Subset Of Colorectal Carcinomas Research Articles

The combination of CDX2 expression status and tumor-infiltrating lymphocyte density as a prognostic factor in adjuvant FOLFOX-treated patients with stage III colorectal cancers.

Colorectal carcinomas (CRCs) with caudal-type homeobox 2 (CDX2) loss are recognized to pursue an aggressive behavior but tend to be accompanied by a high density of tumor-infiltrating lymphocytes (TILs). However, little is known about whether there is an interplay between CDX2 loss and TIL density in the survival of patients with CRC. Stage III CRC tissues were assessed for CDX2 loss using immunohistochemistry and analyzed for their densities of CD8 TILs in both intraepithelial (iTILs) and stromal areas using a machine learning-based analytic method. CDX2 loss was significantly associated with a higher density of CD8 TILs in both intraepithelial and stromal areas. Both CDX2 loss and a high CD8 iTIL density were found to be prognostic parameters and showed hazard ratios of 2.314 (1.050-5.100) and 0.378 (0.175-0.817), respectively, for cancer-specific survival. A subset of CRCs with retained CDX2 expression and a high density of CD8 iTILs showed the best clinical outcome (hazard ratio of 0.138 [0.023-0.826]), whereas a subset with CDX2 loss and a high density of CD8 iTILs exhibited the worst clinical outcome (15.781 [3.939-63.230]). Altogether, a high density of CD8 iTILs did not make a difference in the survival of patients with CRC with CDX2 loss. The combination of CDX2 expression and intraepithelial CD8 TIL density was an independent prognostic marker in adjuvant chemotherapy-treated patients with stage III CRC.

TROP2 in colorectal carcinoma: associations with histopathology, molecular phenotype, and patient prognosis.

Antibody-drug conjugates (ADCs) directed to trophoblast cell surface antigen 2 (TROP2) have gained approval as a therapeutic option for advanced triple-negative breast cancer, and TROP2 expression has been linked to unfavourable outcomes in various malignancies. In colorectal carcinoma (CRC), there is still a lack of comprehensive studies on its expression frequency and its prognostic implications in relation to the main clinicopathological parameters. We examined the expression of TROP2 in a large cohort of 1,052 CRC cases and correlated our findings with histopathological and molecular parameters, tumour stage, and patient outcomes. TROP2 was heterogeneously expressed in 214/1,052 CRCs (20.3%), with only a fraction of strongly positive tumours. TROP2 expression significantly correlated with an invasive histological phenotype (e.g. increased tumour budding/aggressive histopathological subtypes), advanced tumour stage, microsatellite stable tumours, and p53 alterations. While TROP2 expression was prognostic in univariable analyses of the overall cohort (e.g. for disease-free survival, p < 0.001), it exhibited distinct variations among important clinicopathological subgroups (e.g. right- versus left-sided CRC, microsatellite stable versus unstable CRC, Union for International Cancer Control [UICC] stages) and lost its significance in multivariable analyses that included stage and CRC histopathology. In summary, TROP2 is quite frequently expressed in CRC and associated with an aggressive histopathological phenotype and microsatellite stable tumours. Future clinical trials investigating anti-TROP2 ADCs should acknowledge the observed intratumoural heterogeneity, given that only a subset of TROP2-expressing CRC show strong positivity. The prognostic implications of TROP2 are complex and show substantial variations across crucial clinicopathological subgroups, thus indicating that TROP2 is a suboptimal parameter to predict patient prognosis.

Cytokeratin 7 expression as a predictor of an unfavorable prognosis in colorectal carcinoma

Colorectal carcinoma (CRC) is associated with significant morbidity and mortality worldwide. Cytokeratins (CKs) are widely expressed in various types of carcinomas, whereas in CRC it is usually CK7 − and CK20 + . A subset of CRCs is CK7 + . This study aims to determine the prevalence of CK7 expression in CRC and its impact on overall survival. We analyzed 300 randomly selected surgically treated CRC cases using paraffin embedded tumor tissue samples and evaluated CK7 and CK20 expression using the tissue microarray method. Tumors with positivity > 10% and > 25% of tumor cells were considered CK7 and CK20 positive, respectively. Expression of both CKs and several clinical-pathological variables (stage, grade, laterality, mismatch-repair/MMR status) were evaluated using patient follow up data (Kaplan–Meier analysis of cancer-specific survival (CSS)). Significant results include shorter CSS (restricted mean 4.98 vs. 7.74 years, P = 0.007) and 5-year survival (29.4% vs. 64.6%, P = 0.0221) in CK7 + tumors compared to CK7 − tumors, respectively; without significant association with grade, stage or right-sided location. These results were significant in a multivariate analysis. CK20 + tumors are more frequently MMR-proficient and left-sided. MMR-deficient tumors are more frequently right-sided and had longer survival. CK7 expression, right-sided location (rmean CSS 6.83 vs. 8.0 years, P = 0.043), MMR-proficiency (rmean CSS 7.41 vs. 9.32 years, P = 0.012), and UICC stages III + IV (rmean CSS 6.03 vs. 8.92 years, P < 0.001) of the tumor correlated with negative prognostic outcomes, whereas the most significant results concern stage and CK7 positivity. The result concerning negative prognostic role of CK7 differs from those obtained by several previous studies focused on this topic.

