Background Sickle Cell Disease (SCD) is an inherited blood disorder that leads to hemolytic anemia, pain, organ damage and early mortality. It is characterized by polymerized deoxygenated hemoglobin, rigid sickle red blood cells and vaso-occlusive crises (VOC). Recurrent hypoxia-reperfusion injury in the gut of SCD patients could increase tissue injury, permeability, and bacterial translocation. In this context, the gut microbiome, a major player in health and disease, might have significant impact. This study sought to characterize the gut microbiome in SCD. Methods Stool and saliva samples were collected from healthy controls (n = 14) and SCD subjects (n = 14). Stool samples were also collected from humanized SCD murine models including Berk, Townes and corresponding control mice. Amplified 16S rDNA was used for bacterial composition analysis using Next Generation Sequencing (NGS). Pairwise group analyses established differential bacterial groups at many taxonomy levels. Bacterial group abundance and differentials were established using DeSeq software. Results A major dysbiosis was observed in SCD patients. The Firmicutes/Bacteroidetes ratio was lower in these patients. The following bacterial families were more abundant in SCD patients: Acetobacteraceae, Acidaminococcaceae, Candidatus Saccharibacteria, Peptostreptococcaceae, Bifidobacteriaceae, Veillonellaceae, Actinomycetaceae, Clostridiales, Bacteroidacbactereae and Fusobacteriaceae. This dysbiosis translated into 420 different operational taxonomic units (OTUs). Townes SCD mice also displayed gut microbiome dysbiosis as seen in human SCD. Conclusion A major dysbiosis was observed in SCD patients for bacteria that are known strong pro-inflammatory triggers. The Townes mouse showed dysbiosis as well and might serve as a good model to study gut microbiome modulation and its impact on SCD pathophysiology.

Abstract Background: Streptococcus sp. VT_162 (SPV) were identified by our group showed neoplastic transformation in patients with colorectal cancer (CRC). This strain's genome contains 10 virulence factors that match S. pyogenes and 3 different strains of S. pneumomia virulence factors. Hence, we aimed to determine whether (SPV) influences the expression of carcinogenic genes in several human colon cell lines. Methods: colon cell lines (HCT116, SW480 and LoVo) were infected with (SPV) at a multiplicity of infection (MOI) of 10 or 50.RNA extracts were used on an Illumina sequencer and the mRNA expression profiles were compared to cells that were not exposed to (SPV). Sequencing data were processed according to our previous studies. CutAdapt was used to trim Illumina adapters. Reads were aligned to the UCSC hg38 reference sequence using Bowtie2. Sequencing reads were assembled and annotated. To estimate differential expression between different samples, the count data were used in DESeq2 R package and the comparisons were performed. mRNAs with statistically significant (p<0.05) expression difference from untreated cell lines and log 2 FC >3 are reported and the relation to cancer of the most upregulated mRNAs was investigated. Results: An in-silico analysis of (SPV) genome revealed that the strain contains ten (10) putative virulence factors that match those of S. pyogenes and three (3) of different strains of S. pneumomia. A MOI of 10, a total of 427 mRNAs displayed a >3 fold upregulation of their expression. The top 20 upregulated genes were: HSPA6, CSF2, SPRR2D, SERPINB7, CXCL8, LCN2, SBSN, HSPA7, SERPINA3, SLC6A12, RSPH10B, ITGAM, BCRP3, CRYAB, GCM1, TNFRSF9, IGFN1, SNAI2, LUCAT1 and SASH3 with the following respective fold differences: 8.44, 6.75, 6.68, 6.56, 6.23, 5.91, 5.81, 5.56, 5.25, 5.21, 5.19, 5.12, 5.06, 5.03, 5.02, 5.01, 4.99, 4.97, 4.93, 4.91, most have shown involve in many other aggressive cancer, poor survival and prognosis. Three of these mRNA HSPA6, HSPA7 and CRAB are heat shock proteins reflecting that the bacterium is perceived as a stressor to the cells. Some of the upregulated mRNA have no known roles and might correspond to novel, specifically-SPV carcinogenic pathways. Conclusion: SPV infection led to major alterations in the genome expression profiles in the colon cell lines. Many of the upregulated mRNA have known carcinogenic effects and associate with poor prognosis and survival in different organ systems. These findings increase our understanding on the targets by which (SPV) contributes to colorectal cancer transformation and need to be validated in clinical specimens. Citation Format: Hassan Brim, Hamid Boulares, Mohammad Daremipouran, Edward Lee, Hassan Ashktorab. Streptococcus sp. VT_162 infection of colon cancer cell lines induces mRNAs that associate with poor prognosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6102.

