collected demographic/socioeconomic data, clinical data, family history of cancer, medication and toxic substance history, and dietary data using the Block Brief 2000 Food Frequency Questionnaire. Preliminary analyses: To identify potential variables that could influence the cancer disparity between AAs and whites, we performed a preliminary analysis of all these data using a Fisher exact test. A systematic molecular analysis of BRAF and KRAS mutations, microsatellite instability and presence of CpG island methylation phenotype in all CRC tumors is being performed. We are also analyzing protein expression of the mismatch repair genes through immunohistochemistry. We will test for an association between these molecular features and clinical variables. Moreover, 1 year follow-up data is already available and being collected on 23 cases. Therefore, we will soon be able to start investigating the relationship of those molecular features with survival and specific chemotherapy response. Summary: The preliminary analysis has highlighted some significant differences between AA and white CRC patients. In comparison with white patients, a higher proportion of AA patients are less than 50 years at diagnosis, take NSAIDS, and fulfill Bethesda criteria, which is a set of criteria to select out patients to be pre-screened for mismatch repair deficiency and eventually for genetic testing to rule out Lynch syndrome. It has also highlighted that CRC patients have a lower level of education than cancer-free individuals in both ethnicities. This type of sociocultural disparities could potentially be associated with different life styles. In fact, AA CRC cases consumed more carbohydrates than controls reflecting this potential life style difference. Cancer Relevance: Understanding the key molecular features and environmental factors that impact CRC development in the AA population is of paramount importance given the scarce information available. Understanding the role genetic factors will be crucial to help risk stratify patients, recommend screening modalities, and recommend preventive measures. When this information is combined with environmental factors, such as dietary habits, we will be able to offer a comprehensive personalized medicine approach for AA CRC patients. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B73.