Loss of expression of Syndecan-1 is associated with Tumor Recurrence, Metastatic Potential, and Poor Survival in patients with Colorectal carcinoma.

Objective:The loss of expression of syndecansyndecan-1 is associated with poor prognosis in many types of human cancer. The objective of this study was to evaluate the relation between syndecan-1 immunoexpression and several clinicopathological parameters in a subset of colorectal carcinoma (CRC) patients.Methods:Pathology tissue blocks of 202 primary tumors, 41 adenomas, and 37 normal colonic mucosae were used in this study. The cases diagnosed in the period 1995–2015 was included in the study. Immunohistochemistry analysis was performed using anti-CD138/syndecan-1 (B-A38) mouse monoclonal antibody. A semiquantitative method was used to score the syndecan-1 expression based on an evaluation of the percentage and intensity of the membranous and cytoplasmic expression. The data collected from Pathology Department at King Abdulaziz University Hospital, Jeddah, Saudi Arabia. This is a retrospective cohort study that was conducted from July 2018 until August 2019.Results:Loss of syndecan-1 immunoexpression was observed in 72 (42.6%), 5 (12.2%), and 3 (8.1%) cases of CRC, adenomas, and normal mucosae, respectively. Low expression of syndecan-1 showed an association with nodal (p=0.003) and distant (p=0.001) metastasis, lymphovascular invasion (p=0.001), and tumor recurrence (p=0.006). Low syndecan-1 expression were associated with short overall survival (OS) (log rank 4.019, p=0.045) and disease-free survival (DFS) probabilities (log rank 4.748, p=0.029).Conclusion:Loss of syndecan-1 immunoexpression is associated with metastatic potential, tumor recurrence and shorter survival in CRC and is considered a potential biomarker of poor prognosis in CRC patients.

Low expression of MUC2 is associated with longer disease-free survival in patients with colorectal carcinoma.

Background/Aim:The objective of this study was to investigate the relationship between MUC2 immunostaining and clinicopathological characteristics in a subset of colorectal carcinomas (CRCs).Materials and Methods:A total of 128 CRCs, 50 local nodal metastases, and 42 normal colonic mucosae were retrieved from the archives at the Department of Pathology at King Abdulaziz University, Jeddah, Saudi Arabia. Immunohistochemistry was performed using anti-MUC2 antibody. A cut-off of 25% of positive immunostaining was used to define low and high immunostaining. Statistical tests were used to determine the association of MUC2 with clinicopathological characteristics and survival.Results:MUC2 immunostaining was observed in 66.7% in normal colonic mucosa. Low MUC2 immunostaining was higher in primary CRC (P = 0.003) and nodal metastasis (80%) (P < 0.001). There was significant association of low MUC2 immunostaining in CRC with age group below 60 years (P = 0.05) and occurrence of lymphovascular invasion (P = 0.034). Other clinicopathological parameters were not correlated with MUC2 immunostaining. Regression analysis revealed that low MUC2 immunostaining was an independent predictor of lymphovascular invasion (P = 0.041). In the Kaplan–Meier survival analysis, there was a significant longer disease-free survival in patients with low MUC2 immunostaining (P = 0.045). However, there was no association between MUC2 immunostaining and overall survival (P = 0.601).Conclusion:MUC2 immunostaining may have distinct clinical significance and provide valuable information and could be considered as an important independent prognostic factor while planning the adjuvant therapy in CRC. In future perspective, characterization of MUC2 immunostaining on a large number of cases and molecular studies may be needed.