in occludin, ZO-1, JAM, and claudin1 in the MS group, but the change was suppressed in the MS group ingesting Asx.In addition, mRNA expression of tight junction protein in the large intestine was not significantly changed between the MS group and the C group.The principal coordinate analysis of microbiota composition in cecal contents clearly distinguished the Asx group of Group C from the regular diet group.Lactobacillales increased, and Bacteroidales decreased in the MS group compared to the C group, but these changes were canceled by Asx intake.The abundance of Paraprevotella was positively correlated with ZO-1 (r=0.31), and that of Bacteroides was negatively correlated with occludin (r=-0.61)and ZO-1 (r=-0.51)expression in the small intestine.[Conclusion] Changes in the expression of tight junction protein in the small intestine were observed in the MS group, and it was attenuated by Asx ingestion.Moreover, that was correlated with the relative abundance of various taxonomic groups in the gut microbiota.

Background: Prostate cancer (PCa) is a multifactorial disease involving complex interactions between genetic and physiological/environmental factors. Vitamin D receptor (VDR) plays a role in numerous cellular pathways and it has been suggested that VDR genetic variants influence individual susceptibility to PCa. Materials and Methods: Logistic regression analysis was used to assess the association of six VDR single nucleotide polymorphisms (SNPs) and factors such as tanning potential and UV sunlight exposure with PCa risk. Results: Marginal significant interactions were found, with a 2-fold increase risk of PCa between SNP 1 (c278-69G>A) and sunlight UV exposure [odds ratio (OR)=2 .02, 95% confidence interval (CI)=1.036-436; p=0.05]; and a 4-fold increase risk of PCa between SNP 4 (c.907+75C>T) and tanning potential (OR=4.40, 95% CI=0.89-29.12; p=0.0591). In contrast, SNP 5 (rs731236, Taq1) and tanning potential interaction had a protective effect by reducing the risk of PCa by 55% (beta=-0.804; OR=0.448, 95% CI=0.197-9.42; p=0.0427). SNPs 2 (rs61614328) and 6 (rs533037428) did not show any association with PCa even in the presence of UV sunlight exposure. Conclusion: The protective effect of SNP 4 from PCa is lost and modified by tanning potential in African Americans. This finding needs to be verified by larger studies in different ethnic populations.

