Colon Cancer In African Americans Research Articles

Abstract 6696: Microbial signatures and serum markers in colon cancer of African American patients

Abstract Among all the racial/ethnic groups, African American (AA) patients have the highest incidence of CRC and mortality rate. There is growing evidence indicating that the oral bacteria might translocate to the intestine, potentially causing disruptions in the balance of gut microbes. Our study delves into the complex interplay between oral pathogens, gut microbiota, and colon carcinogenesis in AA patients. Methods: 16S rRNA sequencing of tumor and non-tumor tissues, utilized qPCR for detection of oral bacteria Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) relative abundance, performed cytokine profiling via multiplex ELISA, and employed NAPPA for antibody response assessment. Results: Colon tumor tissues had lower microbial diversity, notably in right-sided tumors, with distinct biofilm-associated bacteria. Novel discoveries emerged from differential abundance testing, revealing Prevotella intermedia and Treponema socranski to be associated with early cancer stage and left-sided location, and Fusobacterium necrophorum with advanced cancer stage and right-sided location. qPCR demonstrated Fn and Pg elevation in right-sided tumors and late cancer stages. Both were detected in 45% of tumors versus 37% in non-tumor tissues and 19.15% of tumors and 25.53% in non-tumor tissues, respectively. Fn-positive groups exhibited distinct microbial signatures, with altered bacterial abundances and dysbiosis, including increased CRC-associated bacteria Faecalibacterium and Helicobacter, increased oral pathogen abundance (Fusobacterium, Porphyromonas, Prevotella, Treponema), and reduced beneficial bacteria. Serological analysis highlighted elevated levels of IL-2, IL-10, IFN-γ, and RANK in advanced cancer stages and increased CRP levels in right-sided tumors. By NAPPA analysis, 30 bacterial proteins (anti-IgG) showed a seroprevalence of more than 15% in the samples, with those being the most seroprevalent anti-HP131 and anti-HP-1564 (68% and 61% of all samples) of Helicobacter pylori, and anti-msnK (64%) of Streptococcus gallolyticus. Conclusions: Our study emphasized the reduced diversity in tumor tissues, particularly in right-sided tumors in AA colon cancer. Fn presence in colonic tumors correlated with CRC-associated bacteria and oral pathogen aggregation, suggesting its potential role in cancer pathogenesis. Combining the analysis of cytokines and pathogenic bacteria antibodies may serve as broader serological biomarkers for disease severity and location. These findings advance our comprehension of microbiota-cancer interactions, directing personalized diagnostics and treatments in this population. Citation Format: Sofia Carolina Tortora, Emily Vogtmann, Laura Martello-Rooney. Microbial signatures and serum markers in colon cancer of African American patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6696.

Abstract 994: Single analyte profiling of the mutational, immune and microbiome landscape in african american colon cancer patients

Abstract Colorectal cancer (CRC) disproportionately affects African American (AA) patients who experience a higher incidence and mortality than any other demographic groups in the United States. Yet molecular profiling in this population is scarce. Here, we performed whole-exome sequencing (WES) of 342 immediately snap-frozen tissues comprising AA CRC and matched, distant normal colon and identified distinct genomic drivers, DNA signatures, and, surprisingly, mosaic pathogenic mutations that are not present in the patient-matched CRC. We inferred T cell infiltration and tissue-resident microbiome (TR-M) directly from WES, enabling integrative analyses. Compared to normal colon, T cell infiltration is strongly decreased in tumours with microsatellite stability (MSS), while microsatellite unstable (MSI) and Polymerase Epsilon (POLE) mutant tumours had similar levels, suggesting immune evasion in MSS rather than increased T cell infiltration in MSI/POLE-mutants. Analyses of matched normal and tumour tissues revealed the evolving TR-M at unprecedented granularity and identified distinct niches of interactions with genomic and tumour-microenvironmental features. Together, this study represents the largest comprehensive genomic analysis of CRC in AA and provides a framework for performing a multi-layered analysis from a single analyte, thus, affording a cost-effective approach to expedite medical discoveries in underrepresented ancestries. Citation Format: Carino Gurjao, Edmond Chan, Michael J. Lee, Sho Hangai, Raul Rabadan, Tal Korem, Hanina Hibshoosh, Benjamin Izar. Single analyte profiling of the mutational, immune and microbiome landscape in african american colon cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 994.

Abstract 100: IL-1β pathway promotes cell proliferation and 5-FU resistance in African American colon cancer cell lines