Abstract B011: Immunoediting in untreated mismatch repair deficient colorectal cancer

Abstract A subset of colorectal carcinomas (CRC) with high microsatellite instability (MSI-high) have been shown to respond to immune checkpoint blockade, while the much more common microsatellite stable (MSS) CRC do not usually respond to these immunotherapies. Most MSI-high tumors grow progressively despite a high mutational load, suggesting that the cancer may have developed means to escape from immune recognition, which is termed immunoediting. This immunoediting process includes three phases: an initial phase of nascent cancer cell elimination due to high immunogenicity, then a period of equilibrium with the immune response, finally leading to escape of the cancer cells that have developed genetic or epigenetic mechanisms allowing them to grow progressively in an immunocompetent host. It is likely that MSI-high cancers would go through similar processes to eventually escape from immune recognition. To better understand the differential immunoediting in MSI-high and MSS CRC, we combined tumor exome data from 592 cases from The Cancer Genome Atlas (TCGA) with previously published tumor exome data from 619 cases from the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study to yield an unprecedented sample of 1,211 CRC molecularly characterized cases. The large number of samples, including a very large number of MSI-high cases (N = 179), gives us the power to identify the prevalence of the mechanisms of resistance discussed and thereby prioritize downstream efforts. We utilized the MutSigCV algorithm to identify significantly mutated genes in MSS and MSI-high tumors in the TCGA cohort (496 MSS and 75 MSI-high cases), and the NHS/HPFS cohorts (438 MSS and 104 MSI-high cases). We identified a total of 62 significantly mutated genes, of which 9 were identified in MSS tumors only, 40 in MSI-high tumors only, and 13 in both. Twenty-seven of the 53 significantly mutated genes in MSI-high were novel, likely resulting from a large increase in the number of MSI-high tumors relative to previous studies. We identified 13 significantly mutated genes with a documented role in the immune system, specifically in MSI-high tumors, a subtype shown previously to have a high level of T-cell infiltration; ZFP36L2 and B2M were significantly mutated in both MSS and MSI-high. B2M, HLA-A, and HLA-B are significantly mutated antigen presenting genes that were previously reported to be recurrently mutated in MSI-high. Eleven of the significantly mutated immune-related genes had roles in modulating diverse hematopoietic cell types and effects beyond antigen presentation; those genes include XYLT2, a dendritic cell trafficking gene; ZBTB20, a Toll-like receptor mediating immune response; RNF128, a regulator of IL2 and IL4 mediated T-cell maturation; KLF3, a gene involved in B-cell development; ZFP36L2, a gene involved in thymocyte development; CASP8, a gene involved in innate immunity; and CD58, a gene involved in the activation of both T lymphocytes and NK cells. Together, these mutations indicate positive selection for immune escape through other mechanisms beyond mutations in antigen-presenting machinery. Citation Format: Catherine Grasso, Marios Giannakis, Daniel Wells, David Wheeler, Eve Shinbrot, Syed Zaidi, Jeroen Huyghe, Milan Geybels, Stephen Salipante, Gabriel Abril-Rodriguez, Helena Escuin-Ordinas, Cristina Puig-Saus, Daniel Sanghoon Shin, Shuji Ogino, Antoni Ribas, Ulrike Peters. Immunoediting in untreated mismatch repair deficient colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B011.

K-ras Mutations as the Earliest Driving Force in a Subset of Colorectal Carcinomas.

K-ras oncogene is a key factor in colorectal cancer. Based on published and our data we propose that K-ras could be the oncogene responsible for the inactivation of the tumor-suppressor gene APC, currently considered as the initial step in colorectal tumorigenesis. K-ras fulfills the criteria of the oncogene-induced DNA damage model, as it can provoke well-established causes for inactivating tumor-suppressors, i.e. DNA double-strand breaks (causing allele deletion) and ROS production (responsible for point mutation). The model we propose is a variation of the currently existing model and hypothesizes that, in a subgroup of colorectal carcinomas, K-ras mutation may precede APC inactivation, representing the earliest driving force and, probably, an early biomarker of colorectal carcinogenesis. This observation is clinically useful, since it may modify the preventive colorectal cancer strategy, restricting numerically patients undergoing colonoscopies to those bearing K-ras mutation in their colorectum, either in benign polyps or the normal accompanying mucosa.

KEYNOTE-177: Randomized phase III study of pembrolizumab versus investigator-choice chemotherapy for mismatch repair-deficient or microsatellite instability-high metastatic colorectal carcinoma.

