Sporadic Colorectal Carcinomas Research Articles

Abstract 3872: Multi-ancestral origins of colorectal lesionsMulti-ancestral origins of colorectal lesions

Abstract The widely accepted paradigm that cancer initiation is a monoclonal event has not been definitively resolved. In sporadic cancers this is due to the requirement for longitudinal sampling to study this occult process, which occurs over years to decades. Individuals with familial adenomatous polyposis (FAP) possess a germline APC mutation, leading to hundreds of colonic polyps in adolescence and a 100% lifetime risk of colorectal cancer. Resected colons from FAP patients contain normal mucosa, benign and dysplastic polyps that embed the natural history of colorectal tumorigenesis at different points of malignant progression. Through genome sequencing of 127 independent polyps or mucosa samples from six FAP patients, we characterized the genomic landscape, relative timing of somatic alterations, and clonal dynamics in normal, dysplastic, and invasive colon tissue. Comparison with multi-region sequencing data from FAP patients and sporadic colorectal adenomas and carcinomas indicates similar molecular profiles, with canonical cancer driver genes present across benign and dysplastic premalignant lesions and shared mutational orders. Pairwise sample comparisons and phylogenetic reconstructions revealed extensive heterogeneity and subclonal sharing within a patient’s benign and dysplastic lesions, irrespective of location in the colon. These data imply that polyps were initiated from multiple cells from distinct lineages whose most recent common ancestor arose during development. We corroborate these findings using a mechanistic mathematical model, which infers polyclonality in 100% of benign and 83% of dysplastic samples. Based on these findings, we propose a conceptual model of the multi-ancestral origin of intestinal malignancies. Citation Format: Ryan O. Schenck, Aziz Khan, Aaron Horning, Edward D. Esplin, Debra Van Egeren, HTAN–FAP Consortium, William Greenleaf, James M. Ford, Michael P. Snyder, Christina Curtis. Multi-ancestral origins of colorectal lesionsMulti-ancestral origins of colorectal lesions [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 3872.

Association of Microsatellite Instability and Gene Expression Profile in Colorectal Carcinoma and Potential Implications for Therapy.

Background and Objective: In sporadic colorectal carcinomas (CRC), microsatellite instability (MSI) pathways play important roles. Previously, we showed differences in DNA methylation patterns in microsatellite stable (MSS) colorectal carcinomas and MSI-CRC. In the current study, we explore the similarities and differences in gene expression profiles in MSS and MSI at the gene level and at the pathway level to better understand CRC pathogenesis and/or the potential for therapeutic opportunities. Material and Methods: Seventy-one CRC patients (MSI = 18, MSS = 53) were studied. Paired tumor and adjacent normal tissues were used for genome-wide gene expression assays. Result: At the gene level, we compared the list of differentially expressed genes (fold change (FC) ≥ 3 and FDR < 0.05) in tumor tissues compared to corresponding normal tissue in CRC patients with MSI tumors (190 genes) and MSS tumors (129 genes). Of these, 107 genes overlapped. The list of genes that were differentially expressed in MSI tumors only showed enrichment predominantly in two broad categories of pathways-(a) Inflammation-related pathways including the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, chemokine signaling, nuclear factor kappa B (NFκB) signaling, and cytokine-cytokine interactions, and (b) metabolism-related pathways, including retinol metabolism, steroid hormone biosynthesis, drug metabolism, pentose and glucoronate interconversions, and ascorbate and aldarate metabolism. The genes in inflammation-related pathways were up-regulated whereas genes in metabolism-related pathways were down-regulated in MSI tumor tissue. Pathway-level analysis also revealed similar results confirming the gene enrichment findings. For example, the 150 genes involved in the IL-17 signaling pathway were on average up-regulated by 1.19 fold (CI 1.16-1.21) in MSI compared to 1.14 fold (CI 1.13-1.16) in MSS patients (interaction p = 0.0009). Conclusions: We document an association between MSI status and differential gene expression that broadens our understanding of CRC pathogenesis. Furthermore, targeting one or more of these dysregulated pathways could provide the basis for improved therapies for MSI and MSS CRC.

Functional annotation with expression validation identifies novel metastasis-relevant genes from post-GWAS risk loci in sporadic colorectal carcinomas

BackgroundColorectal cancer (CRC) is the third highest incidence cancer and is the leading cause of cancer mortality worldwide. Metastasis to distal organ is the major cause of cancer mortality. However,...