Abstract Introduction: Colorectal Cancer (CRC) is the third most common cancer and third cause of cancer-related death in the US. When taking race and ethnicity into consideration, African Americans (AA) present with the highest incidence and mortality rates for CRC. Studies have found that AA patients, when compared to Caucasian American (CA) patients, have a lower response to the standard of care chemotherapeutic agent 5-Fluorouracil (5-FU) as well as lower frequency of MSI tumors, making them also less likely to respond to conventional immunotherapies. Our recent findings have shown that tumors from AA patients present higher expression of genes involved in pro-inflammatory processes as well as lower expression of genes promoting anti-tumoral activities. One of the genes that was found upregulated in AA tumors is IL1B, which encodes for the pro-inflammatory cytokine Interleukin-1β (IL-1β). Therefore, we investigated the role of the IL-1β pathway in novel AA colon cancer cell lines. Here, we evaluated changes in cell proliferation, apoptosis, 5-FU response, and activation of inflammatory pathways. Methods: Our approach includes analysis of gene expression in both AA and CA colon cancer cell lines by using RNA sequencing to evaluate pro-inflammatory genes differentially expressed among the cell lines. Furthermore, we used an MTS assay to detect viable cells following treatment with IL-1β, alone or in combination with different concentrations of 5-FU, as well as analyzing changes in apoptosis via Flow Cytometry. To better understand the mechanisms of IL-1β-induced effects, we used IL-1 Receptor Antagonist (IL-1Ra) to block the IL-1β pathway and evaluated changes in cell viability and 5-FU response via MTS assay, as well as activation of NF-kB pathway, via detection of phospho-IкB-α protein using Western Blot analysis. Results: Results from MTS assays indicated that cell proliferation in response to IL-1β differs between the AA and the CA colon cancer cell lines, with the AA colon cancer cells being more sensitive to lower concentrations of the cytokine when compared to CA cell lines. Further, we saw an increased expression of phospho-IкB-α following treatment with IL-1β. This expression was reduced when the cells were treated in combination with IL-1β and the IL-1Ra. The IL-1Ra also appears to reduce the pro-proliferative effects induced by IL-1β. Importantly, our results show that 5-FU sensitivity is drastically reduced in the presence of IL-1β for both AA and CA colon cancer cell lines, suggesting that IL-1β may play a role in defining resistance to the chemotherapeutic drug. Conclusions: Taken together, our results demonstrated a differential response to IL-1β for the AA colon cancer cell lines, suggesting a probable role played by the cytokine in driving inflammation-related cancer progression and reveals a possible new target to exploit in immunotherapy for this population. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Jovanny Zabaleta, Jennie Williams, Laura Martello-Rooney. IL-1β pathway promotes cell proliferation and 5-FU resistance in African American colon cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 100.

Abstract PO-227: Role of IL-1β pathway in colon cancer progression and its therapeutic implications in African American colon cancer cell lines

Abstract Background: Colorectal cancer (CRC) is the third most common cancer and third cause of cancer-related death among African Americans (AA) in the US. When compared to Caucasian Americans (CA), AA patients present with higher incidence and mortality rates for CRC. Recent findings indicate that they have reduced response to the standard of care chemotherapeutic agent 5-Fluorouracil (5-FU) as well as lower frequency of MSI tumors, making them also less likely to respond to conventional immunotherapies. Previous results from human genomic analyses suggest that certain differences seen in AA patients might be due to a decreased antitumor immune response as well as an increased expression of genes involved in inflammatory processes, such as Interleukin-1β (IL-1β). Therefore, we aimed to investigate the role of IL-1β in promoting cell proliferation, cell migration, 5-FU resistance and activation of specific inflammatory pathways in novel AA colon cancer cell lines, and how these responses would compare to well established CA colon cancer cell lines. Methods: Our approach includes using MTS colorimetric assay and Transwell assay to examine the effects of IL-1β treatment on cell proliferation and migration. We performed Western Blot analysis to detect expression of phosphoproteins following treatment with IL-1β. We also investigated how IL-1β affects 5-FU resistance in AA and CA colon cancer cell lines in terms of cell viability. Finally, we tested the ability of IL-1 Receptor antagonist (IL-1Ra) to inhibit the effects of IL-1β on cell proliferation, protein expression, and 5-FU response. Results: Our MTS assay results indicated that cell proliferation in response to IL-1β differs between the AA and the CA colon cancer cell lines, with the AA colon cancer cells being more responsive. Whereas, the migration rate is increased after stimulation with IL-1β for both AA and CA colon cancer cell lines. Protein expression of Phospho-IkB-alpha is increased in AA but not CA colon cancer cell lines following IL-1β treatment, while Phospho-JNK expression was different between the cell lines following treatment. Importantly, our results show that 5-FU efficacy is decreased in the presence of IL-1β for both AA and CA colon cancer cell lines and that treating the cells with IL-1Ra appeared to reestablish 5-FU cell killing effect. IL-1Ra also suppresses the effects of IL-1β on cell proliferation and protein expression. Conclusions: Taken together, our results demonstrated a differential response to IL-1β for the AA colon cancer cell lines, suggesting a probable role played by the cytokine in driving inflammation-related cancer progression and reveals a possible new target to exploit in immunotherapy for this population. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Jovanny Zabaleta, Jennie Williams, Laura Martello-Rooney. Role of IL-1β pathway in colon cancer progression and its therapeutic implications in African American colon cancer cell lines [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-227.

Immune-Related Gene Expression and Cytokine Secretion Is Reduced Among African American Colon Cancer Patients.

