Edward Lee

Distinct Genetic Alterations in Colorectal Cancer

Background Colon cancer (CRC) development often includes chromosomal instability (CIN) leading to amplifications and deletions of large DNA segments. Epidemiological, clinical, and cytogenetic studies showed that there are considerable differences between CRC tumors from African Americans (AAs) and Caucasian patients. In this study, we determined genomic copy number aberrations in sporadic CRC tumors from AAs, in order to investigate possible explanations for the observed disparities. Methodology/Principal Findings We applied genome-wide array comparative genome hybridization (aCGH) using a 105k chip to identify copy number aberrations in samples from 15 AAs. In addition, we did a population comparative analysis with aCGH data in Caucasians as well as with a widely publicized list of colon cancer genes (CAN genes). There was an average of 20 aberrations per patient with more amplifications than deletions. Analysis of DNA copy number of frequently altered chromosomes revealed that deletions occurred primarily in chromosomes 4, 8 and 18. Chromosomal duplications occurred in more than 50% of cases on chromosomes 7, 8, 13, 20 and X. The CIN profile showed some differences when compared to Caucasian alterations. Conclusions/Significance Chromosome X amplification in male patients and chromosomes 4, 8 and 18 deletions were prominent aberrations in AAs. Some CAN genes were altered at high frequencies in AAs with EXOC4, EPHB6, GNAS, MLL3 and TBX22 as the most frequently deleted genes and HAPLN1, ADAM29, SMAD2 and SMAD4 as the most frequently amplified genes. The observed CIN may play a distinctive role in CRC in AAs.

Impact of BRAF, MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study

We have identified an alternative pathway of tumorigenesis in sporadic colon cancer, involving microsatellite instability due to mismatched repair methylation, which may be driven by mutations in the BRAF gene (V600E). Colorectal cancer (CRC) is the most common cancer in the world, and African Americans show a higher incidence than other populations in the United States. We analyzed sporadic CRCs in Omani (of African origin, N = 61), Iranian (of Caucasian origin, N = 53) and African American (N = 95) patients for microsatellite instability, expression status of mismatched repair genes (hMLH1, hMSH2) and presence of the BRAF (V600E) mutation. In the Omani group, all tumors with BRAF mutations were located in the left side of the colon, and for African Americans, 88% [7] of tumors with BRAF mutations were found in the right side of the colon. In African Americans, 31% of tumors displayed microsatellite instability at two or more markers (MSI-H), while this rate was 26% and 13% for tumors in the Iranian and Omani groups, respectively. A majority of these MSI-H tumors were located in the proximal colon (right side) in African American and Iranian subjects, whereas most were located in the distal colon (left side) in Omani subjects. Defects in hMLH1 gene expression were found in 77% of MSI-H tumors in both African Americans and Iranians and in 38% of tumors in Omanis. BRAF mutations were observed in all subjects: 10% of tumors in African Americans (8/82), 2% of tumors in Iranians (1/53), and 19% of tumors in Omanis (11/59). Our findings suggest that CRC occurs at a younger age in Omani and Iranian patients, and these groups showed a lower occurrence of MSI-H than did African American patients. Our multivariate model suggests an important and significant role of hMLH1 expression and BRAF mutation in MSI-H CRC in these populations. The high occurrence of MSI-H tumors in African Americans may have significant implications for treatment, since patients with MSI-H lesions display a different response to chemotherapeutic agents such as 5-fluorouracil.