Detection of Fusobacterium nucleatum in Patients with Colitis-Associated Colorectal Cancer

Fusobacterium nucleatum is supposed to play a critical role in the development of colorectal cancer. The species has also been associated with ulcerative colitis (UC) that can progress into colorectal cancer, however, the involvement of bacteria in this process remains unclear. We analysed 177 colon biopsies obtained from patients during screening, including 20 healthy controls, 56 UC cases and 69 cases at different stages of progression to colitis-associated cancer (CAC); 32 samples of sporadic colorectal carcinoma (sCRC) were also included. The presence of F. nucleatum was detected by quantitative real-time PCR (qPCR). Our data show an association between the presence of the bacteria and the progression of carcinogenesis in UC patients. In 39.5% of CAC samples F. nucleatum was detected, compared to only 1.8% in UC cases. The bacteria were detected in 6.3% of samples with initial neoplastic transformation, so-called low-grade dysplasia (LGD), whereas high-grade dysplasia (HGD) resulted in 33.3% of samples positive for F. nucleatum. The fraction of F. nucleatum-positive samples from sCRC cases was 56.3%, which was not significantly different to the CAC group. We conclude that F. nucleatum is associated with the occurrence and progression of colon carcinogenesis, rather than with UC itself.

Molecular detection of mononucleotide biomarkers of microsatellite instability in sporadic colorectal carcinoma patients with clinicopathological correlation

Objectives: To identify the frequency and types of microsatellite instability among a group of sporadic CRC patients and to correlate the findings with clinicopathological characteristics.&#x0D; Methods: During an 8-month period, all patients with sporadic CRC who attended to two teaching hospitals in Baghdad, Iraq were recruited to this cross-sectional study regardless of age, sex, ethnicity, or tumor characteristics. Demographic, clinical, and histopathological features were recorded. DNA was extracted from FFPE-blocks of the resected tumors and normal tissues. PCR amplification of five microsatellite mononucleotide repeat loci (BAT25, BAT26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeat control markers (Penta C and Penta D) was performed to determine the MSI status. Capillary electrophoresis and Genetic Analyzer 3500 (Applied Biosystem, Japan) were used to separate and examine the products. Data were analyzed by Genescan software (Promega, USA). Instability of two or more loci is considered MSI-H.&#x0D; Result: In this study, ages of the 45 recruited patients ranged between 20-80 years, with a mean±SD of 55±12.3 years; of them, 31(68.9%) were ≥50 years; 25 (55.6%) were males. Rectal bleeding was the most frequent presenting feature [22 (48.9%)] patients; 23 (51.1%) of CRCs were located at recto-sigmoid region, 29 (64.4%) were T3 tumors, 34(75.5%) were non-mucinous adenocarcinoma, 39(86.7%) were moderately differentiated, 17 (37.8%) patients had stage III tumors; and 25 (55.5%) had lymphovascular invasion. MSI-H was seen in 5/45 (11.1%) patients; 3(60%) of them were ≥50 years, 4(80%) were males, 3(60%) were smokers, 2 (40%) presented with intestinal obstruction and altered bowel habits each; 4(80%) had T3 tumors, 3(60%) had mucinous adenocarcinomas [p=0.004], 2(40%) had stage II tumor and stage III each.&#x0D; Conclusion: The frequency of MSI-H among the recruited patients with CRC was 5/45 (11.1%) and it was significantly associated with mucinous adenocarcinoma subtype. NR-24 and NR-21 were the most prevalent instable markers.

Pathways Related to Colon Inflammation Are Associated with Colorectal Carcinoma: A Transcriptome- and Methylome-Wide Study.

