Jan Brabender

ERCC1 and Thymidylate Synthase mRNA Levels Predict Survival for Colorectal Cancer Patients Receiving Combination Oxaliplatin and Fluorouracil Chemotherapy

PURPOSE To test the hypotheses of whether the relative mRNA expression of the thymidylate synthase (TS) gene and the excision cross-complementing (ERCC1) gene are associated with response to and survival of fluorouracil (5-FU)/oxaliplatin chemotherapy in metastatic colorectal cancer. PATIENTS AND METHODS Patients had progressive stage IV disease after unsuccessful 5-FU and irinotecan chemotherapy. All patients were evaluated for eligibility for a compassionate 5-FU/oxaliplatin protocol. cDNA was derived from paraffin-embedded tumor specimens to determine TS and ERCC1 mRNA expression relative to the internal reference gene beta-actin using fluorescence-based, real-time reverse transcriptase polymerase chain reaction. RESULTS The median TS gene expression level from 50 metastasized tumors was 3.4 x 10(-3) (minimum expression, 0.18 x 10(-3);maximum expression, 11.5 x 10(-3)), and the median ERCC1 gene expression level was 2.53 x 10(-3) (minimum, 0.0; maximum, 14.61 x 10(-3)). The gene expression cutoff values for chemotherapy nonresponse were 7.5 x 10(-3) for TS and 4.9 x 10(-3) for ERCC1. The median survival time for patients with TS <or= 7.5 x 10(-3) (43 of 50 patients) was 10.2 months, compared with 1.5 months for patients with TS greater than 7.5 x 10(-3) (P < .001). Patients with ERCC1 expression <or= 4.9 x 10(-3) (40 of 50 patients) had a median survival time of 10.2 months, compared with 1.9 months for patients with ERCC1 expression greater than 4.9 x 10(-3) (P < .001). A TS of 7.5 x 10(-3) segregated significantly into response, stable disease, and progression (P = .02), whereas the association between ERCC1 and response did not reach statistical significance (P = .29). CONCLUSION These data suggest that intratumoral ERCC1 mRNA and TS mRNA expression levels are independent predictive markers of survival for 5-FU and oxaliplatin combination chemotherapy in 5-FU-resistant metastatic colorectal cancer. Precise definition of the best TS cut point will require further analysis in a large, prospective study.

Adenomatous polyposis coli gene promoter hypermethylation in non-small cell lung cancer is associated with survival

Methylation of 5′ CpG islands in promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of this study was to determine whether hypermethylation of the adenomatous polyposis coli (APC) gene promoter occurs in primary non-small cell lung cancer (NSCLC), and whether hypermethylated APC has any relationship with survival. APC promoter 1A methylation was determined in normal and corresponding tumor tissue from 91 NSCLC patients and in a control group of 10 patients without cancer, using a quantitative fluorogenic real-time PCR (Taqman®) system. APC promoter methylation was detectable in 86 (95%) of 91 tumor samples, but also in 80 (88%) of 91 normal samples of NSCLC patients, and in only two (20%) of 10 normal lung tissues of the control group. The median level of APC promoter methylation was 4.75 in tumor compared to 1.57 in normal lung tissue (P<0.001). Patients with low methylation status showed significantly longer survival than did patients with high methylation status (P=0.041). In a multivariate analysis of prognostic factors, APC methylation was a significant independent prognostic factor (P=0.044), as were pT (P=0.050) and pN (P<0.001) classifications. This investigation shows that APC gene promoter methylation occurs in the majority of primary NSCLCs. High APC promoter methylation is significantly associated with inferior survival, showing promise as a biomarker of biologically aggressive disease in NSCLC.

Prognostic significance of cyclooxygenase 2 mRNA expression in non-small cell lung cancer.

