Babak Shokrani

Toward a comprehensive and systematic methylome signature in colorectal cancers

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.

Epigenetic silencing of CHD5, a novel tumor-suppressor gene, occurs in early colorectal cancer stages.

Chromodomain helicase DNA binding protein 5 (CHD5) is a family member of chromatin remodeling factors. The epigenetic silencing mechanisms of CHD5 in colorectal cancer have not been well studied.

Microbiome Analysis of Stool Samples from African Americans with Colon Polyps

Background Colonic polyps are common tumors occurring in ~50% of Western populations with ~10% risk of malignant progression. Dietary agents have been considered the primary environmental exposure to promote colorectal cancer (CRC) development. However, the colonic mucosa is permanently in contact with the microbiota and its metabolic products including toxins that also have the potential to trigger oncogenic transformation. Aim To analyze fecal DNA for microbiota composition and functional potential in African Americans with pre-neoplastic lesions. Materials & Methods We analyzed the bacterial composition of stool samples from 6 healthy individuals and 6 patients with colon polyps using 16S ribosomal RNA-based phylogenetic microarray; the Human intestinal Tract Chip (HITChip) and 16S rRNA gene barcoded 454 pyrosequencing. The functional potential was determined by sequence-based metagenomics using 454 pyrosequencing. Results Fecal microbiota profiling of samples from the healthy and polyp patients using both a phylogenetic microarraying (HITChip) and barcoded 454 pyrosequencing generated similar results. A distinction between both sets of samples was only obtained when the analysis was performed at the sub-genus level. Most of the species leading to the dissociation were from the Bacteroides group. The metagenomic analysis did not reveal major differences in bacterial gene prevalence/abundances between the two groups even when the analysis and comparisons were restricted to available Bacteroides genomes. Conclusion This study reveals that at the pre-neoplastic stages, there is a trend showing microbiota changes between healthy and colon polyp patients at the sub-genus level. These differences were not reflected at the genome/functions levels. Bacteria and associated functions within the Bacteroides group need to be further analyzed and dissected to pinpoint potential actors in the early colon oncogenic transformation in a large sample size.

Identification of novel mutations by exome sequencing in African American colorectal cancer patients

The 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.

Reduced Representation Bisulfite Sequencing Determination of Distinctive DNA Hypermethylated Genes in the Progression to Colon Cancer in African Americans

Background and Aims. Many studies have focused on the determination of methylated targets in colorectal cancer. However, few analyzed the progressive methylation in the sequence from normal to adenoma and ultimately to malignant tumors. This is of utmost importance especially in populations such as African Americans who generally display aggressive tumors at diagnosis and for whom markers of early neoplasia are needed. We aimed to determine methylated targets in the path to colon cancer in African American patients using Reduced Representation Bisulfite Sequencing (RRBS). Methods. Genomic DNA was isolated from fresh frozen tissues of patients with different colon lesions: normal, a tubular adenoma, a tubulovillous adenoma, and five cancers. RRBS was performed on these DNA samples to identify hypermethylation. Alignment, mapping, and confirmed CpG methylation analyses were performed. Preferential hypermethylated pathways were determined using Ingenuity Pathway Analysis (IPA). Results. We identified hypermethylated CpG sites in the following genes: L3MBTL1, NKX6-2, PREX1, TRAF7, PRDM14, and NEFM with the number of CpG sites being 14, 17, 10, 16, 6, and 6, respectively, after pairwise analysis of normal versus adenoma, adenoma versus cancer, and normal versus cancer. IPA mapped the above-mentioned hypermethylated genes to the Wnt/β-catenin, PI3k/AKT, VEGF, and JAK/STAT3 signaling pathways. Conclusion. This work provides insight into novel differential CpGs hypermethylation sites in colorectal carcinogenesis. Functional analysis of the novel gene targets is needed to confirm their roles in their associated carcinogenic pathways.

Association between Diverticular Disease and Pre-Neoplastic Colorectal Lesions in an Urban African-American Population.