Mitochondrial DNA mutations in preneoplastic lesions of the gastrointestinal tract: A biomarker for the early detection of cancer

BackgroundSomatic mutations of mitochondrial DNA (mtDNA) are common in many human cancers. We have described an oligonucleotide microarray (MitoChip) for rapid sequencing of the entire mitochondrial genome (Zhou et al, J Mol Diagn 2006), facilitating the analysis of mtDNA mutations in preneoplastic lesions. We examined 14 precancerous lesions, including seven Barrett esophagus biopsies, with or without associated dysplasia; four colorectal adenomas; and three inflammatory colitis-associated dysplasia specimens. In all cases, matched normal tissues from the corresponding site were obtained as germline control. MitoChip analysis was performed on DNA obtained from cryostat-embedded specimens.ResultsA total of 513,639 bases of mtDNA were sequenced in the 14 samples, with 490,224 bases (95.4%) bases assigned by the automated genotyping software. All preneoplastic lesions examined demonstrated at least one somatic mtDNA sequence alteration. Of the 100 somatic mtDNA alterations observed in the 14 cases, 27 were non-synonymous coding region mutations (i.e., resulting in an amino acid change), 36 were synonymous, and 37 involved non-coding mtDNA. Overall, somatic alterations most commonly involved the COI, ND4 and ND5 genes. Notably, somatic mtDNA alterations were observed in preneoplastic lesions of the gastrointestinal tract even in the absence of histopathologic evidence of dysplasia, suggesting that the mitochondrial genome is susceptible at the earliest stages of multistep cancer progression.ConclusionOur findings further substantiate the rationale for exploring the mitochondrial genome as a biomarker for the early diagnosis of cancer, and confirm the utility of a high-throughput array-based platform for this purpose from a clinical applicability standpoint.

Multilayered epithelium in mucosal biopsy specimens from the gastroesophageal junction region is a histologic marker of gastroesophageal reflux disease

Barrett esophagus (BE) is defined as a columnar metaplasia of the distal esophagus that develops as a result of chronic gastroesophageal reflux disease (GERD). A distinctive type of multilayered epithelium (ME) that exhibits features of both squamous and columnar epithelium has been hypothesized to represent an early, or intermediate, phase in the development of BE. The aim of this prospective study was to evaluate the prevalence and specificity of ME in mucosal biopsies of the squamocolumnar junction (SCJ) from patients who had GERD, either with or without BE. During endoscopic examination of the esophagus, 2 biopsy specimens were obtained from across the SCJ from 27 patients with BE, 12 patients who had GERD without BE, and 14 controls who had no symptoms or endoscopic or histologic signs of GERD. ME was present at the SCJ in 33%, 33%, and 0% of BE, GERD, and control patients, respectively. Compared with control subjects, the prevalence of ME was significantly higher in both GERD and BE patients (P<0.05). In GERD patients without BE, ME was always detected adjacent to areas of cardia-type mucosa composed of mucous glands. ME from GERD patients and BE patients had a similar immunophenotype, showing expression of the intestinal markers MUC2 and cdx-2 in 38% and 77% of cases, respectively. The prevalence of expression of these markers in ME was significantly different from nongoblet epithelium in control patients. Our results provide further evidence that ME may represent an early, transitional form of columnar metaplasia, and that ME may be used as a histologic marker of reflux disease in mucosal biopsies from the gastroesophageal junction region.

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.

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.

SLC5A8 Gene, A Transporter of Butyrate: A Gut Flora Metabolite, Is Frequently Methylated in African American Colon Adenomas

Background Colon cancer is one of the leading causes of cancer related deaths. Its impact on African Americans (AAs) is higher than in the general population both in the incidence and mortality from the disease. Colon cancer aggressiveness in AAs as well as non-frequent check-ups and follow up in this population have been proposed as ways to explain the observed discrepancies. These facts made the detection of early carcinogenesis markers in this population a priority. Materials and Methods Here, we analyzed 50 colon adenomas from AA patients for both microsatellite instability (MSI) and the methylation status of SLC5A8 gene. This genes product is involved in the transport of butyrate that has anti-proliferative properties through its effects on histone acetylation and gene expression. A proteomic analysis to check the expressed histones in adenoma and normal tissues was also performed. Results The analyzed samples displayed 82% (nu200a=u200a41) methylation level of SLC5A8 gene in adenomas. The MSI-H (high) adenoma were about 18% (nu200a=u200a9) while the rest were mostly MSS (microsatellite stable) with few MSI-L (Low). No association was found between SLC5A8 methylation and the MSI status. Also, there was no association between SLC5A8 methylation and the sex and age of the patients. However, there were more right sided adenomas with SLC5A8 methylation than the left sided ones. The proteomic analysis revealed distinct histone expression profiles between normal and adenoma tissues. Conclusion SLC5A8 is highly methylated in AA colon adenomas which points to its potential use as a marker for early detection. The MSI rate is similar to that found in colon cancer tumors in AAs. These findings suggest that both processes stem from the same epigenetic and genetic events occurring at an early stage in colon carcinogenesis in AAs.