ObjectiveTo investigate cyclooxygenase-2 (COX-2) mRNA expression in curatively resected non-small cell lung cancer (NSCLC) and to determine its association with prognosis. Summary Background DataLung cancer is one of the most common malignancies in the world. Despite improvements in the diagnosis and treatment of NSCLC, the 5-year survival rate remains less than 15%. Identification of prognostic predictors based on molecular alterations could lead to additional diagnostic tools and eventually to more effective therapeutic options. Overexpression of COX-2 has been reported in several human malignancies, including lung cancer, but the prognostic importance of this overexpression has not been elucidated. MethodsCOX-2 mRNA expression was analyzed using a quantitative real-time polymerase chain reaction (Taqman) method in surgically resected tumor specimens from 89 patients with curatively resected NSCLC. ResultsCOX-2 mRNA was detectable in all 89 (100%) tumor tissues. High COX-2 expression in tumors was significantly associated with inferior survival. Multivariate analysis showed that high COX-2 expression is an independent predictor of worse survival in patients with NSCLC. ConclusionsHigh COX-2 mRNA expression is an important biomarker for biologically aggressive disease in NSCLC and might be helpful in identifying patients who would benefit from additional therapies for controlling their disease.

High Specificity of Quantitative Excision Repair Cross- Complementing 1 Messenger RNA Expression for Prediction of Minor Histopathological Response to Neoadjuvant Radiochemotherapy in Esophageal Cancer

Purpose: The excision repair cross-complementing 1 (ERCC1) gene is coding for a nucleotide excision repair protein involved in the repair of radiation- and chemotherapy-induced DNA damage. We examined the potential of quantitative ERCC1 mRNA expression to predict minor or major histopathological response to neoadjuvant radiochemotherapy (cisplatin, 5-fluorouracil, and 36 Gy of radiation) followed by transthoracic en bloc esophagectomy in patients with locally advanced esophageal cancer (cT2–4, Nx, M0). Experimental Design: Tissue samples were collected by endoscopic biopsy before treatment. RNA was isolated from biopsies, and quantitative real-time reverse transcriptase PCR assays were performed to determine ERCC1 mRNA expression. Relative mRNA levels (tumor/normal ratios) were calculated as (ERCC1/β-actin in tumor)/(ERCC1/β-actin in paired normal tissue). ERCC1 expression levels were correlated with the objective histopathological response in resected specimens. Histomorphological regression was defined as major response when resected specimens contained <10% of residual vital tumor cells or in case a pathologically complete response was achieved. Results: Twelve of 36 tumors showed a major histopathological response, and 24 of 36 showed a minor histopathological response. Relative expression levels of ERCC1 of >1.09 were not associated with a major histopathological response (sensitivity, 62.5%; specificity, 100%) and 15 of 24 patients with minor histopathological response to the delivered neoadjuvant radiochemotherapy could be unequivocally identified. This association of dichotomized relative ERCC1 mRNA expression and histopathological response was statistically significant (P < 0.001). Conclusions: Relative expression levels of ERCC1 mRNA determined by quantitative real-time reverse transcriptase-PCR appear highly specific to predict minor response to our neoadjuvant radiochemotherapy protocol in patients with locally advanced esophageal cancer and could be applied to prevent expensive, noneffective, and potentially harmful therapies in a substantial number (42%) of patients.

Response Evaluation by Endoscopy, Rebiopsy, and Endoscopic Ultrasound Does Not Accurately Predict Histopathologic Regression After Neoadjuvant Chemoradiation for Esophageal Cancer

Objective:To prospectively assess the sensitivity (sens), specificity (spec), positive predictive value (ppv), negative predictive value (npv), and accuracy (acc) for clinical response evaluation by endoscopy, rebiopsy, and endoscopic ultrasound (EUS) to determine histomorphologic regression UICC T-category downstaging after neoadjuvant chemoradiation for esophageal cancer. Background:Histomorphologic regression is meanwhile established as objective parameter for response and prognosis after neoadjuvant chemoradiation for esophageal cancer. Patients and Methods:Within a prospective observation trial, 80 patients with localized esophageal cancers (cT2-4,Nx,M0) received standardized neoadjuvant chemoradiation (cisplatin, 5-fluorouracil, 36 Gy) and were resected by transthoracic en bloc esophagectomy and two-field lymphadenectomy. Tumor regression was based on the percentage of vital residual tumor cells and classified in 4 categories as reported previously. Evaluation by endoscopy and EUS was performed based on WHO/UICC criteria before starting chemoradiation and before resection and rebiopsies were taken at the time of re-endoscopy. Results:Histomorphologic response was of significant (log rank) prognostic importance (P < 0.001), whereas clinical response evaluation by endoscopy (P = 0.1), rebiopsy (P = 0.34), and EUS (P = 0.35) was not. The results of the 3 diagnostic modalities to assess histomorphologic regression by endoscopy and rebiopsy UICC ypT-category downstaging for EUS are summarized: Endoscopy: sens 60%, spec 34%, ppv 49%, npv 44%, acc 47%. Rebiopsy: sens 36%, spec 100%, ppv 100%, npv 24%, acc 47%. EUS: sens 7%, spec 79%, ppv 18%, npv 57%, acc 50%. Conclusions:Histomorphologic regression is an objective response parameter of significant prognostic importance. The diagnostic accuracy of endoscopy, rebiopsy, and EUS is inadequate for objective response evaluation after neoadjuvant chemoradiation and can be omitted for this purpose in the clinical practice.