Background: It is unclear whether there is a shared pathway in the development of diverticular disease (DD) and potentially neoplastic colorectal lesions since both diseases are found in similar age groups and populations. Aim: To determine the association between DD and colorectal pre-neoplastic lesions in an African-American urban population. Methods: Data from 1986 patients who underwent colonoscopy at the Howard University Hospital from January 2012 through December 2012 were analyzed for this study. The presence of diverticula and polyps was recorded using colonoscopy reports. Polyps were further classified into adenoma or hyperplastic polyp based on histopathology reports. Multiple logistic regression was done to analyze the association between DD and colonic lesions. Results: Of the 1986 study subjects, 1,119 (56%) were females, 35% had DD and 56% had at least one polyp. There was a higher prevalence of polyps (70 vs. 49%; OR = 2.3; 95% CI: 1.9-2.8) and adenoma (43 vs. 25%; OR = 2.0; 95% CI: 1.7-2.5) in the diverticular vs. non-diverticula patients. Among patients who underwent screening colonoscopy, the presence of diverticulosis was associated with increased odds of associated polyps (OR = 9.9; 95% CI: 5.4-16.8) and adenoma (OR = 5.1; 95% CI: 3.4-7.8). Conclusion: Patients with DD are more likely to harbor colorectal lesions. These findings call for more vigilance on the part of endoscopists during colonoscopy in patients known to harbor colonic diverticula.

Giant Peripheral Ossifying Fibroma: A Case Report and Clinicopathologic Review of 10 Cases From the Literature

Peripheral ossifying fibroma (POF) is most often a self-limiting, sessile or pedunculated, gingival nodule that is believed to be a reactive rather than neoplastic pathologic process. The lesion is typically <2cm, however it has been recognized that some examples may grow quite large and may displace teeth. The mass-like clinical presentation and radiographic appearance of soft tissue calcification may lead to misclassification; however the histologic appearance is diagnostic. Giant POFs (GPOF) have been referred to in the literature by several other names (large, atypical, huge, gigantiform). The distinguishing characteristics of GPOFs and the factors that contribute to their growth have primarily been explored through case reports. We present a new case of POF that was giant and review 10 previously reported giant lesions, with focus on the clinical presentation, radiographic features, and outcome to explore the possibility that this represents a distinct clinical subset of lesion, with a unique set of features that warrant recognition for accurate diagnosis.

Abstract 5273: Detection of sessile serrated adenomas/polyps in African Americans

Introduction: The majority of colorectal cancers (CRCs) develop through the adenoma-carcinoma sequence, while 15-20% develop via the serrated pathway. Sessile Serrated Adenoma/Polyps (SSA/P) are more difficult to detect during colonoscopy and generally require a shorter time follow-up than other lesions (3 vs. 5 years). These polyps lead to cancer faster than conventional adenomas. In order to better define patients at risk for these lesions, we performed a retrospective study to evaluate clinicopathological features of patients diagnosed with SSA/P. Methods: We reviewed pathology reports of patients at Howard University Hospital from 2010-2015. We identified 5,900 patients with colorectal lesions, of whom 312 (5.3%) were diagnosed with SSA/P. We analyzed the specific clinical, pathological and demographic features of patients with SSA/P lesions. Results: We identified 312 cases with SSA/P. The incidence of SSA/P over the 5 years period was 5.3%: 198/312 (63.4%) patients had 2 or more polyps, 54.5% of the SSA/P patients were females, 70.5% of patients were 50-64 years of age and 18% were older than 65. SSA/P lesions’ locations were as follows: rectal: 32.1%; rectosigmoid: 18.6%; sigmoid: 16.8%; Ascending Colon: 13.2 % and Descending Colon: 7.1%. Reasons for colonoscopy were as follows: Screening: 43.6 %; GI bleeding: 15.1%; Abdominal Pain: 13.1%and Change in Bowel Habits: 10.2%. Conclusion: Our results show that there is a slightly increased predominance of SSA/P occurrence in females. Most SSA/P occurred in patients 50 to 64 years old. This age range is younger in comparison to patients with conventional adenomas. SSA/Ps were predominantly distal (rectal, rectosigmoid, sigmoid) whereas previous literature reports a proximal location. Most patients in our study were diagnosed in screening colonoscopies; however, other patients presented with symptoms such as GI bleeding, abdominal pain, or change in bowel habits. Citation Format: Nazli Atefi, Sanmet Singh, Babak Shokrani, Edward Lee, Ali Afsari, Mehdi Nouraie, Adeyinka Laiyemo, Joseph Mathews, Carla Williams, Hassan Brim, Hassan Ashktorab. Detection of sessile serrated adenomas/polyps in African Americans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5273. doi:10.1158/1538-7445.AM2017-5273

Abstract 4578: Does poor prognosis in African Americans with MSI-H colorectal cancer associate with altered immune markers

