Expression In Colorectal Carcinoma Research Articles

The prognostic effect of PTEN expression status in colorectal cancer development and evaluation of factors affecting it: miR-21 and promoter methylation.

BackgroundPTEN is a tumor suppressor gene which is involved in cellular proliferation, differentiation, and apoptosis. Loss or down-regulation of PTEN plays an important role in human cancers development. In this study, we investigated the effect of miR-21 and promoter methylation on the PTEN expression status in CRC tissues and analyzed association of the PTEN expression status with clinicopathological features in patients with CRC.ResultsThe PTEN expression was positively detected in 67.2 % CRC tissues and all adjacent non-cancerous samples. PTEN mRNA level was negatively correlated with miR-21 level (r = −0.595, P < 0.001). PTEN expression was also correlated directly with the PTEN mRNA level (r = 0.583, P < 0.001) and conversely with miR-21 level (r = −0.632, P < 0.001). PTEN Promoter methylation was significantly associated with PTEN expression status (p = 0.013). PTEN expression was negatively associated with tumor size (p = 0.007) and advanced tumor stage (P = 0.011). Multivariate analysis indicated that tumor stage, tumor differentiation and PTEN expression status were independent prognostic factors for overall carcinoma in CRC patients (P < 0.05). The Kaplan-Meier curve indicated a negative correlation between PTEN expression levels and survival of CRC patients (P = 0.013).ConclusionsThis study suggests a high frequency of miR-21 overexpression and aberrant promoter methylation in down-regulation of PTEN expression in colorectal carcinoma. Loss of PTEN may be a prognostic factor for patients with CRC.

Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort

Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort

Loss of CDH1 (E-cadherin) expression is associated with infiltrative tumour growth and lymph node metastasis.

Background:Loss of CDH1 (E-cadherin) expression in cancer cells may promote cell migration and invasion. Therefore, we hypothesised that loss of CDH1 expression in colorectal carcinoma might be associated with aggressive features and clinical outcome.Methods:Utilising molecular pathological epidemiology database of 689 rectal and colon cancer cases in the Nurses' Health Study and the Health Professionals Follow-up Study, we assessed tumour CDH1 expression by immunohistochemistry. Multivariate logistic regression analysis was conducted to assess association of CDH1 loss with tumour growth pattern (expansile-intermediate vs infiltrative) and lymph node metastasis and distant metastasis, controlling for potential confounders including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and PIK3CA, BRAF and KRAS mutations. Mortality according to CDH1 status was assessed using Cox proportional hazards model.Results:Loss of tumour CDH1 expression was observed in 356 cases (52%), and associated with infiltrative tumour growth pattern (odds ratio (OR), 2.02; 95% confidence interval (CI), 1.23–3.34; P=0.006) and higher pN stage (OR, 1.73; 95% CI, 1.23–2.43; P=0.001). Tumour CDH1 expression was not significantly associated with distant metastasis or prognosis.Conclusions:Loss of CDH1 expression in colorectal cancer is associated with infiltrative tumour growth pattern and lymph node metastasis.

Clinicopathologic, molecular, and prognostic implications of the loss of EPCAM expression in colorectal carcinoma.

We aimed to comprehensively investigate the clinicopathologic and molecular implications of altered epithelial cell adhesion molecule (EPCAM) expression in colorectal carcinoma (CRC). EPCAM immunohistochemical expression, EPCAM 3′ end deletion, EPCAM promoter methylation, microsatellite instability (MSI), and the CpG island methylator phenotype (CIMP) were analyzed in large cohorts of human CRCs. Among 218 MSI-high CRCs, complete loss (CL) of EPCAM expression was observed in two cases, both of which displayed MSH2 deficiency and EPCAM 3′ deletion. Thirty-one of the 218 MSI-high CRCs demonstrated the partial loss (PL) of EPCAM expression without EPCAM deletion or methylation and were correlated with CIMP-high and poor disease-free survival. Histologically, foci exhibiting EPCAM loss in EPCAM-PL tumors were dominantly distributed in poorly differentiated tumor components and/or in the invasive tumor front. The implications of EPCAM-PL were further validated in a consecutive series of 726 CRCs. EPCAM-PL (n = 50; 6.9%) was also associated with CIMP-high and adverse pathologic factors and was confirmed to be an independent poor prognostic factor in CRC (HR, 1.57; 95% CI, 1.04 to 2.39). EPCAM-CL can be used to screen for EPCAM deletion-induced Lynch syndrome-associated CRC, whereas EPCAM-PL can be used as an indicator of tumor aggressiveness and poor prognosis in CRC.

