Riyad Bendardaf

Intense cytoplasmic ezrin immunoreactivity predicts poor survival in colorectal cancer

Ezrin is a membrane-cytoskeleton anchor, which, in experimental models, regulates tumor cell invasion and metastatic ability. We carried out immunohistochemical analysis of ezrin in 74 advanced colorectal cancer patients and correlated it to clinicopathologic variables and disease outcome. In contrast to the predominantly membraneous immunoreactivity of normal colorectal epithelium, ezrin expression in the colorectal cells was typically cytoplasmic. Altogether, 16.2% (12/74) of the tumors showed negative/weak ezrin staining, 35.1% (26/74) had moderate staining, and 48.6% (36/74) had intense staining. The expression was more intense in colon than in rectal carcinomas (P = .003). Increased ezrin expression was associated with adverse outcome, that is, shorter disease-specific survival; 48.3 months and 36.6 months for negative-weak versus intense expression (P = .041) as well as shorter survival with metastases at 36 months (P = .030); the metastases(36) rates in ezrin(neg/weak), ezrin(moderate), ezrin(intense) are 58.3%, 25.0%, and 18.4%, respectively. In univariate survival analysis, dichotomized (negative/weak versus moderate/strong) ezrin expression significantly predicted both the 5-year disease specific survival (P = .035) and 5-year metastases (P = .018) but lost this predictive power in multivariate (Cox) analysis. High ezrin expression was also related to high E-cadherin (cytoplasmic) expression, DNA aneuploidy, and high thymidylate synthase expression (P = .046, P = .042, P = .046, respectively). These results suggest that ezrin may play a role in colorectal cancer progression and that ezrin expression might provide clinically valuable information in predicting the biological behavior of colorectal cancer.

MMP-9 (Gelatinase B) Expression is Associated With Disease-Free Survival and Disease-Specific Survival in Colorectal Cancer Patients

ABSTRACT Extracellular matrix degradation is required for invasion and metastasis formation in colorectal carcinoma (CRC), therefore, we have examined matrix metalloproteinases MMP-9 expression in tumors from patients with CRC. The study comprises of 360 patients who underwent bowel resection for stage II, III, IV tumors. Paraffin-embedded CRC tissue samples were used for immunohistochemical staining. Negative MMP-9 expression levels correlated with longer survival time as evaluated by disease-free survival and disease-specific survival (p =.023, p =.006). In multivariate survival (Cox) analysis, MMP9 was a significant independent predictor of DFS (p =.014), but not of DSS, which was independently predicted by disease recurrence, stage and localization. The detection of MMP-9 expression may be valuable in finding patients who are at high risk of developing disease recurrence.

Low Collagenase-1 (MMP-1) and MT1-MMP Expression Levels Are Favourable Survival Markers in Advanced Colorectal Carcinoma

Objective: Extracellular matrix degradation is required for invasive growth and metastasis formation in colorectal carcinoma; therefore, we examined matrix metalloproteinases expression (MMP-1, MMP-13 and MT1-MMP) and apoptosis in tumours from 49 patients with advanced colorectal disease. Methods: MMP expression was determined immunohistochemically and apoptotic index (AI) was ascertained using the TUNEL assay. Results: Low levels of MT1-MMP, MMP-1 and AI were found to be favourable markers significantly associated with longer survival. MT1-MMP expression levels below the median (≤14.0% of tumour cells) were associated with better overall survival (median 26.2 vs. 15.6 months, p = 0.02) and MMP-1 expression levels below the median (≤28.7% of tumour cells) correlated with longer survival following metastasis (median 21.5 vs. 13.3 months, p = 0.05). MT1-MMP, MMP-1 and AI were all found to have significant independent effects on survival. Interestingly, MMP-1 expression levels above the median were associated with distal disease of the colon (p = 0.02). Conclusions: These findings reveal that MT1-MMP and MMP-1 expression levels and AI are useful prognostic indicators in advanced colorectal carcinoma and suggest that markers of MMP expression might be used in identifying patients who would benefit from new treatment modalities involving MMP inhibitors.

Mismatch repair status is a predictive factor of tumour response to 5-fluorouracil and irinotecan chemotherapy in patients with advanced colorectal cancer.

Background and Aims: To determine the association between DNA mismatch repair (MMR) protein expression and response to chemotherapy in patients with advanced colorectal cancer (CRC). Methods: Using immunohistochemistry, tumour expression of 2 MMR genes, hMLH1 and hMSH2, was assessed in 86 patients with advanced CRC, who were treated with either irinotecan alone or in combination with 5-flurouracil/folinic acid. Results: Weak/negative staining in the tumours was associated with the presence of metastases at diagnosis (p = 0.026) and with the time for metastases to appear (p = 0.0001). An objective response to treatment was observed in 32/56 (57%) patients who had tumours with negative/weak MMR protein expression (p = 0.001), compared to 17% of patients with tumours with moderate/strong expression. Those who had tumours with weak/absent expression of either hMLH1 or hMSH2 who received the combination therapy were more likely to show an objective response (p = 0.0001). Conclusion: Advanced CRC patients whose tumours have deficient MMR demonstrate a shorter time to metastasis than those with normal hMLH1/hMSH2 expression. Patients with MMR-deficient tumours are also more likely to benefit from combination chemotherapy (irinotecan plus 5-flurouracil/folinic acid).