Background: Colorectal cancer is the third most deadly cancer among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. Here, we investigated if differences in tumor immunology could be contributive to disparities observed between these populations.Methods: We examined gene expression of tumor and non-tumor adjacent tissues from AA and CA by whole transcriptome sequencing, and generated scores for immune cell populations by NanoString. In addition, we utilized “The Cancer Genome Atlas” (TCGA) database from AA and CA as a validation cohort. Finally, we measured the secretion of cytokines characteristic of effector T helper cell (Th) subsets by ELISA using plasma from each AA and CA participant.Results: Colon tumors from AA patients showed significant fold-change increase in gene expression when compared to CA for FOXP3 (6.22 vs. 3.22), IL1B (103 vs. 11.4) and IL8 (220 vs. 28.9) (p < 0.05). In contrast, among CA we observed statistically higher gene expression of markers associated with antitumor activity such as GZMB (Granzyme B), IFNG and the immunotherapy targets PDL1 (CD274) and CTLA4 (p < 0.05). TCGA data validated our observed higher gene expression of GZMB and PDL1 in CA patients when compared to AA. Notably, our observations on immune cell populations show that AA tumors have significantly higher number of exhausted CD8+ cells (p < 0.01), mast cells (p < 0.02) and increased T regulatory cells when compared to CA. AA colon cancer patients differed from CA in cytokine production patterns in plasma (i.e., reduced IL-12).Conclusions: Our study demonstrates significant differences of the immunological profiles of colon tumors from AA compared to CA that suggest a deficiency of appropriate immune defense mechanisms in terms of gene expression, recruitment of immune cells and systemic secretion of cytokines. As such, these immune differences could be mitigated through population-specific therapeutic approaches.

Abstract B134: Tumor biology and cancer health disparity: Gene expression, cytokine secretion, and protein production in African American colon cancer cell lines

Abstract Colorectal cancer (CRC) is the third most common cancer among African Americans (AAs) in the US. When compared to Caucasian Americans (CAs), AAs present with higher incidence and death rates as well as worse prognosis after treatment with 5-Fluorouacil (5-FU). Previous studies have shown a correlation between microsatellite instability (MSI) and response to 5-FU and our recent findings suggest that differences in the tumor immunology and MSI status of AA and CA CRC patients are associated with the observed disparities between these populations. Therefore, we examined if cytokines secretion, protein production and cellular response to 5-FU treatment, differs between colon cancer cell lines from AAs generated in Dr. Williams’ laboratory (SB521, SB501 and CHTN06) and colon cancer cell lines from CA patients (HT29 and HCT116). Methods: We performed whole transcriptome sequencing of colon cancer cell lines (RNAseq), utilizing the NextSeq 500/550 High Output Kit v2.5 (Illumina) and Partek Flow data analysis to correlate gene expression to immune-oncology pathways. ELISA assays (RayBiotech) were used to examine the secretion of cytokines in supernatants from cells treated with interleukin 1β and tumor necrosis factor alpha. We used western blotting for protein detection of the phosphorylated form of c-Jun N-terminal kinases (JNK) as well as cell viability assays for establishing the IC50 of the cell lines to 5-FU. Results: The gene expression results indicated that the immune profiles of AA cell lines differ from CA in genes and cytokines related to cellular anti-tumor activity, including CD8B, IL-1R, IL-1R2, Granzyme B and NFKB. ELISAs of supernatants from CA and AA CRC cell lines revealed a differential cytokines secretion between the two races, namely IL-8. Lastly, the MSI AA cell line showed sensitivity to 5-FU (tenfold less) when compared to the CA cell lines and a distinct protein production pattern. Conclusions: Our gene expression findings demonstrated the differential expression of immunological pathways involved in immune-surveillance and cancer progression in the CRC cell lines between the two races. These results were in accordance with the cytokines’ and protein’s expression patterns observed in the two cohorts of cell lines. Importantly, our data indicates that 5-FU is more efficient in reducing cell viability in the MSI AA cell line than in the two CA cell lines regardless of their MSI status. Altogether, our results illustrate the value of these in vitro models to study 5-FU treatment in AA patients with MSI and MMR mutations and to elucidate the differences in chemotherapy treatment responses between AAs and CAs. In conclusion, we demonstrated distinct immunological profiles and 5-FU sensitivity of AA and CA CRC cell lines. As such, these differences observed could be used to guide new therapeutic strategies. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Ping Ji, Ellen Li, Jovanny Zabaleta, Laura Martello-Rooney, Jennie Williams. Tumor biology and cancer health disparity: Gene expression, cytokine secretion, and protein production in African American colon cancer cell lines [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B134.