TPS815 Background: A subset of colorectal carcinomas (CRCs) are characterized by mismatch repair (MMR) deficiency resulting in microsatellite instability (MSI). MSI-high tumors contain high levels of lymphocyte infiltrates and express the PD-1 immune checkpoint receptor and its ligand, PD-L1. In the phase 2 KEYNOTE-016 study, the anti–PD-1 antibody pembrolizumab demonstrated antitumor activity against MMR-deficient tumors in patients with treatment-refractory metastatic CRC. KEYNOTE-177 (ClinicalTrials.gov, NCT02563002) is an international, randomized, open-label, phase 3 study of pembrolizumab versus standard-of-care (SOC) chemotherapy in first-line MMR-deficient or MSI-high metastatic CRC. Methods: Key eligibility criteria include age ≥18 years, confirmed MSI-high or MMR-deficient metastatic CRC, ECOG PS 0-1, no active autoimmune disease or brain metastases, and no prior therapy for metastatic disease. ~270 patients will be randomly assigned 1:1 to receive either pembrolizumab 200 mg every 3 weeks or investigator's choice of SOC chemotherapy (mFOLFOX6 or FOLFIRI alone or in combination with bevacizumab or cetuximab, chosen before randomization). Treatment is to continue until progressive disease (PD), unacceptable toxicity, patient/investigator decision, or completion of 35 cycles (pembrolizumab only). Response is to be assessed every 9 weeks per RECIST v1.1 by central imaging vendor review and per RECIST adapted for immunotherapy response patterns. Eligible patients may continue pembrolizumab beyond initial RECIST-defined progression. Patients in the SOC arm who have PD and meet crossover criteria may be eligible to receive pembrolizumab for up to 17 treatment cycles. AEs are to be assessed throughout treatment and for 30 days thereafter (90 days for serious AEs) and graded per NCI CTCAE v4.0. Patients are to be followed up for survival every 9 weeks. The primary end point is PFS per RECIST v1.1; key secondary end points include OS and ORR. Enrollment in KEYNOTE-177 is ongoing. Clinical trial information: NCT02563002.

WISP3 is highly expressed in a subset of colorectal carcinomas with a better prognosis.

Outlier genes with marked overexpression in subsets of cancers like ERBB2 have potential for the identification of gene classifiers and therapeutic targets for the appropriate subpopulation. In this study, using the cancer outlier profile analysis strategy, we identified WNT1-inducible-signaling pathway protein 3 (WISP3) as an outlier gene that is highly expressed in a subset of colorectal cancers (CRCs) from The Cancer Genome Atlas dataset. A meta-cancer outlier profile analysis and immunohistochemistry experiment to validate the outlier expression model of WISP3 in CRC was then performed. Our immunohistochemical results indicated that WISP3 was more frequently seen in the small tumors, and there was a significant association between its overexpression with a good prognosis. Furthermore, in the multivariable model, WISP3 outlier expression retained significance for overall survival. In summary, in this study, we identified an outlier gene WISP3 overexpressed in a subset of CRC having less aggressive characteristics and a better prognosis. We suggest WISP3 may provide more accurate and precise information regarding CRC population classification.

Adenoma-like adenocarcinoma: a subtype of colorectal carcinoma with good prognosis, deceptive appearance on biopsy and frequent KRAS mutation.

A subset of colorectal carcinomas (CRCs) architecturally and cytologically resembles adenomatous change, making them difficult to diagnose on biopsy. This subset has not been well characterized to date. For 35 carcinomas with adenomatous-like areas (cytological and surface architectural appearance that would be insufficient to warrant a diagnosis of adenocarcinoma if evaluated on biopsy), we recorded staging information, molecular data, clinical outcome, whether precursor adenoma was present and whether previous biopsy had been diagnosed as malignant. Despite advanced T-category in 23 (66%) tumours, only seven (20%) had nodal metastases, and only five patients (15%) developed distant metastases. Fifteen cases (43%) had been diagnosed as adenoma on biopsy. Twenty-one resections (60%) showed no residual associated adenoma, including nine called adenoma on biopsy. Median follow-up was 44 months. Four patients (12%) died of disease; 22 were alive at last follow-up. KRAS mutation was seen in 14 of 24 (58%) and four of 17 (24%) were microsatellite-unstable. Patients had significantly improved survival compared to a cohort of patients with conventional well-differentiated CRC after controlling for age and stage (P = 0.011). Adenoma-like adenocarcinoma is an uncommon variant of CRC with a low rate of metastasis and good prognosis. Biopsy diagnosis of this lesion may be challenging.

Immunohistochemical detection of ARID1A in colorectal carcinoma: loss of staining is associated with sporadic microsatellite unstable tumors with medullary histology and high TNM stage.