Prognostic Significance of Matrix Metalloproteinase-9 (MMP-9) in Stage II Colorectal Carcinoma

BackgroundApproximately 30% of all colorectal cancer patients are diagnosed with stage II disease. Adjuvant therapy is not widely recommended. However, it is well-established that a subgroup of patients with stage II is at high risk for recurrence within their life time and should be considered for adjuvant chemotherapy. The present work was designed to assess the value of matrix metalloproteinase-9 (MMP-9) as a predictor of disease outcome in a series of 202 stage II colorectal cancer (CRC) patients with long-term follow-up.MethodsThe present study comprises a series of 202 patients who underwent bowel resection for stage II CRC at Turku University Hospital. Archival paraffin-embedded CRC tissue samples were used to prepare tissue microarray blocks for immunohistochemical staining with MMP-9 antibody.ResultsForty-eight percent of all CRC samples were positive for MMP-9. There was no significant correlation between MMP-9 expression and age, depth of invasion, and lymph node status. However, MMP-9 expression was significantly related to histological grade (p = 0.03) and location of the tumor (p = 0.01), therefore, being lower in high-grade tumors and most intense in carcinomas of the descending colon and rectum. Tumors with high MMP-9 expression showed a higher recurrence rate than tumors with low expression (p = 0.02). MMP-9 negative tumors had a more favorable disease-free survival (DFS) than those expressing MMP-9 (p = 0.03). The same was true with disease-specific survival (DSS; p = 0.02) as well, high expression of MMP-9 being associated with shorter survival rates. In multivariate (Cox) survival analysis, MMP-9 expression proved to be an independent predictor of DFS, but not DSS, which was predicted by age and sex only.ConclusionQuantification of MMP-9 expression seems to provide valuable prognostic information in stage II CRC, particularly, in selecting the patients at high risk for recurrent disease who might benefit from adjuvant therapy.

Apoptotic Index and bcl-2 Expression as Prognostic Factors in Colorectal Carcinoma

Objectives: To examine programmed cell death in 57 colorectal carcinomas (49 primary tumours and 8 metastases) and determine the prognostic significance of apoptosis in colorectal cancer. Methods: Apoptotic index (AI) was ascertained by counting apoptotic bodies, using terminal deoxynucleotidyl transferase mediated digoxigenin nick end labelling (Tunel assay) and the expression of bcl-2 was examined immunohistochemically. Statistical analysis was used to test the value of clinical variables, histopathological data, AI and bcl-2 expression in predicting the clinical outcome of these patients and the survival function was calculated using the Kaplan-Meier method. Results: AI was found to have a significant independent effect on survival (p = 0.0006), with lower values of AI conveying better survival. Conclusion: In summary, these findings reveal that AI is a useful prognostic factor in colorectal carcinoma.

MMP-1 (collagenase-1) expression in primary colorectal cancer and its metastases

Objective. The role of MMP-1 (collagenase-1) in the development of a metastatic phenotype in colorectal cancer (CRC) has not been fully studied. The aim of this study was to investigate the mechanisms involved in the dissemination of CRC by examining the expression of MMP-1 in the primary tumours and their metastases, with special reference to standard clinicopathological features and disease outcome. Material and methods. Surgical specimens from the primary tumours (P) and their metastatic (M) lesions were available from 30 patients with Stage II, III and IV CRC, and were subjected to immunohistochemical (IHC) staining for MMP-1. Both cytoplasmic expression in cancer cells (CC) and stromal (ST) expression were related to pertinent clinical and follow-up data. Results. In a pairwise comparison of P-M pairs, CC expression (but not ST expression) in P and M was significantly different (Wilcoxon rank test, p=0.037). Strong CC expression in P was significantly related to the presence of lymph node involvement at diagnosis (p=0.008). CC expression in M was intense only in metachronous metastases (Stage II/III disease) but never in synchronous metastases (Stage IV) (p=0.034). There was a significant down-regulation of CC (p=0.004) in liver metastasis (n=9) in comparison with all other metastatic sites (n=21). ST expression in P (but not in M) showed a linear decrease in parallel with increasing stage (p=0.028 for linear trend). MMP-1 expression was not significantly associated with any other clinicopathological variables, including age, gender, carcinoembryonic antigen (CEA) or patients’ disease-free or overall survival. Conclusions. These data suggest that MMP-1 may play an important role in tumour invasion and metastasis of CRC.