Molecular Biology of Esophageal Cancer

Squamous cell carcinoma (ESCC) is the most frequent histological subtype in esophageal cancer, although the incidence of esophageal adenocarcinoma (EAC) is increasing faster than any other malignancy in the western world. New developments in the understanding of molecular mechanisms in esophageal cancer comprise analysis of the genetic tumor profiles by CGH (comparative genomic hybridization), the detection of tumor suppressor gene inactivation, and the analysis of proto-oncogenes. Especially the inactivation of the p53 gene proved to be of particular importance for the development of esophageal cancer. Also p15 and p16 have been identified to be involved in the pathogenesis of esophageal cancer by influencing the cyclin kinase inhibitor cascade and DNA mismatch repair processes. Amplification of cyclin D1 results in growth advantage for tumor cells and enhances tumorigenesis; gene amplification and overexpression of cyclin D1 were frequently demonstrated especially in ESCC. Regarding the dysplasia-metaplasia-carcinoma sequence of Barrett’s esophagus, inhibition of apoptosis by overexpression of bcl-2 proteins occurs as an early event.

Altered levels of the onco-microRNA 21 and the tumor-supressor microRNAs 143 and 145 in advanced rectal cancer indicate successful neoadjuvant chemoradiotherapy

microRNAs (miRNAs) are small non-coding RNAs with important post-transcriptional regulatory functions. miRNA-21 (miR-21) is upregulated and miR-143 and miR-145 are downregulated in colorectal carcinoma. The aim of our study was to determine if these miRNAs change their expression levels in response to neoadjuvant chemoradiotherapy in advanced rectal cancer. Forty patients with advanced rectal cancer (clinical uT3/T4 Nx) were included. All patients underwent neoadjuvant chemoradiotherapy and surgical resection. Expression of miR-21, -143 and -145 was examined in macrodissected tumor tissue before and after chemoradiotherapy and normal rectal tissue from the resection specimen. RNA was extracted from formalin-fixed and paraffin-embedded tissue by TRIzol method, polyadenylated, reverse transcribed and analyzed by real-time PCR. Therapy response was assessed according to pathological tumor regression. miR-21 was more highly expressed in tumor tissue than in non-tumorous tissue. However, there was a moderately lower expression in post-therapeutic tumor tissue compared to pre-therapeutic tumor tissue. There was a significant upregulation of miR-143 and miR-145 in post-therapeutic tumor tissue compared to pre-therapeutic tumor tissue. According to the predictive and prognostic value of the analyzed miRNAs, a significant correlation between miR-145 expression and tumor regression was seen. Patients with a low intratumoral post-therapeutic expression had significantly more often a worse response to neoadjuvant therapy compared to patients with a high expression of miR145. The present results support the hypothesis that chemoradiotherapy can profoundly alter miRNA expression profiles. miRNAs might play important roles as molecular biomarkers in the prediction of response to treatment and prognosis.