AT-rich interactive domain-containing protein 1A (ARID1A), a chromatin remodeling gene recently discovered to be a tumor suppressor in ovarian cancers, has been found to be mutated at low frequencies in many other tumors including colorectal carcinoma (CRC). An association between ARID1A alteration and DNA mismatch repair (MMR) deficiency has been implicated; understanding this association may facilitate the understanding of the role of ARID1A in the various tumors. In this pilot study, we analyzed the immunohistochemical expression of ARID1A in a consecutive series of 257 CRCs that fulfilled a set of relaxed criteria for Lynch syndrome screening; 59 (23%) were MMR deficient by immunohistochemistry (44 MLH1/PMS2 deficient, 9 MSH2/MSH6 deficient, 4 MSH6 deficient, and 2 PMS2 deficient). ARID1A loss was observed in 9% (22/257) of the cohort: 24% of MMR-deficient tumors (14/59, 13 of the 14 being MLH1/PMS2 deficient) and 4% of MMR-normal tumors (8/198) (P < .05). MLH1 (mutL homolog 1) promoter hypermethylation was observed in 10 of the 13 MLH1/PMS2-deficient/ARID1A-loss tumors, indicating an association between ARID1A loss and sporadic microsatellite unstable CRCs. Among the MMR-deficient cases, ARID1A loss correlated with old age (P = .04), poor tumor differentiation (P < .01), medullary histology (P < .01), and an increased rate of nodal and distant metastasis (P = .03); these patients also trended toward a worse 5-year overall survival. Among MMR-normal tumors, no differences in clinicopathological features were detected between the groups stratified by ARID1A. In conclusion, our results suggest that ARID1A loss may be linked to a specific subset of sporadic microsatellite unstable CRCs that may be medullary but is more likely to present with metastatic disease, warranting further investigation.

Clinical features and outcome of sporadic colorectal carcinoma in young patients: a cross-sectional analysis from a developing country.

Background. Early onset colorectal carcinoma (CRC) is rare and has been hypothesized to be a biologically and clinically distinct entity personifying aggressive disease and worse survival. Methods. Data for 131 patients was collected by retrospective chart review. Cox proportional hazard model was used to compute prevalence ratios and 95% confidence intervals. Results. Early onset sporadic CRC accounted for 32% of all CRC treated in the specified time period. The mean age was 33.3 ± 7.9 years and the male to female ratio was 2 : 1. Colon and rectal cancers accounted for 55% and 45% of patients, respectively. 96% of rectal carcinoma patients received appropriate therapy as opposed to 65% of colon cancers. On multivariable analysis, appropriate reception of therapy (PR 4.99; 95% CI, 1.21–20.6) and signet ring morphology (PR 2.40; 95% CI, 1.33–4.32) were significantly associated with rectal cancers as opposed to colon cancer. Kaplan-Meier analysis revealed a trend towards inferior survival for rectal carcinoma 2 years after diagnosis. Conclusion.A high prevalence of early onset CRC was noted in the study. A trend towards inferior survival was seen in patients with rectal cancer. This finding raises the possibility of rectal carcinoma being an aggressive subset of young CRC.

Loss of Villin Immunoexpression in Colorectal Carcinoma Is Associated with Poor Differentiation and Survival

Background and Aims. Villin is a highly specialised protein and is expressed in intestinal and renal proximal tubular epithelium. It was detected in colorectal carcinomas (CRC) and other nongastrointestinal tumours. The aim of the current study is to investigate the immunohistochemical expression of villin in a subset of primary CRC and determine its relation to tumour differentiation, invasion, nodal metastasis, recurrence, and disease-free survival. Patients and Methods. Paraffin blocks of 93 cases of CRC were retrieved constituting 93 primary CRC and 58 adjacent normal mucosa. Immunohistochemistry was performed using antivillin antibody. The extent (%) of villin immunoexpression was categorised for statistical analysis. Statistical tests were used to determine the association of villin with clinicopathological characteristics: age, sex, tumour location, tumour size, depth of invasion, tumour grade, nodal metastasis, lymphovascular invasion, margin status, recurrence, and survival. Results. Villin immunostaining results showed that villin is downregulated in CRC. Villin has no association with age, sex, tumour location, depth of invasion, nodal metastasis, lymphovascular invasion, margin status, and recurrence. However, villin is expressed in higher rate in CRC less than 5 cm, well- and moderately differentiated CRC. Poor survival was associated with tumour with low villin immunoexpression. Conclusion. Villin was downregulated in CRC. Villin immunoexpression in CRC is associated with better survival, well-differentiated tumours, and small-sized tumours. Villin has no significant association with disease recurrence or nodal metastasis. More in vivo and in vitro studies are required for further elucidation of how villin may be involved in CRC.