Distribution of colorectal cancer in young African Americans: implications for the choice of screening test

We recently reported on a left-sided predominance of colorectal cancers in the young (under age 50). Given the predilection of young African Americans for the disease, we wondered if there may be a difference in the biology of colorectal carcinogenesis between this group and Caucasians. Compare the distribution of colorectal cancer in African American patients and Caucasians under age 50, and describe implications for screening in these groups. Colorectal cancer patients diagnosed under the age of 50 between the years 2000 and 2016. All races other than African American and Caucasian and all patients with hereditary colon cancer or inflammatory bowel disease were excluded. race, age at diagnosis (5 subgroups: < 20, 20-29, 30-39, 40-44, and 45-49years) and cancer location; right (cecum, ascending colon, hepatic flexure, transverse colon, splenic flexure), left (descending colon and sigmoid colon), or rectal. 759 patients were included; 695 (91.6%) were Caucasian and 64 (8.4%) were African American. Most cases were diagnosed between ages 40 and 49 (African American = 75%, Caucasian = 69.5%). Rectal cancer was most common in both races, although significantly more common in Caucasian than in African American patients (64.2% vs 39.1%). Right colon cancer was more commonly found in African Americans (37.5%) compared with Caucasians (18%) (p = 0.0002). The ratio of rectal to right-sided colon cancer in African Americans was 1:1 compared with 3.6:1 in Caucasians. Relatively low number of African American patients CONCLUSION: The high rate of right-sided cancer in young African American patients means that they should be screened with colonoscopy. The increased incidence of right-sided cancers may represent a different biology of carcinogenesis in African Americans and deserves further study.

Single nucleotide polymorphisms in the MYLKP1 pseudogene are associated with increased colon cancer risk in African Americans.

IntroductionPseudogenes are paralogues of functional genes historically viewed as defunct due to either the lack of regulatory elements or the presence of frameshift mutations. Recent evidence, however, suggests that pseudogenes may regulate gene expression, although the functional role of pseudogenes remains largely unknown. We previously reported that MYLKP1, the pseudogene of MYLK that encodes myosin light chain kinase (MLCK), is highly expressed in lung and colon cancer cell lines and tissues but not in normal lung or colon. The MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. In this study, we further validate MYLKP1 as an oncogene via elucidation of the functional role of MYLKP1 genetic variants in colon cancer risk.MethodsProliferation and migration assays were performed in MYLKP1-transfected colon and lung cancer cell lines (H441, A549) and commercially-available normal lung and colon cells. Fourteen MYLKP1 SNPs (MAFs >0.01) residing within the 4 kb MYLKP1 promoter region, the core 1.4 kb of MYLKP1 gene, and a 4 kb enhancer region were selected and genotyped in a colorectal cancer cohort. MYLKP1 SNP influences on activity of MYLKP1 promoter (2kb) was assessed by dual luciferase reporter assay.ResultsCancer cell lines, H441 and A549, exhibited increased MYLKP1 expression, increased MYLKP1 luciferase promoter activity, increased proliferation and migration. Genotyping studies identified two MYLKP1 SNPs (rs12490683; rs12497343) that significantly increase risk of colon cancer in African Americans compared to African American controls. Rs12490683 and rs12497343 further increase MYLKP1 promoter activity compared to the wild type MYLKP1 promoter.ConclusionMYLKP1 is a cancer-promoting pseudogene whose genetic variants differentially enhance cancer risk in African American populations.

Abstract IA06: Changes in the composition and activity of the colonic microbiome that may explain the extreme risk of colon cancer in Alaskan Native People

Abstract Alaska Native People (AN) suffer the highest recorded incidence of CRC globally (100:100,000) and thus have the greatest risk of dying from this disease. Diet drives genetic susceptibility risk factors, and there is convincing evidence that high consumption rates of red and processed meats and fat increase risk, while a diet rich in fiber suppresses risk. We have shown that the high risk of colon cancer in African Americans (~65:100,000) is suppressed by increasing the consumption of high-fiber foods, in part driven by the health-promoting and antineoplastic influence of colonic microbiome cometabolites (e.g., short chain fatty acids [SCFA] and butyrate) and the suppression of inflammatory and procarcinogenic cometabolites (e.g., conjugated bile acids, nitrogenous end products). This study aimed to determine if dietary-microbiome metabolism explains the extreme risk of colon cancer in AN. 20 adults living in Anchorage and 9 in Barrow (age range 40-65 years) were prospectively recruited and dietary data were recorded. This cohort was compared to 25 native Africans who consume a high-fiber diet (&amp;gt;50g/day) and have an extremely low risk of colon cancer (&amp;lt;5:100,000). Meta-taxonomic analysis of stool was performed by 16s rRNA sequencing and by 1H NMR (nuclear magnetic spectroscopy) for analysis of the metabolome. Results confirmed the AN diet was rich in protein and fat, but low in fiber-rich foods. Measurements of the microbiome composition showed clear separation between AN and Africans on PCA and MDS plots (p=0.0001). The microbiotas were dominated at the genus level by Blautia, Bacteroides, and Lachnospiraceae, which characterize Western populations (enterotype I) while the Africans overexpressed Prevotella, Ruminococcus, and Succinovibria, organisms that play a key role in fiber breakdown and butyrogenesis (enterotype II). The fecal metabolome showed clear separation on OPLS-DA cross-validated scores plot (p=4x10-11) driven in part by higher levels of SCFA and butyrate in Africans. Subgroup analysis showed that these differences were exaggerated in the Barrow cohort. In conclusion, these findings are consistent with our hypothesis that the extreme risk of colon cancer in AN is related to insufficient intake of fiber-rich foods and that fiber supplementation of their current diet may reduce the development of cancer. Citation Format: Stephen O'Keefe, Annette Wilson, Kathryn Koller, Christie Flanagan, Flora Sapp, Gretchen Day, Peter Holck, Barbara Methe, Alison Morris, Jia Li, James Kinross, Timothy Thomas. Changes in the composition and activity of the colonic microbiome that may explain the extreme risk of colon cancer in Alaskan Native People [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 IA06.