Abstract We have previously demonstrated that Adiponectin, leptin, TNF-alpha, and IGF-1 were highly prevalent in patients with colon adenomas when compared to healthy individuals. In the present study, we aimed to establish specific miRNA signatures in obese and diabetic African American patients developing colorectal adenomas. Patients & Methods: Serum samples from 72 individuals (18 normal, 18 adenoma-obese, 18 adenoma-diabetic, and 18 adenoma-obese-diabetic) seen at Howard University Hospital were collected. All selected patients were age and gender-matched across the 4 groups. Serum RNA extracts were used for library preparation and sequencing on an Illumina NextSeq 500 using SR75 High Output kit. Sequencing data were processed through the miasma-Seq pipeline. CutAdapt was used to trim Illumina adapters and remove reads that were less than 15 bp long. Reads were aligned to the UCSC hg38 human genome reference sequence using Bowtie2. Annotation data in the form of a GFF file from mirBase was used to count the number of reads falling in each hairpin locus. To estimate differential expression between different groups of samples, the count data were used in the DESeq2 R package and the 4 patient groups comparisons were performed. MiRNAs with statistically significant (p < 0.05) expression difference from normal and log 2 FC > 2 and/or < -2 were selected for functional analysis using Ingenuity Pathway Analysis (IPA). Results: Comparisons to normals revealed that 11 miRNAs [hsa-mir-34a, hsa-mir-133a-1, hsa-mir-127, hsa-mir-99b, hsa-mir-485, hsa-mir-378d-2, hsa-mir-3168, hsa-mir-3679, hsa-mir-378e, hsa-mir-4635, hsa-mir-6509] were down-regulated in Adenoma-Obese; 3 miRNAs were down-regulated [hsa-mir-1228, hsa-mir-3912, hsa-mir-4665] and 3 up-regulated [hsa-mir-455, hsa-mir-219b, hsa-mir-8072 ] in Adenoma-Diabetic patients; and 2 down-regulated [hsa-mir-487b, hsa-mir-6777] and 1 up-regulated miRNA [hsa-mir-4525] in Adenoma-Obese-Diabetic patients. The IPA analysis revealed that many of the altered miRNA have known roles in carcinogenic and metabolic syndrome pathways. Conclusion: From the pairwise comparison, diabetes seems to be associated with more alterations of the miRNA profile in the analyzed samples when compared to patients developing colon adenomas on an obesity background. MiRNA-4525 can be used as a potential serum marker in obese patients while miRNA-455, miRNA-219b, and miRNA-8072 can serve as such in diabetic patients. Except for miRNA-455 that was previously described as a promotor of migration and invasion in breast cancer, the other mi-RNAs are novel and their mechanisms of action need further study. Citation Format: Hassan Brim, Mohammad Daremipouran, Gail Nunlee-Bland, Adeyinka Laiyemo, Hassan Ashktorab. Serum analysis of obese and diabetic African American colon adenoma patients reveals novel potential miRNA markers of carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2578.

Background/Aim: Vitamin D receptor (VDR) is present in numerous cellular pathways and it has been suggested that VDR genetic variants influence individual susceptibility to prostate cancer. Also, analyses of single nucleotide polymorphisms (SNPs) in VDR revealed ethnicity-associated polymorphisms. The aim of this study was to identify VDR SNPs in African American men with and without prostate cancer. Materials and Methods: The entire VDR gene was screened for germline mutations in a casecontrol study by denaturing high performance liquid chromatography and DNA sequencing. Logistic regression was used to estimate the association of SNPs, age, family history, and Gleason score with prostate cancer risk. Results: Six SNPs in the non-coding regions, and one SNP in the coding region, were detected. SNP 1 (c.278-69G>A) and SNP 4 (c.907+75C>T) have not been previously reported. SNP 4 had a significant protective effect (beta=-0.6, p<0.05); whereas, SNP 7 (rs7975232) showed an increase association with prostate cancer risk and high Gleason score (beta=0.32, p<0.05). SNP 4, SNP 7 and age were better predictors of prostate cancer risk than family history with a high degree of sensitivity (74.7%) and specificity (92.4%). Conclusion: SNP 4 and SNP 7 could be promising markers for prediction of reduced or increased prostate cancer risk, respectively.