Diagnostic Significance of Serum HMGB1 in Colorectal Carcinomas

High mobility group box 1 protein (HMGB1), a nuclear protein, can be translocated to the cytoplasm and secreted in colon cancer cells. However, the diagnostic significance of HMGB1 has not been evaluated in colorectal carcinomas. For this purpose, we have screened the expression and secretion of HMGB1 in 10 colon cancer cell lines and 1 control cell line and found that HMGB1 was detected in the culture medium. To evaluate the diagnostic value of HMGB1, we performed an enzyme-linked immunosorbent assay to measure HMGB1 levels and compared them to carcinoembryonic antigen (CEA) levels in the serum samples of 219 colorectal carcinoma patients and 75 healthy control subjects. We found that the serum HMGB1 level was increased by 1.5-fold in patients with colorectal carcinoma compared to those in healthy controls. When HMGB1 and CEA levels were compared, HMGB1 had similar efficacy as CEA regarding cancer detection (the sensitivity was 20.1% for HMGB1 vs. 25.6% for CEA, and the specificity was 96% for HMGB1 vs. 90.7% for CEA). Moreover, the diagnostic accuracy of HMGB1 for stage I cancer was significantly higher than that of CEA (sensitivity: 41.2% vs. 5.9%; specificity: 96% vs. 90.7). When we combined HMGB1 and CEA, the overall diagnostic sensitivity was higher than that of CEA alone (42% vs. 25.6%), and the diagnostic sensitivity for stage I was also elevated (47% vs. 5.9%). However, the prognosis of patients was not related with serum HMGB1 concentrations. Our findings indicate that serum HMGB1 levels are increased in a subset of colorectal carcinomas, suggesting their potential utility as a supportive diagnostic marker for colorectal carcinomas.

Relevance, Pathogenesis, and Testing Algorithm for Mismatch Repair–Defective Colorectal Carcinomas: A Report of the Association for Molecular Pathology

Loss-of-function defects in DNA mismatch repair (MMR), which manifest as high levels of microsatellite instability (MSI), occur in approximately 15% of all colorectal carcinomas (CRCs). This molecular subset of CRC characterizes patients with better stage-specific prognoses who experience no benefit from 5-fluorouracil chemotherapy. Most MMR-deficient (dMMR) CRCs are sporadic, but 15% to 20% are due to inherited predisposition (Lynch syndrome). High penetrance of CRCs in germline MMR gene mutation carriers emphasizes the importance of accurate diagnosis of Lynch syndrome carriers. Family-based (Amsterdam), patient/family-based (Bethesda), morphology-based, microsatellite-based, and IHC-based screening criteria do not individually detect all germline mutation carriers. These limitations support the use of multiple concurrent tests and the screening of all patients with newly diagnosed CRC. This approach is resource intensive but would increase detection of inherited and de novo germline mutations to guide family screening. Although CRC prognosis and prediction of 5-fluorouracil response are similar in both the Lynch and sporadic dMMR subgroups, these subgroups differ significantly with regard to the implications for family members. We recommend that new CRCs should be classified into sporadic MMR-proficient, sporadic dMMR, or Lynch dMMR subgroups. The concurrent use of MSI testing, MMR protein IHC, and BRAF c.1799T>A mutation analysis would detect almost all dMMR CRCs, would classify 94% of all new CRCs into these MMR subgroups, and would guide secondary molecular testing of the remainder.

Differential roles of EPS8 in carcinogenesis: Loss of protein expression in a subset of colorectal carcinoma and adenoma