The association of chronic inflammation with colorectal carcinoma (CRC) development is well known in ulcerative colitis (UC). However, the role of inflammatory changes in sporadic CRC pathogenesis is less widely appreciated. In this study, in the first step using RNA-seq, we identified gene-pathway-level changes in UC-associated CRC (UC CRC, n = 10) and used the changes as a proxy for inflammation in human colon to ask if there were associations of inflammatory pathway dysregulations in sporadic CRC pathogenesis (n = 8). We found down-regulations of several inflammation-related metabolic pathways (nitrogen metabolism, sulfur metabolism) and other pathways (bile secretion, fatty acid degradation) in sporadic CRC. Non-inflammation-related changes included up-regulation of the proteasome pathway. In the next step, from a larger number of paired samples from sporadic CRC patients (n = 71) from a geographically and ethnically different population and using a different platform (microarray), we asked if the inflammation-CRC association could be replicated. The associations were significant even after stratification by sex, tumor stage, grade, MSI status, and KRAS mutation status. Our findings have important implications to widen our understanding of inflammatory pathogenesis of sporadic CRC. Furthermore, targeting of several of these dysregulated pathways could provide the basis for improved therapies for CRC.

Mismatch repair system in colorectal cancer. Frequency, cancer phenotype, and follow-up

Introduction and aimsA frequent task in the study of colorectal carcinomas (CRC) is to identify tumors harboring deficient DNA mismatch repair systems (dMMR), which are associated with microsatellite instability. Given that there is scant information on those tumors in Mexican patients, our aim was to describe their frequency, clinical and pathologic characteristics, and results, which are necessary for future trials. Materials and methodsA consecutive series of CRC patients, treated and followed at a tertiary care center was performed. The clinical and pathologic variables and the risk of hereditary or familial cancer syndrome were retrieved. The original slides and hMLH1, hPMS2, hMSH2, hMSH6 immunohistochemistry were evaluated. Tumors with an absence of at least one protein were considered dMMR. Differences were contrasted, utilizing non-parametric tests. ResultsOne hundred and forty-four patients were included, with a median age of 65 years. A total of 134/93% patients presented with sporadic CRC, 8/5.6% had a family history of CRC, and 2/1.4% met the diagnostic criteria for hereditary non-polyposis colon cancer, according to the Amsterdam and Bethesda criteria. dMMR tumors were found in 39 patients, distributed among the three groups. They were locally advanced (p<0.001), right-sided, had the mucinous phenotype, and harbored a Crohn’s-like lymphoid reaction (all three features, p<0.04). Adjuvant or palliative chemotherapy was administered to 57 (39.6%), concomitant chemoradiotherapy to 24 (16.7%), but 63 (43.8%) patients received no additional treatment to surgery. Five-year follow-up was completed in 131 of the patients and the outcomes alive-with-disease or died-of-disease were more frequently observed in the proficient (pMMR) lesions. ConclusionsIn the present pre-FOLFOX case series, outcomes were better in dMMR CRC than in proficient lesions.

Sistema de reparación de errores de emparejamiento en carcinoma colorrectal. Frecuencia, fenotipo y seguimiento

Introduction and aimsA frequent task in the study of colorectal carcinomas (CRC) is to identify tumors harboring deficient DNA mismatch repair systems (dMMR), which are associated with microsatellite instability. Given that there is scant information on those tumors in Mexican patients, our aim was to describe their frequency, clinical and pathologic characteristics, and results, which are necessary for future trials. Materials and methodsA consecutive series of CRC patients, treated and followed at a tertiary care center was performed. The clinical and pathologic variables and the risk of hereditary or familial cancer syndrome were retrieved. The original slides and hMLH1, hPMS2, hMSH2, hMSH6 immunohistochemistry were evaluated. Tumors with an absence of at least one protein were considered dMMR. Differences were contrasted, utilizing non-parametric tests. ResultsOne hundred and forty-four patients were included, with a median age of 65 years. A total of 134/93% patients presented with sporadic CRC, 8/5.6% had a family history of CRC, and 2/1.4% met the diagnostic criteria for hereditary non-polyposis colon cancer, according to the Amsterdam and Bethesda criteria. dMMR tumors were found in 39 patients, distributed among the 3groups. They were locally advanced (P<.001), right-sided, had the mucinous phenotype, and harbored a Crohn's-like lymphoid reaction (all 3features, P<.04). Adjuvant or palliative chemotherapy was administered to 57 (39.6%), concomitant chemoradiotherapy to 24 (16.7%), but 63 (43.8%) patients received no additional treatment to surgery. Five-year follow-up was completed in 131 of the patients and the outcomes alive-with-disease or died-of-disease were more frequently observed in the proficient (pMMR) lesions. ConclusionsIn the present pre-FOLFOX case series, outcomes were better in dMMR CRC than in proficient lesions.