Response to Chemotherapy (Irinotecan plus 5-Fluorouracil) in Colorectal Carcinoma Can Be Predicted by Tumour DNA Content

Objective: The aim of this study was to identify markers that might predict response to chemotherapy. Postoperative chemotherapy improves the outcome in stage III colon cancer and is widely accepted as a standard therapy, but there are currently no reliable predictors to identify and select patients that will benefit. Methods: Using DNA image cytometry, the DNA content was determined from the isolated nuclei of 56 primary colorectal carcinomas of patients who received chemotherapy (either irinotecan or irinotecan plus 5-fluorouracil and folinic acid) for advanced disease. Response to chemotherapy could be reliably evaluated in 53 patients. Results: The modal DNA content (ploidy status) of the tumour correlated with the observed response to chemotherapy (p = 0.01). An objective response was observed in 56% of patients whose tumour histograms displayed tetraploid, peri-tetraploid or multiploid patterns of peaks, compared with 19% in patients with diploid, peri-diploid or aneuploid peaks. Notably, 86% (6/7) of patients whose tumours displayed a multiploid peak pattern showed an objective response and 1 patient had stable disease. Conclusion: This study suggests that modal DNA content can be used to predict a patient’s response to chemotherapy in advanced colorectal carcinoma. This may help in identifying patients who will benefit most from therapy for advanced colorectal cancer.

Thymidylate synthase and microsatellite instability in colorectal cancer: Implications for disease free survival, treatment response and survival with metastases

Background. Colorectal cancer (CRC) cell lines displaying microsatellite instability (MSI) are resistant to 5-fluorouracil (5-FU) in vitro, which can be overcome by restoring DNA mismatch repair (MMR) competence. Thymidylate synthase (TS) is inhibited by 5-FU, being another potential mediator of therapeutic resistance to 5-FU. The clinical relevance of these observations remains unclear. Objective. We examined the expression of TS and two MMR proteins (hMLH1 and hMSH2) in advanced CRC patients, to determine a) their mutual relationship, b) association to therapeutic response and c) impact on disease outcome. Material and methods. Tumour samples from 73 patients CRC who were treated in advanced stage with either irinotecan alone or in combination with 5-FU/leucovorin, were analysed for expression of TS, hMLH1 and hMSH2 using immunohistochemistry (IHC). Results. TS expression was closely correlated with hMLH1 expression (negative-weak/moderate-strong) (p=0.0001). TS-MMR expression was significantly (p=0.029 for whole series; p=0.004 for the 5-FU treated cases) related to response to treatment; tumours with low levels of both TS and MMR responded better (n=14/27, 51.8%) than those with high TS and MMR (n=3/18, 16.6%). Patients with high TS-MMR expression had a significantly longer DFS (47 months vs. 9months, n=26) than those with low TS-MMR index (p=0.015), while the reverse was true concerning survival with metastases (WMS) (p=0.018) in all the patients (n=73). Conclusions. The present data suggest that MSI patients with low TS and deficient MMR demonstrate a significantly shorter DFS and longer WMS than patients with high expression of both markers, and they are also more likely to obtain the greatest benefit from 5-FU based chemotherapy.

Thymidylate synthase expression in primary colorectal tumours is correlated with its expression in metastases

Objective. Thymidylate synthase (TS) is the rate-limiting enzyme in the synthesis of pyrimidine nucleotides and as such a critical target for fluoropyrimidines, which are widely used in the treatment of colorectal cancer (CRC). The purpose of this study was to investigate TS expression in the primary tumours (PTs) and their metastases (M) in advanced CRC. Material and methods. TS expression was determined immunohistochemically in paraffin-embedded biopsies of PT-M pairs in 39 CRC patients, as related to the clinical data. Results. There was no difference in the mean TS index of PTs compared with that of M, 1.25 and 1.14, respectively (p=0.12). TS expression of PTs was above the mean more often than that of M (61.5% and 41.0%, respectively, p=0.035). High TS expression in PTs was significantly related to high expression in M (the Fisher exact test, p=0.001). Using the absolute index values, TS expression in PT and M was significantly correlated (Pearson R=0.501, p=0.001). In 29/39 (74.3%) pairs, PT and M had concordant expression levels (Cohens kappa 0.508, 95% CI 0.260–0.756, p=0.001; intraclass correlation coefficient (ICC) = 0.679, 95% CI 0.358–0.836, p=0.0001). No significant association was found between TS expression and any of the clinicopathological variables, disease outcome (DFS, DSS) or its response to treatment in univariate or multivariate analysis. Conclusions. Albeit usually higher, TS expression in PT was closely correlated with TS expression in M. This suggests that measurement of TS in primary CRC accurately predicts TS expression in subsequent metastases, which may help in selecting those patients most likely to respond to 5-FU-based regimens.