Abstract Background: The recent introduction of the cancer hotspot panel can help enhance targeted cancer therapy, but requires further refinement to identify which genes have significant clinical value for the population of interest. We aim to determine such genes by identifying somatic alterations in the 48 cancer gene panel in African Americans. Methods: Tumor matched normal tissues (n=16) from African American colorectal cancer patients were analyzed using a 48 hotspot cancer panel by Ion Torrent sequencing. Variants of the genes found in TruSeq Amplicon - Cancer Panel (TSACP) were identified and validated. The type of variant was determined using bioinformatic pipelines including Ensembl's Variant Effect Predictor. The genes with relevant clinical value were determined based on frequency of somatic variants and results from previous studies. Results: Somatic variants were detected in 39/48 (82%) genes. Of the 39 genes, the 7 with the most clinical value were selected for further analysis. Overall, 46 novel and known hotspot somatic variants were determined in the following 7 genes: KRAS (7/46 [15%]), NRAS (2/46 [4%]), MLH1 (1/46 [2%]), PIK3CA (8/46 [18%]), SMAD4 (6/46 [13%]), RET (9/46 [20%]), and TP53 (13/46 [28%]). Of the KRAS variants, 57% (4/7) were found in codons 12 and 13 while the other 43% (3/7) were found in codons 117 and 146. Out of the 46 somatic variants, 30% (14/46) were in hotspot regions. Of the KRAS hotspot variants, 100% (4/4) were in codons 12 and 13. The 2 NRAS variants were in codons 59 and 61. Lastly, the 9 genes without variants in our population are CDKN2A, SMARCB1, AKT1, ALK, FGFR2, IDH2, CSF1R, HNF1A and MPL. Conclusion: We have identified novel and known hotspot somatic variants in African Americans with colorectal lesions. Patients with significant hotspot variants, primarily missense alterations, had a worse prognosis. As a result, cancer gene panel hotspot profile should be taken into consideration when treating African American CRC patients. Citation Format: Hassan Brim, Payaam Tavakoli, Mohammad Reza Daremipouran, Babak Shokrani, Edward Lee, Mehdi Nouraie, Adeinko O. Laiyemo, Hassan Ashktorab. Identification of novel and known hotspot variants in African Americans with colorectal cancer [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 647.

Background/Aim: Denaturing high-performance liquid chromatography (DHPLC) is a technique that is used to detect mutations. The aim of the present study was to determine whether DHPLC elution patterns of vitamin D receptor (VDR) gene PCR products can serve as indicators of susceptibility to prostate cancer (PCa) risk. Materials and Methods: DNA samples of PCa cases and controls were screened for mutations and/or polymorphisms in coding exons of VDR gene using DHPLC analysis. Logistic regression, phi-coefficient (f), and Backward Wald models were used to analyze the data. Results: Similar elution patterns of exons 1, 6, 7 and 9 along with higher prevalence of heteroduplex DNA were observed in PCa samples than in controls. Exons 4 and 8 had highly significant protective effects (p<0.05). Whereas, exons 5, 7, and 9 were perfectly positively correlated with PCa risk (phi=1), thus presenting candidate exons significantly associated with susceptibility to PCa. Conclusion: DHPLC elution patterns of the selected exons could be useful to predict susceptibility to develop PCa.

Background: In order to promote positive health behavior of the population, public health outreach is often carried out in various forms to reach the target stakeholders. However, the effectiveness of these outreach programs is unclear. Aim: To determine up-to-date adherence to colorectal cancer (CRC) screening guidelines among US adults according to the trust they have in various sources of health information. Methods: We used the 2007 Health Information National Trends Survey (HINTS) and identified 4342 respondents (weighted population size = 82,811,829) who were at least 50 years old. Respondents expressed the degree of trust they have in various sources of health related information. We considered a source as trusted when it is trusted “a lot” or had “some trust” and regarded as not trusted if it was trusted “a little” or “not at all”. We defined being current with CRC screening as the use of fecal occult blood testing (FOBT) within 1 year, sigmoidoscopy within 5 years, or colonoscopy within 10 years. We used logistic regression models to calculate odds ratios (OR) and 95% confidence intervals (CI). Survey weights were used in all analyses. Results: Approximately 94.3% of respondents trusted their doctors, 59.3% trusted family or friends, 50.2% trusted newsmagazines, 32.7% trusted radio, 66.2% trusted internet, 40.6% trusted television, 68.5% trusted government sources, 44.3% trusted charity organization and 34.5% trusted religious organizations. When compared with those who did not trust the source of health information, trust in radio (64.7% vs 60.7%; OR = 1.24; 95%CI: 1.01-1.52), internet (64.0% vs 58.3%; OR = 1.26; 95%CI: 1.04-1.53), television (64.4% vs 60.9%; OR = 1.23; 95%CI: 1.02-1.48), and government sources (64.0% vs 58.2%; OR = 1.32; 95%CI: 1.09-1.60) were associated with being up-to-date with colorectal cancer screening whereas trust in doctors (63.2% vs 49.2%; OR = 1.53; 95%CI: 0.92-2.55), family or friends (61.5% vs 63.1%; OR = 0.98; 95%CI: 0.84-1.15), newsmagazines (64.0% vs 60.8%; OR = 1.14; 95%CI: 0.98-1.32), charity organizations (62.0% vs 62.1%; OR = 1.04; 95%CI: 0.86-1.24) and religious organizations (59.7% vs 63.3%; OR = 0.90; 95%CI: 0.74-1.09) were not. Conclusion: Even with modest trust in the source of health information, outreach modalities which engage people repeatedly and those that they can readily access over and over again may exert substantial influence on the uptake of colorectal cancer screening. Multiple outreach methods should be used to promote colorectal cancer screening. Citation Format: Adeyinka O. Laiyemo, Hamidat Segunmaru, Jessica Rogers, Mohammad Daremipouran, Clinton Burnside, Florencia Gonzalez, Cherie Spencer, Carla D. Williams. Trust in health information source and colorectal cancer screening uptake among U.S. adults. [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 2578.