Advances in colorectal cancer genomics and transcriptomics drive early detection and prevention.

Colorectal carcinoma (CRC) is a high incidence cancer and leading cause of cancer mortality worldwide. The advances in genomics and transcriptomics in the past decades have improved the detection and prevention of CRC in familial CRC syndromes. Nevertheless, the ultimate goal of personalized medicine for sporadic CRC is still not within reach due no less to the difficulty in integrating population disparity and clinical data to combat what essentially is a very heterogenous disease. This minireview highlights the achievement of the past decades and present possible direction in the hope of early detection and metastasis prevention for reducing CRC-associated morbidity and mortality.

Tetranucleotide and Low Microsatellite Instability Are Inversely Associated with the CpG Island Methylator Phenotype in Colorectal Cancer.

Simple SummaryA type of DNA mismatch repair defect known as “elevated microsatellite alterations at selected tetranucleotide repeats” (EMAST) is found across many different cancers. Tetranucleotide microsatellite instability, which is caused by MSH3 mismatch repair gene/protein loss-of-function, shares a molecular basis with “low microsatellite instability” (MSI-L) in colorectal cancer. Tetranucleotide microsatellite instability is also a byproduct of “high microsatellite instability” (MSI-H) that arises from deficiency of mismatch repair due to MSH2, MSH6, MLH1 or PMS2 gene alterations. MSH3-related EMAST is emerging as a biomarker of poor prognosis in colorectal cancer and needs to be clearly differentiated from MSI-H. Here, we show that tumours with non-MSI-H-related EMAST or MSI-L rarely show concordant promoter methylation of multiple marker genes. Colorectal tumours that are positive for a single (1/5) tetranucleotide repeat marker are an important subset of the EMAST spectrum.MSH3 gene or protein deficiency or loss-of-function in colorectal cancer can cause a DNA mismatch repair defect known as “elevated microsatellite alterations at selected tetranucleotide repeats” (EMAST). A high percentage of MSI-H tumors exhibit EMAST, while MSI-L is also linked with EMAST. However, the distribution of CpG island methylator phenotype (CIMP) within the EMAST spectrum is not known. Five tetranucleotide repeat and five MSI markers were used to classify 100 sporadic colorectal tumours for EMAST, MSI-H and MSI-L according to the number of unstable markers detected. Promoter methylation was determined using methylation-specific PCR for MSH3, MCC, CDKN2A (p16) and five CIMP marker genes. EMAST was found in 55% of sporadic colorectal carcinomas. Carcinomas with only one positive marker (EMAST-1/5, 26%) were associated with advanced tumour stage, increased lymph node metastasis, MSI-L and lack of CIMP-H. EMAST-2/5 (16%) carcinomas displayed some methylation but MSI was rare. Carcinomas with ≥3 positive EMAST markers (13%) were more likely to have a proximal colon location and be MSI-H and CIMP-H. Our study suggests that EMAST/MSI-L is a valuable prognostic and predictive marker for colorectal carcinomas that do not display the high methylation phenotype CIMP-H.

Regulation of KRAS protein expression by miR-544a and KRAS-LCS6 polymorphism in wild-type KRAS sporadic colon adenocarcinoma.

Colorectal carcinoma (CRC) results from the accumulation of genetic mutations and alterations in signaling pathways. KRAS is mutated in 40% of CRC cases and is involved in increased tumor cells proliferation and survival. Although KRAS mutations are a dominant event in CRC tumorigenesis, increased wild-type KRAS expression has a similar effect on accelerated tumor growth. In this study, we investigated the KRAS status in correlation with clinicopathological features in sporadic CRC and more importantly the role of let-7a-5p and miR-544a-3p in the regulation of wild-type KRAS protein expression in the tumor center (T1) and invasive tumor front (T2). Analysis showed that 39.1% of tumor samples had KRAS mutations. In wild-type KRAS tumors, 62.0% were positive for KRAS protein expression and there was a higher percentage of KRAS-positive tumor cells and a higher intensity of immunohistochemical reaction in T2 than in T1 samples. This could not be attributed to differences in KRAS mRNA levels, suggesting regulation via miR-544a-3p expression which was significantly decreased in T2 samples. Furthermore, we demonstrated that tumor samples carrying the KRAS-LCS6 variant allele had significantly higher protein expression of the wild-type KRAS. Our results suggest the role of the KRAS-LCS6 polymorphism and miR-544a-3p expression in the regulation of wild-type KRAS protein expression in sporadic CRC.