Mucinous Colorectal Adenocarcinoma: Influence of EGFR and E-Cadherin Expression on Clinicopathologic Features and Prognosis.

Previous studies have shown conflicting results on epidermal growth factor receptor (EGFR) and E-cadherin expression in colorectal carcinoma and their prognostic significance. To the best of our knowledge, this study is the first to investigate EGFR and E-cadherin expression, interrelation and relation to clinicopathologic, histologic parameters, and survival in rare colorectal mucinous adenocarcinoma (MA). In this study, we studied tumor tissue specimens from 150 patients with colorectal MA and nonmucinous adenocarcinoma (NMA). High-density manual tissue microarrays were constructed using modified mechanical pencil tips technique, and immunohistochemistry for EGFR and E-cadherin was performed. All relations were analyzed using established statistical methodologies. NMA expressed EGFR and E-cadherin in significantly higher rates with significant heterogenous pattern than MA. EGFR and E-cadherin positivity rates were significantly interrelated in both NMA and MA groups. In the NMA group, high EGFR expression was associated with old age, male sex, multiplicity of tumors, lack of mucinous component, and association with schistosomiasis. However, in the MA group, high EGFR expression was associated only with old age and MA subtype rather than signet ring carcinoma subtype. Conversely, high E-cadherin expression in MA cases was associated with old age, fungating tumor configuration, MA subtype, and negative intratumoral lymphocytic response. However, in the NMA cases, none of these factors was statistically significant. In a univariate analysis, neither EGFR nor E-cadherin expression showed a significant impact on disease-free or overall survival. Targeted therapy against EGFR and E-cadherin may not be useful in patients with MA. Neither EGFR nor E-cadherin is an independent prognostic factor in NMA or MA.

Aberrant expressions of c-KIT and DOG-1 in mucinous and nonmucinous colorectal carcinomas and relation to clinicopathologic features and prognosis.

c-KIT and DOG-1 are 2 highly expressed proteins in gastrointestinal stromal tumors. Few studies had investigated c-KIT, but not DOG-1, expression in colorectal carcinoma (CRC). This study aims to investigate expressions of c-KIT and DOG-1 in colorectal mucinous carcinoma and nonmucinous carcinoma using manual tissue microarray technique. In this work, we studied tumor tissue specimens from 150 patients with colorectal mucinous (MA) and nonmucinous adenocarcinoma (NMA). High-density manual tissue microarrays were constructed using modified mechanical pencil tip technique, and immunohistochemistry for c-KIT and DOG-1 was done. We found that aberrant c-KIT expression was detected in 12 cases (8%); 6 cases (4%) showed strong expression. Aberrant DOG-1 expression was detected in 15 cases (10%); among them, only 4 cases (2.7%) showed strong expression. Nonmucinous adenocarcinoma showed a significantly high expression of c-KIT, but not DOG-1, than MA. Aberrant c-KIT and DOG-1 expressions were significantly unrelated but were associated with excessive microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. In conclusion, aberrant c-KIT and DOG-1 expressions in CRC are rare events, either in NMA or MA. Nonmucinous adenocarcinoma showed a significantly higher expression of c-KIT, but not DOG-1, than MA. The expressions of both in CRC are significantly unrelated but are associated with microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. So, c-KIT and DOG-1 immunostaining is not a cost-effective method of identifying patients with CRC who may benefit from treatment with tyrosine kinase inhibitors.

The effects of focal adhesion kinase on the motility, proliferation and apoptosis of Caco2 and SMMC-7721 cells.