Abstract Background: The uptake of Human Papillomavirus (HPV) vaccination needs improvement. However, efforts to improve uptake of HPV vaccine is often concentrated on women. Although men can influence the uptake of the vaccine by their children, the opinion of adult men in the United States on HPV vaccination has not been well studied. Aim: To evaluate the willingness of adult men to vaccinate their daughters for HPV Methods: Health Information National Trends Survey (HINTS) 2007 was used to capture the opinion of 1,927 men (weighted population size = 72,633,670) who answered questions on their willingness to let their daughters receive HPV vaccine. We compared the characteristics of those who were willing to let their daughters receive HPV vaccine with those who were not. We used survey weights in all analyses, and variance estimations were performed using Taylor series linearization to account for the complex survey design. We used logistic regression models to calculate odds ratios (OR) and 95% confidence intervals (CI). Results: A total of 1,554 (80.7%) men were willing to let their daughters receive HPV vaccine while 373 (19.3%) would not let their daughters receive the vaccine. The most common reasons for disagreeing with vaccination were: not knowing enough about it (27.7%); vaccine safety concerns (16.5%); and “my child is not sexually active” (13.7%). Of note, less than 1% felt the vaccine will promote promiscuity. Demographic (age, race, marital status, education status, and health insurance) and lifestyle characteristics (smoking and body mass index) did not predict men who would let their daughters get HPV vaccine. Conclusion: A high percentage of men are willing to let their daughters receive HPV vaccine suggesting that all men should be engaged in the conversation to improve HPV vaccine uptake in the United States. Primary care physicians should make it a priority to discuss the HPV vaccine with men and women more frequently to enlighten the population and allay their fears about the safety of the vaccine. Citation Format: Jessica Rogers, Kolapo A. Idowu, Rahmatalai Coker, Hamidat Segunmaru, Mohammad Daremipouran, Clinton Burnside, Carla D. Williams, Adeyinka O. Laiyemo. Characteristics of adult men who are willing to give HPV vaccine to their daughters in the United States: a national survey. [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 851. doi:10.1158/1538-7445.AM2015-851

BACKGROUND/AIM:Vitamin D deficiency in African-Americans is common due to the high melanin content of the skin that reduces the absorption of UV radiation. To determine if there is a correlation between UV exposure, tanning potential and vitamin D with prostate cancer (PC) risk, we conducted a case-control study of 183 African-American men aged 40 years and older residing in the Washington, DC area.PATIENTS AND METHODS:PC status was described as a binary variable as the presence or absence of cancer and the environmental factors as continuous variables. We used a logistic regression model describing PC as the response, while age, tanning potential, sunlight and vitamin D were treated as the predictors.RESULTS:Men aged 60 years and older had a seven-fold increased risk for developing PC compared to those aged 50 years and less (p<0.003). Tanning potential was a significant (p=0.05) risk factor for PC, while sunlight exposure and vitamin D were not. Tanning potential was also significant (p=0.044) when adjusted for vitamin D and age. However, tanning potential was only marginally significant when adjusted for sunlight exposure (p=0.064) CONCLUSION: The findings of this study indicate that tanning potential may be a predictor for PC risk in African-American men.