A 50-year review of colorectal cancer in African Americans: implications for prevention and treatment.

BackgroundAfrican-Americans (AA) have the highest rate of colorectal cancer (CRC) incidence and mortality in the US. CRC in AA is more advanced and right-sided. Although screening has been shown to reduce mortality from CRC in the general US population, AA continue to experience a disproportionately higher CRC death compared to other ethnic groups. This study aimed at assessing the trend of CRC in AA, focusing on the changing pattern of in situ tumors in this ethnic group and how observed trends may guide current and future preventive and treatment strategies.Materials and MethodsAll pathologic reports from 1959 to 2006 in Howard University Hospital (nxa0=xa0150,000) were reviewed manually. The pathology reports showing colorectal cancer were carefully reviewed and selected by a GI pathologist. Intraepithelial or intramucosal carcinomas were diagnosed as in situ carcinoma. Reviewed pathological information were entered into Microsoft Excel and checked for duplication and missing data. Differences in situ and advanced cancer by sex, histology, location, and years of diagnosis were assessed by Chi-square test.ResultsA total of 1,753 CRC cases were diagnosed in this period. About 56% of the cases were female and 51% of the tumors were left-sided. Mean (SD) age was 66 (13)xa0years. The frequency of in situ tumor was 5.8% in this period. There was no statistically significant difference between in situ and advance tumor by age, sex, and tumor location. The rate of in situ tumor peaked in the 1990s at 8.5% (Pxa0=xa00.0001). We observed a decade-to-decade increasing rate of right-sided tumors, which started at 36% in the period 1959–1970 and peaked in the period of 2001–2006 at 60% (Pxa0=xa00.0001).ConclusionsThe recent increasing number of advanced and right-sided tumor in our study is concordant with SEER data and has great importance in developing CRC prevention and treatment strategies for AA population.

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.

Prevalence of Colorectal Neoplasia Among Young African Americans and Hispanic Americans

BackgroundThe disproportionately higher incidence of and mortality from colorectal cancer (CRC) among African Americans (AA) led the American College of Gastroenterology to recommend screening starting at age 45 in 2005.AimThe purpose of this study was to determine the prevalence of colorectal neoplasia among 40–49-year-old inner city AA and Hispanic Americans (HA).MethodsWe reviewed the medical records of 2,435 inner city AA and HA who underwent colonoscopy regardless of indication and compared the prevalence of colorectal neoplasia between AA and HA patients. We used logistic regression models to calculate odds ratios (OR) and 95xa0% confidence intervals (CI).ResultsThere were 2,163 AAs and 272 HA. There were 57xa0% women in both groups. A total of 158 (7xa0%) AA and 9 (3xa0%) HA (Pxa0=xa00.014) underwent the procedures for CRC screening. When compared to HAs, AAs had higher prevalence of any polyp (35 vs. 18xa0%, ORxa0=xa02.53; 95xa0% CI 1.82–3.52). Overall, AA had higher prevalence of colorectal neoplasia (adenoma and cancer) when compared to HAs (16 vs. 10xa0%; ORxa0=xa01.68; 95xa0% CI 1.10–2.56).ConclusionWe observed a higher frequency of colorectal neoplasia among 40–49-year-old AAs as compared to HAs suggesting an increased susceptibility to CRC risk in this population.