Focal adhesion kinase (FAK) plays important roles in cancer development. However, the significance of FAK expression in colorectal carcinoma and hepatocellular carcinoma has not been clarified. This study aims to explore the roles FAK played in the progression of colorectal carcinoma and hepatocellular carcinoma. RNAi method was used to inhibit the expression of FAK in Caco2 and SMMC-7721 cells. Reverse transcriptase polymerase chain reaction analysis and Western blot analysis were used to examine mRNA and protein expression of FAK. Then, the proliferation, motility and apoptosis of both types of cells were detected using MTT assay, wound healing/transwell assay and nuclear staining assay. The microstructure changes (F-actin, β-tubulin and lamin B1) of SMMC-7721 cells were visualized by immunofluorescence. FAK was overexpressed in both cell lines and down-regulation of FAK resulted in suppression of cell proliferation, inhibition of cell migration and invasion. The apoptosis of cells was increased significantly following the FAK expression inhibition. Moreover, actin polymerization, β-tubulin and lamin B1 expression of cells were significantly decreased. The results highlight the role of FAK in the progression of cancers. These findings suggest FAK serve as a potential therapeutic target for cancer therapy.

Semaphorin 4D and hypoxia-inducible factor-1α overexpression is related to prognosis in colorectal carcinoma.

To investigate semaphorin 4D (Sema4D) and hypoxia-inducible factor-1α (HIF-1α) expression in colorectal carcinoma and evaluate their clinicopathological and prognostic significance. Eighty-six curatively resected colorectal carcinoma patients at different stages of disease were randomly selected from the group of patients who underwent surgery, and none of them received preoperative radiochemotherapy. Normal proximal adjacent bowel tissue, which served as an internal control, was obtained from 52 randomly selected patients. Immunohistochemistry was performed to analyze the expression of Sema4D and the tumor angiogenesis-related protein HIF-1α in normal colorectal tissues and colorectal carcinoma tissues. The relationships between the expression and clinical characters and prognosis were analyzed. HIF-1α and Sema4D were positively expressed in 58% and 60% of colorectal carcinoma tissues, respectively. Significantly lower expression levels were observed in normal mucosa (8% and 12%, respectively). HIF-1α and Sema4D expression was closely correlated with histological tumor type, tumor-node-metastasis (TNM) stage, and lymphatic metastasis (P<0.05), but not with age or tumor size (P>0.05). HIF-1α and Sema4D protein expression was significantly correlated with prognosis of colorectal carcinoma, as determined by Spearman rank correlation analysis (r=0.567; P<0.01). Multivariate Cox analysis revealed that only Sema4D expression played a significant role in predicting patient prognosis (P<0.05). These findings suggest that HIF-1α and Sema4D expression correlates with histological tumor type, TNM stage, and lymphatic metastasis in colorectal carcinoma and that Sema4D is a prognostic indicator of colorectal carcinoma.

Characterization of HER2 Gene/Protein and Ki67 Protein Expressions in Colorectal Carcinoma Variants With Relation To Clinicopathological Parameters and Prognosis: An Immunohistochemical and Fluorescence In Situ Hybridization Study

Objective The data on the frequency and pattern of HER2 expression in colorectal carcinoma (CRC) and its clinical signifi¬cance are ambiguous. In addition, little is known about HER2 status in CRC variants and its relation with proliferative activity and clinical outcome. Such knowledge may be of potential value for therapeutic decision making in CRCs. Methods The HER2 gene/protein status was assessed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in a tissue microarray of 150 CRCs and correlated with the expression of the proliferation marker Ki67, clinicopathological factors, and prognosis. Results CRCs were categorized into conventional adenocarcinoma (CA), 47 cases; adenocarcinoma with mucinous component (AMC), 28 cases; mucinous adenocarcinoma (MA), 56 cases; and signet ring cell carcinoma (SRCC), 19 cases. Compared to other variants, CA was significantly associated with favorable clinicopathological features, higher overall survival (OS) and disease-free survival (DFS), while SRCC and MA were significantly associated with ominous clinicopathological features, lower OS and DFS. Cytoplasmic HER2 overexpression was detected in 14.2% of CRC cases and showed a significant agreement with gene amplification. HER2 overexpression was significantly associated with favorable clinicopathological features notably early stages. High Ki67 expression was detected in 48% of CRC cases. HER2 and Ki67 were significantly different among CRC variants with AMC showing the greatest frequency of HER2 overexpression and Ki67 high expression than the other variants. The interrelation between HER2 and Ki67 expression was statistically insignificant and neither had any significant relation to OS or DFS in any of the CRC variants. Conclusions We conclude that mucinous histology infers an adverse prognosis in CRC. A subset of early stage CRC patients, with HER2 overexpression and possibly a distinct variant, may benefit from HER2 targeted therapy. IHC can be used as a method for screening of HER2 gene amplification in CRCs.