BACKGROUND: Microsatellite instability high (MSI-H) of sporadic colorectal carcinomas (CRC) is usually associated with improved prognosis and a high density of tumor-infiltrating lymphocytes. However, African Americans with MSI-H have poor prognosis. AIM: To evaluate whether or not expression of different immune and tumor markers individually or in combination in African American MSI-H CRC associate with the prognosis status. METHODS: Tissues Microarray (TMA) were prepared by microdissection from FFPE (Formalin Fixed Paraffin Embedded) blocks of 15 MSI-H patients. Immune markers (CD8+, Granzymes, Perforins, CD4+, STAT1, IRF1 and IRF5) and PD-L1 status were analyzed by immunohistochemistry (IHC). Demography and clinical data including TNM, tumor grading (WHO standard), histological type of the tumor, date of diagnosis, date of the last follow-up examination, treatment, comorbidities, metastasis, recurrence, 5-year disease-free survival and death were collected. RESULTS: There were 4 patients with improved prognosis (27%) with relatively high CD4 density (2 with >50% and 2 with 11-50%) regardless of PD-L1 status (3+/1-), stage (II,III), and other immune markers (CD8, CD4, Granzymes, Perforins, IRF1, IRF5 and STAT1). One of the improved prognosis patients with KRAS mutation has elevated expression of all considered immune markers besides CD4. There were 9 patients (6 dead, 3 alive) with poor prognosis, with different immune and tumor markers level (3 were PD-L1+ and 5 have relatively high CD4 count; but with low level of STAT1. There were also 2 patients with expected improved prognosis based on their immune markers level (stage II), however, they died within the 5 year period post-diagnosis, due to their age (98 and 87 years old). Conclusion: MSI-H Colorectal cancer from African American patients has poor prognosis which may correlate with the nature of tumor-associated immune response. Other factors such as MSH3 defects might cancel the positive prognosis of MSI-H status. Nonetheless, low STAT1 and low CD4 may be indicators of poorer prognosis. Citation Format: Umamaheshwari Golconda, Lena Sokol, Babak Shokrani, Edward Lee, Donna Hansel, Oluwole Fadare, Sandip Patel, Mehdi Nouraie, Hooman Soleimani, Zaki Sharif, Ali Afsari, Fareed Daremipouran, Hassan Brim, Hassan Ashktorab. Does poor prognosis in African Americans with MSI-H colorectal cancer associate with altered immune markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4578. doi:10.1158/1538-7445.AM2017-4578

Abstract 4488: Targeted sequencing revealed distinctive and pathogenic mutations in African Americans with colorectal cancer

Background & Aim: We recently analyzed 12 pairs of African American CRC tumors and their matched normal tissue using whole exome sequencing and established a panel of novel mutations that are potentially useful for the detection of CRC in this population. In this study, we examined APC, MSH3, and MSH6 mutations using targeted exome sequencing (TES) to determine distinctive mutations frequencies and their chronology in the neoplastic transformation. Materials & Methods: A total of 140 colon samples: (30 normal, 21 adenomas, 33 advanced adenomas & 56 tumors collected from African Americans were used as our discovery set on an Ion Torrent platform. A subset of 36 samples were used as validation set on an Illumina platform. Bioinformatic analysis was performed on both sets of data. Common mutations were considered validated. Results: Two novel MSH6 mutations were validated. Four known mutations in MSH3 were validated and were located in nonsynonymous (exon 10) and 1synonomous mutation (exon 18). As for the APC, 20 mutations were validated include 4 novel mutations. The novel mutations were 3 stopgain and 1 nonsynonymous located at the EB1 binding site, in the mutational cluster region (MCR), and at the 15 Amino Acid repeat. Conclusion: We here defined novel mutations that target DNA MMR and APC genes in African Americans with colorectal lesions. Greater frequency of mutations in cells defective for DNA repair and APC genes is an advantage in cell growth and genetic instability and relevant to the initiating events of colon tumorigenesis. Citation Format: Hassan Ashktorab, Hamed Azimi, Mike Nickerson, Sara Bass, Joseph Boland, Meredith Yeager, Sudhir Varma, Mohamed Daremipouran, Zaki Sherif, Shima Ghavimi, Babak Shokrani, Edward Lee, Adeyinka Laiyemo, Hassan Brim. Targeted sequencing revealed distinctive and pathogenic mutations in African Americans with 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 4488.