Abstract 2829: Identifying germline variants for metastatic risk assessment in sporadic colorectal carcinomas

Abstract Colorectal carcinoma (CRC) is the third highest incidence cancer and leading cause of cancer mortality worldwide. Metastasis to distal organ is the major cause of cancer mortality. This trend is expected to be exacerbated by an aging population in the developed world. Genetic predisposition plays a greater role in disease progression than disease etiology in sporadic CRC. However, the underlying genetic factors for metastasis are currently unclear. We identified germline single nucleotide polymorphism (SNP) variants contributing to metastasis risk from a case-case (metastasis-positive vs metastasis-negative) genome-wide association study (GWAS) comprising 3,000 sporadic Chinese CRC cases with definitive metastasis status. Metastasis-positive case is defined as one with distal-organ involvement attributable to primary CRC; metastasis-negative case is defined as metastasis-free with 5 years or more follow-up. By interrogating summarized SNP statistics from the case-case GWAS using the Functional Mapping and Annotation (FUMA) software, we identified 5 genomic risk loci and 74 disease-relevant genes based on gene set enrichment analysis and the Molecular Signatures Database. These genes are in various pathways implicated in different metastasis steps, from epithelial-mesenchymal transition (EMT) to implantation at distal organs. They are different from the risk variants identified by case-control GWAS for disease occurrence. We will expression-profile 250 metastasis-positive vs metastasis negative cases from the GWAS panel to validate the bioinformatics analysis by quantitative real-time PCR. We will focus first on the five genes implicated in glycolysis as this is a biochemical fingerprint of malignant cells that represents one of the ‘hallmarks of cancer'. Expression profile of these genes will be validated in a second independent panel. Kaplan Meier survival analysis as function of the median expression value will be performed to assess prognostic value of these genes. The ultimate goal is to improve metastasis risk assessment for secondary prevention to reduce cancer mortality and morbidity for individual patient as well as reducing public health expenditure. Citation Format: PehYean Cheah, Lai Fun Thean, Michelle Wong, Michelle Lo, Emile Tan, Choong Leong Tang. Identifying germline variants for metastatic risk assessment in sporadic colorectal carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2829.

Relationship between Metalloprotease-7 and -14 and Tissue Inhibitor of Metalloprotease 1 Expression by Mucosal Stromal Cells and Colorectal Cancer Development in Inflammatory Bowel Disease.

Colorectal carcinoma (CRC) associated with inflammatory bowel disease (IBD) is an example of an inflammation-related cancer. Matrix metalloproteases (MMP) are known to be associated with both processes. The aim of the study was to compare the expression of MMP-7, MMP-14 and tissue inhibitor of metalloproteases-1 (TIMP-1) in sporadic CRC- and IBD-associated CRC, and to compare the expression in inflamed and non-inflamed colonic tissue samples from IBD patients without or with associated CRC. An immunohistochemical study of MMP-7, -14 and TIMP-1 was performed on sporadic CRC (n = 86), IBD-associated CRC (n = 23) and colorectal mucosa of non-tumor samples from IBD patients without (n = 47) and with (n = 23) associated CRC. These factors were more frequently expressed by cancer-associated fibroblasts (CAF) from IBD-associated CRC than by CAF from CRC not associated with IBD. Regarding the inflamed tissue of IBD patients, Crohn’s disease (CD) patients with CRC development showed a higher expression of MMP-14 by fibroblasts and by mononuclear inflammatory cells (MICs) than CD patients without CRC development. In non-inflamed tissue samples, MMP-7 associated with fibroblasts and MICs, and TIMP-1 associated with MICs, were more frequently expressed in CD patients with CRC development than in CD patients without CRC development. Our data suggest that these factor expressions by stromal cells may be biological markers of CRC development risk in IBD patients.