The significance of sonic hedgehog immunohistochemical expression in colorectal carcinoma

Colorectal carcinoma is a significant source of major morbidity and mortality. Sonic hedgehog (Shh) is expressed in normal gastrointestinal tract mucosa and in many malignancies. The purpose of the present study is to investigate the relationship between Shh immunoexpression in CRC and clinicopathological characteristics. Paraffin blocks of 155 primary CRCs and 37 nodal metastases were retrieved and tissue microarrays were constructed. Immunohistochemistry was performed using anti-Shh antibody. Immunostaining was scored and results were analysed in relation to the clinicopathological parameters. Shh was overexpressed in primary CRC (p=0.02) and in nodal metastasis (p=0.004). There was no difference between Shh immunoexpression in primary CRC and in nodal metastasis (p=0.941). High Shh immunoexpression was associated with well differentiated tumours (p=0.004). However, there was no association with other clinicopathological parameters. Shh overexpression was not associated disease free survival (log-rank=0.079, p=0.778). Shh is overexpressed in well differentiated CRC. However, Shh is not associated with other clinicopathological and prognostic factors. Loss of Shh may be associated with proliferation and loss of differentiation in CRC. Further molecular studies are required to address the potential importance of Shh signalling in CRC and to test Shh inhibitors and activators as potential therapeutic targets in CRC.

Syndecan-1 expression is associated with tumor size and EGFR expression in colorectal carcinoma: a clinicopathological study of 230 cases.

Background: Syndecan-1 (SDC1) is reported to modulate several key processes of tumorigenesis and has variable expression in many cancers. To date, the cause of altered expression has not been elucidated. In this study, we compared SDC1 expression with various clinicopathological parameters and molecular markers to evaluate its clinical significance in colorectal carcinoma.Methods: We screened for SDC1 expression using immunohistochemistry in 230 surgical specimens of primary colorectal carcinoma from patients consecutively treated between 2008 and 2011 at Seoul St. Mary's Hospital, The Catholic University of Korea. The relationship between SDC1 expression and various clinicopathological parameters and molecular markers was analyzed.Results: The tumors were principally located in the left colon (71.3%) and rectum (33.5%). There were 216 (93.9%) adenocarcinomas, 10 (4.3%) mucinous adenocarcinomas, and 4 other tumors. Most of the carcinomas were pT3 (68.3%) and pT4 (22.2%). There was regional lymph node metastasis in 140 patients. SDC1 expression was identified in the cancer cells of 212 (96.8%) colon cancer cases. Of the SDC1-positive cases, 131 showed predominantly membranous immunopositivity, and 81 showed a predominantly cytoplasmic staining pattern. Mixed membranous and cytoplasmic staining was observed in 154 cases. In 93 cases, stromal SDC1 reactivity was noted. Epithelial SDC1 immunopositivity was significantly associated with tumor size (p = 0.016) and epidermal growth factor receptor expression (p = 0.006). However, it was not significantly correlated with lymph node metastasis, distant metastasis, lymphatic or vascular invasion, or KRAS mutation. In addition, stromal SDC1 immunopositivity was significantly associated with the male sex (p = 0.018).Conclusions: The expression profile of SDC1 may be of clinical value in colorectal cancer and may help in identifying aggressive forms of colorectal carcinoma. Further studies are needed in order to better understand the role of SDC1 in the progression and invasiveness of colorectal carcinoma.