Abstract A73: Role of microsatellite stability and mismatch DNA repair mechanism in the response to 5-fluorouacil treatment in African American colon cancer cell lines

Abstract Despite progress in closing the gap, health disparities still persist among African American (AA) colon cancer patients both in incidence and death rates as well as worse prognosis after treatment with 5-fluorouacil (5-FU) when compared to Caucasian American (CA) patients. In addition, AA colon cancer patients have higher incidence of mutations on the mismatch DNA-repair mechanism (MMR) than CA patients, and previous studies have shown a correlation between mutations in MMR and microsatellite instability (MSI) to resistance to 5-FU. Therefore, we aim to examine if differential MSI and MMR play a role in the efficacy of 5-FU treatment in AAs. Methods: We examined the microsatellite status and MMR mutations found in the tumors from AA colon cancer patients at our institution from 2010 to 2017 and utilized the AA tumor-derived colon cancer cell line SB-521, generated in Dr. Williams's laboratory, and the CA colon cancer cell line HT-29 for in vitro studies. In order to assess the response to 5-FU, we used the protein levels of the ribonucleotide reductase catalytic subunit M1 (RRM1) protein as a marker for drug resistance, the cleavage of caspase-3 for apoptosis initiation, c-Jun N-terminal kinases (JNK) phosphorylation as indicator of cellular stress, and histone 3 protein levels as reference of gene methylation changes. Results: Our retrospective data (N=83 patients) demonstrated that up to 20% of our colon cancer patients have MSI and up to 30% showed MMR mutations. As hypothesized, the protein levels of the chosen targets in the two cell lines exhibited differences after treatment with 5-FU. The AA cell line SB-521 appears to be more sensitive to the chemotherapeutic in terms of apoptosis, stress response, and drug resistance when compared to HT-29 cells. Conclusions: Altogether, our results illustrate the range of MSI, MMR, and protein patterns in AA tumors and cell line. The cell line SB-521 is a potential model to study 5-FU treatment for patients with MSI and MMR mutations as it has high MSI, does not express MLH1, has reduced MSH2, and overexpresses MSH6 (MMR proteins). Previous data suggest that lack of a functional MMR mechanism may play a role in the response to this chemotherapeutic. Further studies are needed to elucidate the differences in chemotherapy treatment responses between AAs and CAs and their role in colon cancer health disparities. Citation Format: Jenny E. Paredes, Ping Ji, Maria Munoz-Sagastibelza, Malhaar Agrawal, Jennie Williams, Laura Martello-Rooney. Role of microsatellite stability and mismatch DNA repair mechanism in the response to 5-fluorouacil treatment in African American colon cancer cell lines [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 A73.

Abstract C68: Decreased antitumor cytotoxic immunity in African American colon cancer

Abstract Background: African Americans (AA) have a higher incidence of colorectal cancer (CRC) and more aggressive disease than patients of other ethnicities. Several studies have suggested that biological/genetic factors contribute to this health disparity. Recent work has shown the importance of the immune system to cancer patient prognosis, for CRC in particular. Anti-tumor T helper 1 (TH1) cell activation of cytotoxic T lymphocytes (CTL) increases CRC patient disease-free survival while deficiencies in these responses correlate with a worse prognosis. We hypothesize that differences in anti-tumor immunity by race contribute to the disparity and lead to more aggressive CRC in AA patients. Methods: Over 250 human colon tumors samples were stained using immunohistochemistry to evaluate in situ immune cell infiltration (CD8 and CD57), CTL activity [granzyme B (GzmB)], and IL-17 production. Also, using GzmB immunostaining, we analyzed the histological location of CTL/Natural Killer (NK) cell activity at intratumoral vs. invasive border sites of the CRC samples. In addition, a colon cancer mouse model is being used to identify mechanisms that may alter TH1 and CTL activity in developing tumors. The mouse model is the TS4Cre x APCfl-468 conditional Adenomatous Polyposis Coli (APC) knockout. Exons 11 and 12 are deleted from one APC gene in the colon and distal ileum due to the expression of CRE under a Fatty acid binding protein promoter leading to polyposis. These colonic tumors are being evaluated using qPCR on immune and inflammatory gene products. Results: Our studies on human microsatellite stable colon cancer samples showed that the number of CD8+ and CD57+ tumor-infiltrating cells doesn't differ by race (p=0.83 and 0.32, respectively). The level of GzmB+ cells, i.e. CTLs and activated NK cells, however, does vary significantly by race (p=0.01), particularly at the invasive border (p=0.0006). The numbers of IL-17A producing cells do not vary (p=0.63) but IL-17 may modulate GzmB correlation with CD8 and CD57 by race. For example, in AA, the correlation between GzmB and CD8 drops from 0.80 in IL-17Ahigh samples to 0.53 in IL-17Alow samples. For GzmB and CD57, the correlation level drops from 0.51 to 0.28 between IL-17A-high and -low samples, respectively. No such drop in correlation is observed in Caucasian American samples. In our mouse model, our preliminary studies show the TS4CRExAPCfl-468 mice initiate colonic polyposis at ~18 weeks of age and invasive cancer is observed in 20-25% of mice older than 31 weeks. The mean number of colonic polyps significantly increases between 18 and 24 weeks (from 2.3±0.9 to 8.0±1.9 tumors/mouse). Interestingly, the number declines by 36 weeks of age (5.5±0.5 tumors/mouse). However, the mean size of the colonic polyps significantly increases from 18 weeks to 36 weeks of age (from 1.54mm±0.55 to 4.23mm±0.56). To determine what mechanisms might result in lower anti-tumor CTL/NK activity, mouse tumors at different stages of development are currently being evaluated for IL-27, GzmA, IL-23, ƔIFN, CD8, Perforin, CX3CL1, IL-17a, H2DMB2, and Tbet gene expression using qPCR. Primers sets are also being developed to include IL-4, IL-6, IL-10, IL-12p40, and FoxP3 in our analyses. Conclusion: Our data to date indicate that the number of AA patients with high numbers of GzmB expressing cells infiltrating their tumors is significantly lower than Caucasians. In addition, IL-17 may have a modulatory effect on anti-tumor cytotoxicity by race. These observations support our hypothesis that immune processes contribute to the racial disparities observed in CRC. Current studies in our mouse model of CRC should identify mechanisms decreasing anti-tumor CTL/NK cell activity that will be tested in our Caucasian and AA colon cancer patient tumor samples in future studies. Note: This abstract was not presented at the conference. Citation Format: Mohammad W. Khan, MengXi Tian, John M. Carethers, Kathleen L. McGuire. Decreased antitumor cytotoxic immunity in African American colon cancer. [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 C68.