BACKGROUNDThe purpose of this study was to identify genome‐wide single nucleotide variants and mutations in African American patients with colorectal cancer (CRC). There is a need of such studies in African Americans, because they display a higher incidence of aggressive CRC tumors.METHODSWe performed whole exome sequencing (WES) on DNA from 12 normal/tumor pairs of African American CRC patient tissues. Data analysis was performed using the software package GATK (Genome Analysis Tool Kit). Normative population databases (eg, 1000 Genomes SNP database, dbSNP, and HapMap) were used for comparison. Variants were annotated using analysis of variance and were validated via Sanger sequencing.RESULTSWe identified somatic mutations in genes that are known targets in CRC such as APC, BRAF, KRAS, and PIK3CA. We detected novel alterations in the Wnt pathway gene, APC, within its exon 15, of which mutations are highly associated with CRC.CONCLUSIONSThis WES study in African American patients with CRC provides insight into the identification of novel somatic mutations in APC. Our data suggest an association between specific mutations in the Wnt signaling pathway and an increased risk of CRC. The analysis of the pathogenicity of these novel variants may shed light on the aggressive nature of CRC in African Americans. Cancer 2015;121:34–42. © 2014 American Cancer Society.

endoscopy with gastric biopsies and sPGI and sG 17 determination at baseline.All patients underwent eradication therapy.Serum sPGI and sG 17 were measured againa after 6 months, and at 1, 2, 3, 5 and 6 yrs after eradication therapy.RESULTS: Mean sPGI levels prior to eradication were 13,4 microgr/L (range: 1,5-24 microgr/L).6 months after eradication therapy, mean sPGI levels significantly increase to 16,6 microgr/L (p=0.05).At the completion of the study, 6 yrs after eradication, sPGI levels increased to 27,3 microgr/L (p=0.01).Conversely, the sG 17 dropped out from 84,8 at baseline to 67,6 pmol/L after the 6 yrs follow up period (p,0.01).5 patients (4 female, mean age 56, range 49-66 yrs) out of the 74 sample atrophic gastritis subjects experienced a 3 month period of treatment with Acetium 100 mg at dosage of 3 capsules daily before eating.In all, both sPGI and sG 17 were reduced at baseline and after 3 months period of treatment.The mean levels of sPGI increases from 5,3 microgr/L at baseline to 7,8 after 90 days of Acetium intake as well as sG 17 drop out from 74,6 to 79,8 pmol /L.CONCLUSION: After H.Pylori eradication subjects with body atrophic gastritis showed long-lasting improvement of physiological gastric function, reflected by significantly and stabile increasing of sPGi levels and a parallel decreased of sG 17 over a 6-year period of follow up.

Abstract Background: Several driver mutations have been discovered in colorectal cancer (CRC) progressions including KRAS and BRAF that has a practical significant therapeutic and prognostic value. Sequencing technology has advanced and now provides a tool for the discovery of novel driver mutations. In addition, sequencing technologies now provide a tool for whole genome methylation analysis. Here, we performed whole exome sequencing (WES) and whole genome methylation analysis to elucidate the involvement of novel candidate genes the KRAS pathway that may effect colorectal carcinogenesis. Patients and Methods: WES was carried out on genomic DNA extracted from 8 normal-tumor pairs of frozen biopsies from African Americans patients with CRC. WES and Reduced Representation Bisulfite Sequencing (RRBS) were performed. Pyrosequencing and sanger sequencing used for validation of methylation and single nucleotide variants (SNV) respectively. For WES, base call quality recalibration, realignment around indels, SNV calling and variant call recalibration were carried out using Genome Analysis Tool Kit. Variants were then annotated using Annovar. Results: WES uncovered somatic mutations alteration in many genes that are known be mutated in CRC including APC, BRAF, KRAS, Notch1, PIK3C2A, and NDRG4. We discovered a number of Novel SNVs in EID3, RGS3, HNRNPF, and GNAS in tumor samples. One GNAS missense mutation was discovered among KRAS mutated tumors. In addition, the tumor with the GNAS mutation was significantly hypomethylated (P<0.05) in the CpG sites of GNAS promoter as compared with paired normal tissue. The tumor was located in the cecum and identified to be invasive adenocarcinoma with stage IV1b (T4b, N2a, M1). Ingenuity pathway analysis (IPA) showed that GNAS were involved in CRC metastasis signaling via APC, BRAF, GSK3A, KRAS, MLH1, MLH2, MLH3, Notch family, and PIK3C family. Conclusion: This work provides insight into identification of novel somatic mutations in GNAS coupled with promoter hypomethylation at the same locus using WES and RRBS. GNAS SNV resulted in gain of function of G-protein signaling that may play a pivotal role in CRC Identification the biological significance of GNAS in CRC may introduce a new target for colorectal cancer diagnosis and treatment. Citation Format: Hamed Rahi, Sohaila Soltani, Mohammad Daremipouran, Hassan Brim, Eward L. Lee, Alfreda Woods, Wayne Frederick, Adeyinka O. Laiyemo, Joe Devaney, Ron Leavitt, Xueguang Sun, Hassan Ashktorab. Novel mutation and hypomethylation define distinct biological subgroups of altered KRAS colon tumors. [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 1934. doi:10.1158/1538-7445.AM2013-1934