To analyze the epidermal growth factor receptor pathway substrate 8 (EPS8) expression status and role in colorectal carcinogenesis given that EPS8 has a conserved actin barbed-end capping function that is required for proper maturation in intestinal cells. We studied 8 colon cancer cell lines and 58 colorectal tumors (19 adenomas and 39 carcinomas). We performed expression microarray analysis of colon cancer cell lines followed by loss of heterozygosity (LOH) analysis and immunohistochemistry for EPS8 expression in colon tumors. Subsequently, we performed mutation analysis by direct sequencing and methylation analysis by bisulfite sequencing and methylation-specific polymerase chain reaction assays. Expression microarray analysis of colon cancer cell lines showed overexpression of EPS8 transcript in all lines but RKO. Genome wide loss of heterozygosity (LOH) analysis of colon tumors, showed considerable LOH at the EPS8 gene locus. Immunohistochemically, EPS8 was constitutively expressed in normal colonic mucosa with a dot-like supranuclear localization with accentuation at the luminal surface supporting its proposed role in epithelial maturation. Nineteen colon tumors (4 adenoma, 15 carcinoma) out of 51 (37%) showed strikingly tumor specific EPS8 protein loss. Of the remaining tumors, 5/51 (2 adenoma, and 3 carcinoma, 10%) showed marked overexpression, while 27/51 tumors (53%) showed retained expression. Mutation analysis revealed a missense mutation (c.794C>T, p.R265C) in exon 8 in RKO. The EPS8 promoter was also methylated in RKO, but there was no significant methylation in other cell lines or carcinoma specimens. The loss of EPS8 expression in colorectal adenomas and carcinomas suggests that down regulation of this gene contributes to the development of a subset of colorectal cancers, a finding which could have applications in diagnosis and treatment.

Abstract 2147: Co-expression of cyclooxygenase-2 and FoxM1 is an independent prognostic marker for poor outcome in Stage II and III colorectal carcinoma

Abstract Cox-2 and FoxM1 are extensively studied and both have been implicated in colorectal carcinogenesis. Therefore, the FoxM1 may represent a novel therapeutic target, and thus the development of agents that will target FoxM1 is likely to have significant therapeutic impact on human cancer. We studied COX2 and FoxM1 expression in a tissue microarray cohort of more than 400 colorectal carcinomas, Cox-2 and FoxM1 signaling in in vitro in CRC cell lines. Incidence of Cox-2 and FoxM1 expression in all CRC cases was 66.2 %(245/370) &amp; 53.8%(211/392) respectively. Cox-2 expression in all CRC's(Stage I – IV) was associated with an aggressive phenotype characterized by poorly differentiated tumors, lymph node metastasis, histology type of mucinous carcinoma and proliferative marker Ki-67. Interestingly Cox-2 expression was associated with expression of FoxM1 (p=0.0226). Considering the fact that both Cox-2 and Foxm1 have been implicated in colorectal carcinogenesis and the potential therapeutic utility of both these markers we grouped our cases based on expression on Cox-2 and FoxM1 in Stage II and Stage III CRC's only. Co-expression of both Cox-2 and FoxM1 was seen in 42.4% (104/245) of CRC's and was again associated with an aggressive phenotype characterized by by poorly differentiated tumors, lymph node metastasis, histology type of mucinous carcinoma, proliferative marker Ki-67, overexpression of MMP9(p=0.0061) and KRAS mutations(p=0.0011). Kaplan Meier survival analysis in the Stage II-III subgroup showed that both over-expression of Cox-2(p=0385) and co-expression of Cox-2 and FoxM1 (p=0.0228) was associated with poor overall survival. In the multivariate analysis using Cox Proportional Hazard model for multiple factors like age, gender, tumor differentiation and co-expression of Cox-2 and FoxM1 the relative risk was 1.77 for co- expression of Cox-2 and FoxM1 (95% CI 1.05-3.03); p = 0.0317) and 1.58 for tumor differentiation. (95% CI 0.85-2.80; p =0.1417). Thus, co-expression of Cox-2 and FoxM1 was an independent prognostic marker for poor overall survival in the Stage II-III subset of colorectal carcinoma. Our in vitro studies showed that co-inhibition of FoxM1 and Cox-2 with pharmacological inhibitors resulted a synergistic effect on apoptosis in CRC cell lines. Cox-2 siRNA and NS398 treatment had no effect on the expression of FoxM1. Similarly treatment of Xenografts tumors in nude mice had no effect on FoxM1 expression suggesting that FoxM1 is functional upstream of Cox-2. These findings demonstrates that Co-expression of Cox-2 and FoxM1 play critical role in the pathogenesis of CRC and a novel therapeutic target for the treatment of stage II and III CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2147. doi:10.1158/1538-7445.AM2011-2147

Genome-Wide Expression Analysis of Middle Eastern Colorectal Cancer Reveals FOXM1 as a Novel Target for Cancer Therapy