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.

Transcriptomes and shRNA suppressors in a TP53 allele-specific model of early-onset colon cancer in African Americans.

African Americans are disproportionately affected by early-onset, high-grade malignancies. A fraction of this cancer health disparity can be explained by genetic differences between individuals of African or European descent. Here the wild-type Pro/Pro genotype at the TP53Pro72Arg (P72R) polymorphism (SNP: rs1042522) is more frequent in African Americans with cancer than in African Americans without cancer (51% vs. 37%), and is associated with a significant increase in the rates of cancer diagnosis in African Americans. To test the hypothesis that Tp53 allele-specific gene expression may contribute to African American cancer disparities, TP53 hemizygous knockout variants were generated and characterized in the RKO colon carcinoma cell line, which is wild type for TP53 and heterozygous at the TP53Pro72Arg locus. Transcriptome profiling, using RNAseq, in response to the DNA-damaging agent etoposide revealed a large number of Tp53-regulated transcripts, but also a subset of transcripts that were TP53Pro72Arg allele specific. In addition, a shRNA-library suppressor screen for Tp53 allele-specific escape from Tp53-induced arrest was performed. Several novel RNAi suppressors of Tp53 were identified, one of which, PRDM1β (BLIMP-1), was confirmed to be an Arg-specific transcript. Prdm1β silences target genes by recruiting H3K9 trimethyl (H3K9me3) repressive chromatin marks, and is necessary for stem cell differentiation. These results reveal a novel model for African American cancer disparity, in which the TP53 codon 72 allele influences lifetime cancer risk by driving damaged cells to differentiation through an epigenetic mechanism involving gene silencing. TP53 P72R polymorphism significantly contributes to increased African American cancer disparity.

Prevalence Pattern of Right-Sided Colon Cancer in African Americans (AA) from 2000-2011

Prevalence Pattern of Right-Sided Colon Cancer in African Americans (AA) from 2000-2011

3'-UTR and functional secretor haplotypes in mannose-binding lectin 2 are associated with increased colon cancer risk in African Americans.

Because chronic intestinal inflammation is a risk factor for colorectal cancer, we hypothesized that genetic variants of inflammatory mediators, such as mannose-binding lectin 2 (MBL2), are associated with colon cancer susceptibility. Here, we report the association of 24 MBL2 single-nucleotide polymorphisms (SNP) and corresponding haplotypes with colon cancer risk in a case-control study. Four SNPs in the 3'-untranslated region (UTR) of the gene (rs10082466, rs2120132, rs2099902, and rs10450310) were associated with an increased risk of colon cancer in African Americans. ORs for homozygous variants versus wild-type ranged from 3.17 [95% confidence interval (CI), 1.57-6.40] to 4.51 (95% CI, 1.94-10.50), whereas the 3'-UTR region haplotype consisting of these four variants had an OR of 2.10 (95% CI, 1.42-3.12). The C allele of rs10082466 exhibited a binding affinity of miR-27a and this allele was associated with both lower MBL plasma levels and activity. We found that 5' secretor haplotypes known to correlate with moderate and low MBL serum levels exhibited associations with increased risk of colon cancer in African Americans, specifically as driven by two haplotypes, LYPA and LYQC, relative to the referent HYPA haplotype (LYPA: OR, 2.60; 95% CI, 1.33-5.08 and LYQC: OR, 2.28; 95% CI, 1.20-4.30). Similar associations were not observed in Caucasians. Together, our results support the hypothesis that genetic variations in MBL2 increase colon cancer susceptibility in African Americans.