Abstract Background: Whole Exome sequencing (WES) is a tool that is revolutionizing screening for pathogeni single nucleotide variations (SNV) in complex disorders such as cancers. Using matched normal-tumor pairs, it is possible to identify exome-wide germline and somatic variant alterations that may have an effect on disease progression or response to different interventions. The existing analysis pipelines are under continuous enhancements. In analyzing matched pairs, there is a critical assumption that the sequenced data are matched, without any quality check. Identification of germline and rare somatic variants depend on the normal sample being the qualified matched pair. Our aim was to determine if Identity By State (IBS), a genetics concept on measuring relatedness between individuals (matched tumor-normal pairs) can assess somatic landscape between individual using WES data. Materials and Methods: Genomic DNA was extracted from 8 normal-tumor pair tissues from African Americans (males n=3, females n=5) with colorectal cancer (CRC). WES was performed for identification of SNV according to manufacturer recommendations (Illumina HiScan SQ). The low quality reads were discarded and the clean reads were aligned against the human reference genome (HG19) and base quality calibration was completed using SAMTools, and GATK for variant calling and annotation. Pairs with no shared allele, one shared allele, and two shared allele assigned as IBS 0, IBS 1, and IBS 2 respectfully. Alleles in the dataset were coded using A and B and pairwise IBS was computed between all samples. Display of the IBS landscape was done using GenomeRelator. Results: The frequency of IBS from WGS data sets showed that most of the changes were IBS-1 which were heterozygous variants; i.e. AA/BB>AB (somatic) or AB>AA/BB (LOH). Frequency of LOH varied from 4.36% to 73.61%. Homozygous variant (i.e. IBS-0, AA>BB) on the other hand was not common, from 0.00% to 4.17%. Distribution of IBS across genome for matched pairs reveals that most of the IBS were IBS-2, indicating that both alleles between matched normal and tumor were similar. Frequency of IBS-1 and IBS-0 varied amongst the samples. Sample CC1053 had the most allele changes in chr1-22, and Xq, and sample CC1054 showed the second highest allele changes in chr1,3,4,6,8,9,10,11,13, 16, 17,19,20 and 22. Conclusion: Our results showed that the SNV difference between normal-tumor matched pair is relatively small consistent with the assumption of low mutation rate in cancer. IBS approach provides the tumor contents as well as assurance in the selection of samples for sequencing using SNP data. In our samples 88% similarly was found, as shown in IBS landscape across the chromosomes. We concluded that our matched pair samples are appropriately selected and are suitable for analysis of mutation across the samples. Citation Format: Mohammad Daremipouran, Hassan Hassanzadeh Namin, Sohaila Soltani, Joe Devaney, Wayne Frederick, Edward L. Lee, Hassan Brim, Hassan Ashktorab. Paired analysis of matched colon normal and tumor using whole exome sequencing. [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 5307. doi:10.1158/1538-7445.AM2013-5307