Most colitis associated carcinomas lack expression of LGR5: a preliminary study with implications for unique pathways of carcinogenesis compared to sporadic colorectal carcinoma

BackgroundLeucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), a component of the Wnt receptor complex, is thought to lineage label gastric and intestinal stem cells. LGR5 expression is increased in colorectal carcinoma (CRC) compared to normal tissue. Colitis associated colorectal adenocarcinoma (CAC) often shows distinct morphologic and molecular phenotypes compared to sporadic cases. However, the expression profile of LGR5, and by extension the potential role of an intestinal stem cell phenotype, has not been well described in a series of human CAC.MethodRNA in situ hybridization (ISH) for LGR5 expression on 30 CACs (12 cases with conventional morphology and 18 cases with non-conventional type morphology) from 29 inflammatory bowel disease (IBD) patients was performed and compared the expression profile to a control group of 10 sporadic CRCs. Immunohistochemistry for beta-catenin and SATB2 was performed on the 30 CACs.ResultLGR5 was positive in 30% (9/30) of CAC cases and 90% (9/10) of sporadic CRCs (p = 0.002). A large majority (89%) of LGR5 positive CACs were of the conventional histologic type, and conventional type CAC showed a significantly higher LGR5 score (median 3.0; interquartile range 1.75–3.25) than non-conventional type CAC (median 1.5; interquartile range 1.00–2.00) (p = 0.034). CAC with conventional morphology did have a lower level of LGR5 expression than sporadic CRC. Sporadic CRCs showed a significantly higher LGR5 level score than non-conventional type CACs (p < 0.001). Nuclear translocation of beta-catenin was strongly associated with LGR5 expression (p = 0.003), however no significant association was identified between SATB2 expression and LGR5 expression status in CACs.ConclusionThese findings suggest that the wider spectrum of tumor morphology in CAC may be associated with absence of a LGR5-expressing intestinal stem cell phenotype.

Tissue mRNA for S100A4, S100A6, S100A8, S100A9, S100A11 and S100P Proteins in Colorectal Neoplasia: A Pilot Study.

S100 proteins are involved in the pathogenesis of sporadic colorectal carcinoma through different mechanisms. The aim of our study was to assess tissue mRNA encoding S100 proteins in patients with non-advanced and advanced colorectal adenoma. Mucosal biopsies were taken from the caecum, transverse colon and rectum during diagnostic and/or therapeutic colonoscopy. Another biopsy was obtained from adenomatous tissue in the advanced adenoma group. The tissue mRNA for each S100 protein (S100A4, S100A6, S100A8, S100A9, S100A11 and S100P) was investigated. Eighteen biopsies were obtained from the healthy mucosa in controls and the non-advanced adenoma group (six individuals in each group) and thirty biopsies in the advanced adenoma group (ten patients). Nine biopsies were obtained from advanced adenoma tissue (9/10 patients). Significant differences in mRNA investigated in the healthy mucosa were identified between (1) controls and the advanced adenoma group for S100A6 (p = 0.012), (2) controls and the non-advanced adenoma group for S100A8 (p = 0.033) and (3) controls and the advanced adenoma group for S100A11 (p = 0.005). In the advanced adenoma group, differences between the healthy mucosa and adenomatous tissue were found in S100A6 (p = 0.002), S100A8 (p = 0.002), S100A9 (p = 0.021) and S100A11 (p = 0.029). Abnormal mRNA expression for different S100 proteins was identified in the pathological adenomatous tissue as well as in the morphologically normal large intestinal mucosa.

Genomic amplification of chromosome 20q13.33 is the early biomarker for the development of sporadic colorectal carcinoma