Characterization and analysis of claudin-1 expression in colorectal cancer and its metastases: A pilot study

Claudins are the main sealing protein of the intercellular tight junctions and play an important role in cancer progression and dissemination. Several authors have reported conflicting results of claudin-1 expression in colorectal carcinoma. The goal of this study is to examine claudin-1 expression in a tissue microarray of colorectal cancer and metastases, simultaneously, in order to assess the prognostic value of this protein in colorectal cancer. This study analysed the expression of claudin-1 by immunohistochemistry in 99 tissue samples (including 33 colorectal cancer cases with 33 concordant lymph node metastases and 33 concordant cancer-adjacent normal tissues, all from the same patients). The tumours included adenocarcinomas from the colon (n=25) and from the rectum (n=8). This is the first study to demonstrate the expression of claudin-1 protein across a sample of colorectal cancer and its metastases, simultaneously. We report loss of claudin-1 expression in the lymph node metastases in the majority of cases. We also report differential expression of claudin-1 among colorectal cancers of different grades, with the highest expression being in well-differentiated, low-grade tumours; and complete loss of expression in the majority of cases of poorly-differentiated, high grade tumours.

Significance of β-catenin and vitamin D receptor expression in colorectal carcinoma

Significance of β-catenin and vitamin D receptor expression in colorectal carcinoma

Smac/DIABLO expression in human gastrointestinal carcinoma: Association with clinicopathological parameters and survivin expression.

Lack of apoptosis is a key factor in carcinogenesis and tumor progression. Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) is an antagonist of IAPs. Recently, Smac/DIABLO was identified as a potent therapeutic target. However, the clinical significance of Smac/DIABLO in gastrointestinal carcinomas remains unclear. In the present study, Smac/DIABLO expression was analyzed by immunohistochemistry in 72 gastric adenocarcinomas and 78 colorectal adenocarcinomas. The expression of Smac/DIABLO was significantly higher in colorectal carcinoma than in gastric carcinoma. Additionally, a correlation was found between the expression of Smac/DIABLO and nuclear survivin in well- to moderately-differentiated colorectal adenocarcinomas (r=0.245; P<0.01). Based on these results, it was hypothesized that gastric and colorectal carcinomas differ in the level of Smac/DIABLO expression. Our previous studies revealed that the expression of cleaved caspase-9 was significantly lower in colorectal carcinoma than in gastric carcinoma (P<0.0001). Conversely, the expression levels of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and survivin were significantly higher in colon cancer than in gastric cancer (P<0.0001 and P<0.01, respectively). Taken together, these results indicate that not only LC3 and survivin expression, but also Smac/DIABLO expression, are significantly higher in colorectal carcinoma than in gastric carcinoma. We hypothesize that the analysis of Smac/DIABLO, survivin and LC3 expression in colorectal carcinoma is likely to aid cancer therapy due to the involvement of these markers in apoptosis and/or autophagy.

B7-H1 and B7-H4 expression in colorectal carcinoma: Correlation with tumor FOXP3+ regulatory T-cell infiltration

B7-H1 and B7-H4 are newly discovered members of the B7-CD28 family. They can inhibit T cell activation and proliferation and regulate T cell immune response negatively. Both B7-H1 and B7-H4 are expressed in many tumors and are classified as co-inhibitors of cell-mediated immunity. FOXP3+ regulatory T cells (Tregs) play an important role in the maintenance of tumor immunity tolerance. However, the implication of B7-H1 and B7-H4 expression and their interaction with Tregs infiltration in colorectal cancer are unknown. The present study aimed to determine the expression of B7-H1 and B7-H4 as well as Tregs infiltration in colorectal cancer and to explore the clinical and pathological implication of suppressor immune cells and molecules. Frozen sections and immunohistochemical assay were undertaken to assess B7-H1, B7-H4 expression and Tregs infiltration in fresh specimens collected from 56 patients with colorectal carcinoma. The results showed that expression of B7-H1 and B7-H4 in colorectal carcinoma tissues was significantly higher than in adjacent normal mucosa (P<0.001). B7-H1 expression was positively correlated to the infiltration depth, lymph node metastasis and advanced Duke's stage (P<0.05, P<0.05 and P<0.05, respectively), whereas B7-H4 expression was positively related to the infiltration depth and lymph node metastasis (P<0.01 and P<0.05, respectively). Furthermore, Tregs infiltration was more frequent in tumor tissue than in adjacent normal mucosa and was associated with poor differentiation and positive lymph node metastasis (P<0.01, and P<0.01, respectively). The statistical analysis indicated a significant correlation between Tregs infiltration and B7-H1 or B7-H4 expression respectively. These results suggest that over-expression of B7-H1 and B7-H4 has stronger prognostic significance and promote tumor tolerance, and they might contribute to Tregs development in the colorectal carcinoma tolerogenic milieu.