CpG Island Methylator Phenotype (CIMP) is one of the underlying mechanisms in colorectal cancer (CRC). This study aimed to define a methylome signature in CRC through a methylation microarray analysis and a compilation of promising CIMP markers from the literature. Illumina HumanMethylation27 (IHM27) array data was generated and analyzed based on statistical differences in methylation data (1st approach) or based on overall differences in methylation percentages using lower 95% CI (2nd approach). Pyrosequencing was performed for the validation of nine genes. A meta-analysis was used to identify CIMP and non-CIMP markers that were hypermethylated in CRC but did not yet make it to the CIMP genes' list. Our 1st approach for array data analysis demonstrated the limitations in selecting genes for further validation, highlighting the need for the 2nd bioinformatics approach to adequately select genes with differential aberrant methylation. A more comprehensive list, which included non-CIMP genes, such as APC, EVL, CD109, PTEN, TWIST1, DCC, PTPRD, SFRP1, ICAM5, RASSF1A, EYA4, 30ST2, LAMA1, KCNQ5, ADHEF1, and TFPI2, was established. Array data are useful to categorize and cluster colonic lesions based on their global methylation profiles; however, its usefulness in identifying robust methylation markers is limited and rely on the data analysis method. We have identified 16 non-CIMP-panel genes for which we provide rationale for inclusion in a more comprehensive characterization of CIMP+ CRCs. The identification of a definitive list for methylome specific genes in CRC will contribute to better clinical management of CRC patients.

Abstract Background: The identification and validation of genes that are frequently methylated in CRC will have potential value for both diagnostic and therapeutic implications. However, the DNA methylation patterns in CRC especially in African Americans have not been well investigated. Here we aimed to determine the methylation status of CpG islands of candidate genes in critical pathways important in the initiation and development of CRC. Materials and Methods: Genomic DNA from 7 individuals (1 normal, 2 adenomas, and 4 adenocarcinomas) was used for global methylation analysis using Reduced Representation Bisulfite Sequencing (RRBS). Based on literature review, differential methylation ratio, gene ontology, WNT, Notch, EGFR, RAS-MPK, PI3K, TGF-b, and P53 pathways analysis, RGS3, EID3, GNAS, ATXN7L1, GAS7, HNRNPF, GPR7, SOX15, and TNFAIP2 genes were selected for methylation validation . Established methylation status of NDRG4, BMP3, Septin 9, Vimentin, and B-Actin were used as validation controls. Forty-four paired colorectal tumors and normal adjacent colonic tissue samples were used for validation. Samples were first bisulfite converted and then amplified at specific loci using 48.48 Access Array (Fluidigm). Amplicon pools were barcoded and adapterized prior to 150-bp paired-end sequencing on the Illumina MiSeq Reads were de-multiplexed using the Fluidigm indexes and mapped back to the reference genome (Hg18). A paired t-test was used for p-value and meth-diff estimation. Results: We validated the methylation status of 355 CpG sites located in 16 gene promoter regions associated with CpG islands. Fifty-nine CpG sites located on CpG islands of ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), NDRG4 (2), Sept9 (6), and TNFAIP2 (1), were significantly methylated in tumor vs. normal (p<0.05). Most of these genes showed significant hypermethylation in tumors compared to normal mucosa, According to gene ontology analysis, GAS7 methylation is associated with the cadherin signaling (WNT) pathway. Conclusion: Methylation profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. We identified novel methylated genes that are potential biomarkers for CRC. In consistence with other reports, our study showed that GAS7 is also hypermethylated in African American CRC patients. Investigations into the possible roles of this gene as a potential biomarker in the context of early diagnosis and prognosis of CRC are underway. Citation Format: Hassan Ashktorab, Mohammad Daremipouran, Hamed Rahi, Eward L. Lee, Wayne Frederick, Adeyinka O. Laiyemo, Ron Leavitt, Xueguang Sun, Sudhir Varma, Hassan Brim. Identification of new hypermethylated candidate genes in colorectal cancer using reduced representation bisulfite next generation sequencing. [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 4247. doi:10.1158/1538-7445.AM2013-4247