To identify genes potentially playing an important role in the progression of colorectal carcinoma (CRC), we screened global gene expression using cDNA expression array on 41 CRC tissue samples and 25 noncancerous colorectal tissue samples. Among the up-regulated genes, forkhead box M1 (FOXM1) has been shown to play a critical role in pathogenesis of various malignancies. Using immunohistochemistry on 448 Saudi CRC samples in tissue microarray format, FoxM1 protein overexpression was seen in 66% of CRC tissues and was significantly associated with poorly differentiated and highly proliferative tumors (P = 0.0200 and 0.0018, respectively). FoxM1 expression was also significantly associated with MMP-9 protein expression (P = 0.0002). In vitro data using CRC cell lines showed that inhibition of FoxM1 by thiostrepton resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner. Overexpression of FoxM1 potentiated cell proliferation, cell transformation, and migration/invasion of CRC cells via up-regulation of FoxM1 target genes MMP2 and MMP9 and protected these cells from thiostrepton-mediated antiproliferative effects. Finally, in vivo, overexpression of FoxM1 promoted growth of CRC-cell line xenograft tumors in nude mice. Altogether, our data indicate that FoxM1 signaling contributes to aggressiveness in a subset of CRC and that the FOXM1 gene may serve as a useful molecular biomarker and potential therapeutic target.

Clinicopathologic study of 85 colorectal serrated adenocarcinomas: further insights into the full recognition of a new subset of colorectal carcinoma

Colorectal serrated adenocarcinoma represents a subtype of colorectal carcinoma that originates from serrated adenomas. Previous studies have suggested a more aggressive course, but this has not been verified. The aim of this work was to test the diagnostic reproducibility of previously proposed histologic criteria for serrated adenocarcinoma and to analyze the clinicopathologic features and outcome that would warrant its recognition as a new subtype of colorectal cancer. Nine hundred twenty-seven consecutive colorectal cancer specimens were used to search for cases fulfilling the criteria of serrated adenocarcinoma and matched controls. Clinicopathologic findings of 85 serrated adenocarcinomas were compared with a matched control group of conventional cancers. Serrated adenocarcinomas were encountered in 9.1% (n = 85) of cases. Residual serrated adenoma was present in 44 (51.7%). Absence of residual adenoma did not have any influence on the parameters studied. Interobserver variation between 2 Spanish and a Finnish pathologist showed moderate agreement (κ = .5873). Compared with their matched controls, serrated adenocarcinomas were more often accompanied by synchronous residual serrated adenomas (P < .0001), remote serrated adenomas (P = .0035), and serrated adenocarcinomas or cancers representing partial features of these tumors (P = .002). They had a less favorable 5-year survival than conventional cancers (P = .048 Breslow, Kaplan-Meier), and left-sided ones had the worst prognosis (P = .001). Serrated adenocarcinoma is an identifiable subset of colorectal cancer; and the histopathologic differences, in addition to its less favorable prognosis, may justify its recognition as a distinct subset of colorectal cancer warranting the search for specific clinical management strategies.

Class I Histone Deacetylase Expression Has Independent Prognostic Impact in Human Colorectal Cancer: Specific Role of Class I Histone Deacetylases In vitro and In vivo

Recently, several studies reported a strong functional link between histone deacetylases (HDAC) and the development of tumors of the large intestine. However, despite the importance of these molecules, comparably little is known on expression patterns and functions of specific HDAC isoforms in colorectal cancer. We characterized class I HDAC isoform expression patterns in a cohort of 140 colorectal carcinomas by immunohistochemistry. In addition, effects of HDAC inhibition by valproic acid and suberoylanilide hydroxamic acid, and specific HDAC isoform knockdown by short interfering RNA, were investigated in a cell culture model. We found class I HDACs highly expressed in a subset of colorectal carcinomas with positivity for HDAC1 in 36.4%, HDAC2 in 57.9%, and HDAC3 in 72.9% of cases. Expression was significantly enhanced in strongly proliferating (P = 0.002), dedifferentiated (P = 0.022) tumors. High HDAC expression levels implicated significantly reduced patient survival (P = 0.001), with HDAC2 expression being an independent survival prognosticator (hazard ratio, 2.6; P = 0.03). Short interfering RNA-based inhibition of HDAC1 and HDAC2 but not HDAC3 suppressed growth of colon cancer cells in vitro, although to a lesser extent than chemical HDAC inhibitors did. The strong prognostic impact of HDAC isoforms in colorectal cancer, the interactions of HDACs with tumor cell proliferation and differentiation in vivo, and our finding that HDACs are differentially expressed in colorectal tumors suggest that the evaluation of HDAC expression in clinical trials for HDAC inhibitors might help to identify a patient subgroup who will exceptionally profit from such a treatment.