Abstract A86: Investigation into racial/gender disparities in postsurgery survival of colorectal cancer: A retrospective study from a community hospital in New York City

Abstract Background: Racial disparities and gender differences in the prevalence and mortality of colorectal cancer have been reported. African Americans (AA) have the highest incidence and worst prognosis of colorectal cancer amongst ethnic groups. Men also have a younger age at diagnosis than women along with a poorer prognosis. It is uncertain whether differences persist when patients are of similar biological status at diagnosis, and they have equal opportunity to receive surgery. The aim of this study is to investigate post-surgery survival between AAs and Whites as well as between men and women in the setting of a community hospital in which a majority of the population is AA. Methods: This is a retrospective analysis of data from The Brooklyn Hospital Center's cancer registry from 1997 to 2010. Of all of 1068 registered patients, 806 (75.46%) are AA, 243 (22.75%) are White and 19 (1.8%) are Asian. 445 are male and 623 are female. Among all of the subjects, 817 (76.5%) underwent surgery. Among the 817 surgical patients, 619 are African American, 174 are White, and 34 are Asian (not included in the data analysis). 330 are men, and 403 are women (exclude Asian). Statistical analyses were done by SPSS and Epi info software. Chi square test, Fisher's exact test, and one-way ANOVA analysis are used for the baseline characteristics. Kaplan–Meier survival probabilities are calculated and multivariate Cox proportional hazards models were applied to estimate hazard ratios (HR) with 95% confidence intervals (95% CI). Results: The average age at diagnosis is 67.62±13.23 in AAs and 69.54±14.25 in Whites with no significant difference. Average age at diagnosis in men is 65.81±11.78 and in women is 68.09±13.5 (P=0.014). There is significant difference in location of the presenting tumor; AAs right sided colon cancer in AAs is 40.87% as compared with Whites in whom right sided colon cancer occurs 30.46% of the time. 50.57% of Whites have colorectal cancer located in rectosigmoid region vs. only 38.93% of AAs (p=0.002). There was no significant difference in tumor location between men and women. No significant difference existed in the distribution of histological grade (p=0.19), pathologic stages (p=0.82), people who received surgery, and percentage of patients who received chemotherapy between ethnic groups as well as between gender groups. Hemicolectomy is the most common procedure performed. Significant difference for the distribution of the surgery types was present with 51.4% of AAs receiving hemicolectomy vs. 39.1% of Whites (p=0.022). Also, a significant difference in surgery type was measured between genders with 47% of men receiving hemicolectomy versus 49.9% of women (p=0.044). Kaplan Meier survival analysis: no significant differences presented between these two ethnic groups (Log-rank p=0.2958) with regards to the 5 years survival probability; 5 year survival in AAs is 61.07% and in Whites is 57.36 %. There was a significant difference between men and women in 5 year survival with Log rank p=0.035 and a 5-year survival probability for men of 54.38% vs. 64.24% for women with Cox hazard ratio 1.248. Conclusions: Much attention has been paid to racial disparities and gender differences for the past two decades. Our study shows that AAs are younger at diagnosis and are more likely to present with proximal tumors than Whites, but there is no significant difference in survival between these two ethnic groups post-surgery. Based on similar biological background and with equal opportunity to receive surgery, the racial disparity is diminished perhaps even ameliorated. However, a gender difference is present in our data analysis even when taking into account no difference in the biological characteristics. Men have a younger age at diagnosis and a poorer prognosis. As some studies state, that gender difference may be attributed to hormone levels. Further study is needed to uncover the underlying mechanisms. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A86.

Abstract B48: Do the gut flora play a role in the high incidence of colon cancer in African Americans?

Abstract Colonic adenomatous polyps are common tumors occurring in 50% of Western populations with 10% risk of malignant progression. To date, the environmental exposures proposed to promote the development of colorectal cancer (CRC) have been primarily dietary agents. However, the local environment to which the colonic mucosa is exposed is created by the bacterial microbiota of the colon and their metabolic products that include toxins. African Americans are known to have a higher incidence of colon cancer than other groups. We analyzed the gut flora from African Americans patients by both 16S rDNA sequencing and HIT (Human Intestinal Tract) Chip. Our findings reflected a high prevalence of Bacteroidetes and Clostridia groups with the Bacteroidetes slightly more represented in patients with colon polyps than healthy ones. While Bifidobacteria were not detected at all in all samples, Lactobacilliwere present at a very low frequency. Both bacteria are referred to as “good bacteria” and are generally associated with healthy colon. Helicobacter bacteria, associated with gastric cancer, were also detected in all analysed samples. Bacteroides fragilis and relatives were present in all samples at a high frequency. It is noteworthy that an enterotoxigenic Bacteroides fragilis have been found to trigger colon polyps formation in Min mice through a pSTAT3 pathway. A tissue microarray immunohistochemistry experiment on colon adenoma and matched normals revealed a strong expression of pSTAT3 both in colonic epithelial cells and in colonic immune cells in adenoma samples but not as much in normal ones. These findings highlight the presence of a possible ETBF-like induction mechanism in the analysed samples. Enzyme immunoassay for the detection of antibodies against ETBF toxin would confirm such findings and a metagenomic analysis of the analysed samples would shed light on predominant bacterial functions in adenoma patients that might play a role in colon oncogenic transformation in this population. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B48.