BackgroundColorectal carcinoma (CRC) is the third most common cancer in the world and also the third leading cause of cancer-related mortality in Taiwan. CRC tumorigenesis is a multistep process, starting from mutations causing loss of function of tumor suppressor genes, canonically demonstrated in adenomatous polyposis coli pathogenesis. Although many genes or chromosomal alterations have been shown to be involved in this process, there are still unrecognized molecular events within CRC tumorigenesis. Elucidating these mechanisms may help improve the management and treatment.MethodsIn this study, we aimed to identify copy number alteration of the smallest chromosomal regions that is significantly associated with sporadic CRC tumorigenesis using high-resolution array-based Comparative Genomic Hybridization (aCGH) and quantitative Polymerase chain reaction (qPCR). In addition, microsatellite instability assay and sequencing-based mutation assay were performed to illustrate the initiation event of CRC tumorigenesis.ResultsA total of 571 CRC patients were recruited and 377 paired CRC tissues from sporadic CRC cases were used to define the smallest regions with chromosome copy number changes. In addition, 198 colorectal polyps from 160 patients were also used to study the role of 20q13.33 gain in CRC tumorigenesis. We found that gain in 20q13.33 is the main chromosomal abnormalities in this patient population and counts 50.9 and 62.8% in CRC and colon polyps, respectively. Furthermore, APC and KRAS gene mutations were profiled simultaneously and co-analyzed with microsatellite instability and 20q13.33 gain in CRC patients. Our study showed that the frequency of 20q13.33 copy number gain was highest among all reported CRC mutations.ConclusionAs APC or KRAS mutations are currently identified as the most important targets for CRC therapy, this study proposes that 20q13.33 copy number gain and the associated chromosomal genes function as promising biomarkers for both early stage detection and targeted therapy of sporadic CRCs in the future.

Immunohistochemistry as a Surrogate Marker of Underlying Molecular Derangements in Sporadic Colorectal Carcinoma in Children – A Series of Three Cases

Background: Colorectal carcinomas (CRCs) are uncommon tumors in children. Here, we elucidate three cases of childhood CRCs with their underlying molecular derangements using immunohistochemistry (IHC) with emphasis on BRAF mutation. Case summary: All three CRCs were sporadic tumors involving the left colon with two of them having a mucinous phenotype. We performed IHC for BRAF, p53 and β-catenin along with markers of microsatellite instability (MSI) in all three tumors. All the tumors had diffuse strong cytoplasmic BRAF positivity, with focal p53 positivity in two cases and cytoplasmic β-catenin staining in one case. One case showed CpG island hypermethylation with isolated loss of PMS2 staining. None of the cases had any family history of CRC. Conclusions: IHC can be used as a surrogate marker for determining the underlying molecular derangements in CRC. Sporadic CRCs in children are a cumulative effect of multiple mutations, of which BRAF mutation is significant and critical for planning targeted therapy.

Calponin 3 promotes invasion and drug resistance of colon cancer cells.

BACKGROUNDCalponin 3 (CNN3) is an actin-binding protein expressed in smooth muscle and non-smooth muscle cells. It is required for cytoskeletal rearrangement and wound healing.AIMTo dissect the role of CNN3 in carcinogenesis with a focus on colon cancer.METHODSA total of 20 cancer cell lines (8 breast, 11 colon, and HeLa cervical cancer cell as a positive control for mesenchymal phenotype) and 57 formalin-fixed, paraffin-embedded sections from archived sporadic colorectal carcinomas were included in this study. CNN3 expression analysis by western blot or immunohistochemistry was followed by functional analyses. The CNN3 gene was silenced by specific small interfering RNA (commonly known as siRNA), followed by confirmation of the silencing efficiency by western blotting. Then, the silenced cells and control siRNA-transfected cells were analyzed for changes in epithelial and mesenchymal markers, invasion, and response to 5-fluoruracil treatment. We also performed proteomics analysis using a phospho-kinase array-based panel of 45 proteins.RESULTSCNN3 showed positive expression in 6/8 breast and 9/11 colon cancer lines and in HeLa cells. Interestingly, the colorectal adenocarcinoma line SW480 was negative, while the cell line developed from its matching lymph node metastasis (SW620) was positive for CNN3. CNN3 expression was fairly consistent with the metastatic phenotype in colon cancer because it was absent in one other colon cell line from a primary site and expressed in all others. We selected SW620 for subsequent functional analyses. CNN3-silenced SW620 cells showed a reduction in collagen invasion and loss of mesenchymal markers. CNN3 silencing caused an increase in the SW620 colon cancer cell sensitivity to 5-fluorouracil. Phospho-kinase array-based proteomics analysis showed that CNN3 silencing in SW620 reduced extracellular signal-regulated kinase, β-Catenin, mutant p53, c-Jun, and heat shock protein 60 activities but increased that of checkpoint kinase 2. CNN3 was expressed in 20/57 (35%) colon cancer cases as shown by immunohistochemistry. CNN3 was associated with a decrease in overall survival in colon cancer in silico.CONCLUSIONThese results show the involvement of CNN3 in lymph node metastasis and resistance to chemotherapy in colon cancer and suggest that significant oncogenic pathways are involved in these CNN3-related actions.