Drug resistance reversed by silencing LIM domain-containing protein 1 expression in colorectal carcinoma.

The role of LIM domain-containing protein 1 (LIMD1) in the multidrug resistance of colorectal carcinoma (CRC) has not yet been established. The aim of the current study was to investigate the chemosensitivity of CRC multidrug-resistant (MDR) cells following the silencing of LIMD1. The MDR phenotypic Colo205 and HCT-8 cell lines were examined, which were established by exposure to increasing doses of 5-fluorouracil (5-FU) over a period of one year. LIMD1 siRNA constructs were transfected into CRC MDR cells and the phenotypic effects were determined comprehensively. The Colo205 and HCT-8 cell lines were more resistant to 5-FU compared with their respective parental cell lines. In addition, the two MDR cell types expressed significantly more LIMD1 compared with their parental lines. The stably transfected cells showed various degrees of reversal of the MDR phenotype, and 5-FU-induced apoptosis was increased in the transfected cells compared with the controls. In conclusion, RNA interference targeting LIMD1 may present a novel therapeutic option for CRC.

Loss of Anterior Gradient-2 expression is an independent prognostic factor in colorectal carcinomas

AimsThe human Anterior Gradient-2 (AGR2) protein is strongly expressed in various human cancers, and it has been described to promote aggressive tumour features in some entities. So far, a comprehensive analysis of AGR2 expression in colorectal carcinomas has not been described. MethodsNormal intestinal cells and colorectal carcinoma cell lines were analysed for AGR2 expression. AGR2 protein expression was immunohistochemically analysed in 28 normal tissue samples and 1068 tissue samples of clinically well characterised colorectal carcinomas. For statistical analysis, chi square test, spearman rank correlations, Kaplan–Meier estimates (Log rank test) and Cox regression were applied to test for diagnostic or prognostic associations. ResultsIn the normal intestinal cell line and in normal colon mucosa AGR2 was found in all cases (n=28). In contrast, loss of AGR2 was found in all six analysed colorectal cancer cell lines and in 833/1068 (78%) of the colorectal carcinoma tissue samples analysed, and it was significantly associated with a higher tumour grade and tumour localisation in the left-sided colon. In addition to the conventional prognostic tumour parameters pT category, nodal status, metastasis and histological tumour grade the loss of AGR2 expression was significantly associated with reduced overall survival times in univariate and multivariate analyses, thus suggesting AGR2 as an independent prognostic factor in primary colorectal carcinoma. ConclusionsAGR2 is frequently lost in colorectal carcinomas and might be a novel independent prognostic factor for overall patient survival.

BRAF-mutated microsatellite stable colorectal carcinoma: an aggressive adenocarcinoma with reduced CDX2 and increased cytokeratin 7 immunohistochemical expression.