Clinicopathological Features of Colon Polyps from African-Americans

Among the ethnic groups, the age-standardized incidence rate of colorectal cancer (CRC) is highest among African-Americans. The majority of CRC arise from preexisting adenoma. It is shown that 30% of the US adult population has adenomas. The potential risk of malignant transformation in adenomas differs by specific pathologic and clinical characteristics that we aimed to study in AAs. All pathologic reports (150,000) in Howard University Hospital from 1959 to 2006 were reviewed manually. Those pathology reports compatible with the colorectal polyps were carefully reviewed and selected by a GI pathologist. All cases with cancer were then excluded from the list. Data were then entered into Microsoft Excel and checked for missing data and duplications. Differences in right-side and left-side polyps for sex, histology, and clinical symptoms were assessed by Chi-2 test. A total number of 5,013 colorectal polyps were diagnosed in this period that include 47% male, with mean age (SD) of 63 (12). Half of the cases were diagnosed in 2001-2006. Tubular adenoma was the most frequent pathology (73%). The highest frequency of right-sided polyps was observed in the 1990s (56%). Left-sided polyps were younger (p < 0.0001), more hyperplasic (23 vs. 5%; p < 0.0001), and more frequent in female (56 vs. 52%; p = 0.02) compared to right-sided polyps. The frequency of right-sided adenoma significantly increases from 18% in the 1960s to 51% in the period of 2001-2006 (p < 0.0001). The most frequent symptom in both sides was GI bleeding (21%). There was a ratio of 8:1 for neoplastic to hyperplastic polyps in our study, which is more than what has been reported in Caucasians (7:1). Our data shows a shift in polyps from the left side to the right side of the colon in recent years. This data is consistent with the lack of a reduction in the incidence of colon cancer in African-Americans. Screening is thus very important in AA to reduce the incidence of colon cancer.

GSTM1 and GSTT1 Polymorphisms, Cigarette Smoking, and Risk of Colon Cancer: A Population-based Case-control Study in North Carolina (United States)

Cigarette smoke is a risk factor for colon cancer, but the importance of dose and interaction with genetic susceptibility remain poorly understood. We used data from a population-based case control study, to examine the association between cigarette smoking and colon cancer in African Americans and whites, and colon cancer and polymorphisms in GSTM1 and GSTT1. A total of 554 cases of primary colon cancer and 874 controls were included in this analysis. We found no association between cigarette smoking (ever versus never) and colon cancer in African Americans (odds ratio (OR)=0.93, 95% confidence interval (CI)=0.65-1.33). In contrast, there was an increased risk of cigarette smoking in whites (OR=1.43, CI=1.05-1.94). There was a small increased risk of colon cancer for individuals with GSTM1 null (African Americans, OR=1.43, CI, 0.98-2.09; whites, OR=1.19, CI, 0.90-1.58) and a decreased risk of colon cancer for individuals with GSTT1 null (African Americans, OR=0.59, CI: 0.40-0.86; whites, OR=0.72, CI: 0.53-1.00). There were weak interactions between GSTT1 null and cigarette smoking in whites, and GSTM1 null genotype and cigarette smoking in African Americans. GSTT1 and GSTM1 polymorphisms may be weakly related to colon cancer risk and there may be racial differences in gene-smoking interactions.

COX-2 Polymorphism, Use of Nonsteroidal Anti-Inflammatory Drugs, and Risk of Colon Cancer in African Americans (United States)

The inducible Cyclooxygenase (COX)-2 enzyme plays an important role in inflammation and carcinogenesis. Recent reports suggest that single nucleotide polymorphisms (SNPs) in the COX-2 gene may alter enzyme function and in turn modify an individual's risk of colon cancer. We explored the association between the COX-2 Val511Ala SNP and risk of colon cancer among 240 African American cases and 326 African American controls in a population-based, case-control study in North Carolina. We used unconditional logistic regression models to determine the odds ratios (ORs) for genotype and risk of colon cancer. We observed a non-statistically significant inverse association between any Ala COX-2 genotype and risk of colon cancer (OR = 0.62, 95% CI: 0.33, 1.16) among African Americans. The inverse association was present among non-regular NSAID users, use < or = 3 times/week, (OR = 0.66; 95% CI: 0.32, 1.37) and regular NSAID users, use > or =3 times/week for > or =3 months, (OR = 0.41; 95% CI: 0.11, 1.54). Our results suggest that the COX-2 Val511Ala SNP does not antagonize the effect of NSAIDs on colon cancer risk and provides support that NSAID use and the COX-2 Val511Ala SNP may contribute to a reduced risk of colon cancer among African Americans.