Telomere Length Maintenance and Its Transcriptional Regulation in Lynch Syndrome and Sporadic Colorectal Carcinoma.

Background: Activation of telomere maintenance mechanisms (TMMs) is a hallmark of most cancers, and is required to prevent genome instability and to establish cellular immortality through reconstitution of capping of chromosome ends. TMM depends on the cancer type. Comparative studies linking tumor biology and TMM have potential impact for evaluating cancer onset and development.Methods: We have studied alterations of telomere length, their sequence composition and transcriptional regulation in mismatch repair deficient colorectal cancers arising in Lynch syndrome (LS-CRC) and microsatellite instable (MSI) sporadic CRC (MSI s-CRC), and for comparison, in microsatellite stable (MSS) s-CRC and in benign colon mucosa. Our study applied bioinformatics analysis of whole genome DNA and RNA sequencing data and a pathway model to study telomere length alterations and the potential effect of the “classical” telomerase (TEL-) and alternative (ALT-) TMM using transcriptomic signatures.Results: We have found progressive decrease of mean telomere length in all cancer subtypes compared with reference systems. Our results support the view that telomere attrition is an early event in tumorigenesis. TMM gets activated in all tumors studied due to concerted overexpression of a large fraction of genes with direct relation to telomere function, where only a very small fraction of them showed recurrent mutations. TEL-related transcriptional state was dominating in all CRC subtypes, showing, however, subtype-specific activation patterns; while contribution of the ALT-TMM was slightly more prominent in the hypermutated MSI s-CRC and LS-CRC. TEL-TMM is mainly activated by over-expression of DKC1 and/or TERT genes and their interaction partners, where DKC1 is more prominent in MSS than in MSI s-CRC and can serve as a transcriptomic marker of TMM activity.Conclusions: Our results suggest that transcriptional patterns are indicative for TMM pathway activation with subtle differences between TEL and ALT mechanisms in a CRC subtype-specific fashion. Sequencing data potentially provide a suited measure to study alterations of telomere length and of underlying transcriptional regulation. Further studies are needed to improve this method.

1527 Ulcerative Colitis With Concomitant Serrated Polyposis Syndrome

INTRODUCTION: Ulcerative colitis (UC) is associated with increased risk of colon cancer. Current knowledge of UC with concomitant serrated polyposis syndrome (SPS) is limited and clinical guidelines regarding management is lacking. Sessile serrated adenomas and traditional serrated adenomas are precursors of at least 20% of sporadic colorectal carcinomas. Patients with SPS have higher risk of developing dysplastic lesions. We describe an interesting case of concurrent UC with SPS and a brief review of literature surrounding this association. CASE DESCRIPTION/METHODS: A 45-year old female with no significant past medical history presented to clinic for a 2-month history of change in bowel habits with bloating and occasional hematochezia. She denied a family history of colon cancer or inflammatory bowel diseases (IBD). Colonoscopy revealed three flat polyps measuring 2 mm, 7 mm and 8 mm in size at the cecal base, and two flat polyps each 11 mm in size in the ascending colon. All these polyps were removed and found to be of sessile serrated type on histopathology thereby meeting the WHO criteria for SPS. Patient was also found to have a 20 cm segment of inflammation in the rectum, biopsies showed diffuse chronic active proctitis with severe activity, meeting the criteria for ulcerative proctitis. Patient was started on mesalamine 1000 mg rectal suppository daily with plan for a repeat colonoscopy in 6 months to assess response. Laboratory analysis at time of colonoscopy showed C reactive protein &lt;0.40 mg/dL, WBC count 6.12 × 10(9)/L and Hgb 12.5 g/dL. DISCUSSION: Frequency of occurrence of SPS among IBD patients seems to parallel that of general population. But the risk of colon cancer in this setting is unclear since IBD itself can give rise to colonic dysplasia. It has been suggested that a serrated pathway of carcinogenesis in inflammatory bowel disease is characterized by silencing of MGMT by gene promoter methylation, KRAS mutations, and potentially molecular alterations that are yet to be characterized and suspected to eventually lead to adenocarcinoma. Further studies are needed to define optimal management of these patients. This case highlights the complexity of managing this concurrent syndrome in patients with IBD.