Reduced CDX2 and cytokeratin 20 (CK20) expression in colorectal carcinoma with BRAF mutation and high-level microsatellite instability (MSI-H) has been well documented. The immunophenotype of BRAF-mutated microsatellite stable (MSS) colorectal carcinoma has not been reported. We analyzed 205 colorectal carcinomas including 28 BRAF-mutated MSS, 53 BRAF-mutated MSI-H, and 124 BRAF wild-type MSS tumors for CDX2, cytokeratin 7 (CK7), and CK20 immunohistochemical expression. CDX2 was scored semiquantitatively for both staining intensity and percent of tumor cells staining and a modified CDX2 H-score was calculated. Patients with BRAF-mutated MSS colorectal carcinomas were more frequently stage IV at presentation compared to patients with BRAF-mutated MSI-H colorectal carcinomas and BRAF wild-type MSS colorectal carcinomas (32% versus 8% versus 15%, P < .001). BRAF-mutated MSS colorectal carcinoma displayed reduced CDX2 expression compared to BRAF wild-type MSS colorectal carcinoma (75% versus 94%; mean CDX2 H-score 98 versus 150, P < .001). CK7 expression was more often identified in BRAF-mutated MSS colorectal carcinoma compared to both BRAF-mutated MSI-H colorectal carcinoma and BRAF wild-type MSS colorectal carcinoma (39% versus 6% versus 6%, P = .0001). BRAF-mutated MSI-H colorectal carcinomas were less often CK20 positive compared to BRAF-mutated MSS and BRAF wild-type MSS tumors (70% versus 93% versus 90%, P = 0.001). In summary, BRAF-mutated MSS colorectal carcinoma often displays reduced CDX2 and increased CK7 expression. Knowledge of this altered immunophenotype is important as patients with BRAF-mutated MSS colorectal carcinoma often present with metastatic disease and the altered tumor immunophenotype may lead to the erroneous assumption that origin from the colon/rectum is unlikely.

Loss of ARID1A expression in colorectal carcinoma is strongly associated with mismatch repair deficiency.

ARID1A is a tumor suppressor gene involved in chromatin remodelling. ARID1A mutations and loss of protein expression occur commonly in endometrioid and gynecological clear cell carcinoma where they are associated with mismatch repair (MMR) deficiency. We assessed ARID1A expression in a large cohort of colorectal carcinomas (CRCs). Immunohistochemistry for ARID1A was performed on whole sections from 100 CRCs and on 1876 CRCs in tissue microarray format. There was complete concordance between the staining on whole slides and tissue microarray sections. Loss of staining was found in 110 (5.9%) of 1876 CRCs and was strongly associated with older age, right sided location, large size, BRAF V600E mutation, MMR deficiency, high histological grade and medullary morphology, (all P < .01). There was a trend towards loss of expression being more common in females (P = .06). When subclassified by combined BRAF V600E mutation and MMR status, loss of ARID1A expression was found most commonly in CRCs with the BRAF V600E mutated, MMR- deficient phenotype (58 of 232 cases, 25%, P < .01). In univariate and multivariate analysis, loss of ARID1A expression was not associated with overall survival-hazard ratio 1.05 (0.68-1.64) and 0.60 (0.24-1.44), respectively. All carcinomas arising in patients with known Lynch syndrome (n = 12) were ARID1A positive. We conclude that loss of ARID1A expression occurs in a small but significant proportion of CRCs where it is strongly correlated with mismatch repair deficiency and other clinical and pathological features associated with somatic hypermethylation.

PTEN mutation, loss of heterozygosity, promoter methylation and expression in colorectal carcinoma: Two hits on the gene?

The phosphatase and tensin homologue (PTEN) gene is considered to be a tumour-suppressor gene in various types of cancer, colorectal carcinoma among them. According to the 'two-hit' tumour-suppressor gene concept, inactivation occurs by any combination of the following three pathogenetic processes: mutation, loss of one allele [i.e. loss of heterozygosity (LOH)] or promoter methylation. To determine the frequencies of PTEN tumour-suppressor gene features in colorectal carcinoma, we used DNA from colorectal carcinoma xenografts/primary tumour cell lines (N=22) or neoplastic glands isolated by laser-capture microdissection (N=20). Sequencing exons 1-9 of the gene revealed a total of 8 somatic mutations in 5 tumours (3 with high-degree microsatellite instability). In 1 tumour, a truncating mutation of one allele was combined with two missense mutations of the other allele. Polymorphic microsatellite marker analyses (D10S5412, D10S579 and D10S1765) showed complete loss of one allele (i.e. LOH sensu stricto) in 3 tumours, but combined LOH and mutation was found only once. Promoter methylation, tested by MethyLight technology, was found in only 1 of the tumours, not combined with mutation or LOH. In contrast, by immunohistochemistry (mAb 6H2.1), reduction or even loss of PTEN expression was found in 18 tumours. Taken together, PTEN downregulation is a fairly frequent event in colorectal carcinoma, but this apparently is not usually caused by two hits on the gene.