Osteopontin but not osteonectin messenger RNA expression is a prognostic marker in curatively resected non-small cell lung cancer

Purpose: The purpose of this study was to better define the role of osteopontin (OPN) and osteonectin [also known as secreted protein acidic and rich in cysteine (SPARC)] in lung tumorigenesis by comparing the expressions of these genes in lung tumor tissue and matched normal tissue and by determining the prognostic significance of the gene expressions. Experimental Design: Quantitative real-time reverse transcription-PCR was used to analyze OPN and SPARC mRNA expression in normal lung tissue and matching tumor samples from 82 patients with non-small cell lung cancer. Gene expression data for each patient were matched to survival data. Results: The overall median mRNA expression level of OPN was about 20-fold higher in tumor tissues than in matching normal lung tissues (P < 0.001), whereas SPARC gene expression was not significantly different in both tissue types. Forty of 82 patients had high (≥4.1) intratumoral OPN expression, and 15 of 82 patients had high (≥15.5) SPARC expression. High OPN expression in the tumor tissue was associated with inferior survival (P = 0.014), whereas high SPARC expression showed a trend toward longer survival (P = 0.095). The impact of high OPN and low SPARC expression on patient survival was additive (P = 0.001). Conclusions: The large increase in OPN expression in tumors compared with normal tissue and its association with survival suggest a role for OPN in lung tumorigenesis.

A multigene expression panel for the molecular diagnosis of Barrett's esophagus and Barrett's adenocarcinoma of the esophagus.

In order to identify genes or combination of genes that have the power to discriminate between premalignant Barretts esophagus and Barretts associated adenocarcinoma, we analysed a panel of 23 genes using quantitative real-time RT–PCR (qRT–PCR, Taqman®) and bioinformatic tools. The genes chosen were either known to be associated with Barretts carcinogenesis or were filtered from a previous cDNA microarray study on Barretts adenocarcinoma. A total of 98 tissues, obtained from 19 patients with Barretts esophagus (BE group) and 20 patients with Barretts associated esophageal adenocarcinoma (EA group), were studied. Triplicate analysis for the full 23 gene of interest panel, and analysis of an internal control gene, was performed for all samples, for a total of more than 9016 single PCR reactions. We found distinct classes of gene expression patterns in the different types of tissues. The most informative genes clustered in six different classes and had significantly different expression levels in Barretts esophagus tissues compared to adenocarcinoma tissues. Linear discriminant analysis (LDA) distinguished four genetically different groups. The normal squamous esophagus tissues from patients with BE or EA were not distinguishable from one another, but Barretts esophagus tissues could be distinguished from adenocarcinoma tissues. Using the most informative genes, obtained from a logistic regression analysis, we were able to completely distinguish between benign Barretts and Barretts adenocarcinomas. This study provides the first non-array parallel mRNA quantitation analysis of a panel of genes in the Barretts esophagus model of multistage carcinogenesis. Our results suggest that mRNA expression quantitation of a panel of genes can discriminate between premalignant and malignant Barretts disease. Logistic regression and LDAs can be used to further identify, from the complete panel, gene subsets with the power to make these diagnostic distinctions. Expression analysis of a limited number of highly selected genes may have clinical usefulness for the treatment of patients with this disease.

High Cyclooxygenase-2 Expression Following Neoadjuvant Radiochemotherapy Is Associated with Minor Histopathologic Response and Poor Prognosis in Esophageal Cancer

Purpose: High expression of cyclooxygenase-2 (COX-2) was shown to inhibit chemotherapy- and radiotherapy-induced apoptosis. We analyzed the association of COX-2 mRNA and protein expression with histomorphologic response to neoadjuvant radiochemotherapy in esophageal cancer. Experimental Design: Fifty-two patients with resectable esophageal cancers (cT2-4, Nx, and M0) received neoadjuvant radiochemotherapy (cisplatin, 5-5-fluorouracil, 36 Gy) followed by transthoracic en bloc esophagectomy. Histomorphologic regression was defined as major response when resected specimens contained less than 10% of residual vital tumor cells. RNA was isolated from endoscopic biopsies (paired tumor and normal tissue) before neoadjuvant treatment and quantitative real-time reverse transcriptase-PCR (Taqman) assays were done to determine COX-2 mRNA expression levels standardized for β-actin. COX-2 protein expression in pretreatment biopsies and post-therapeutic resection specimens was analyzed by immunostaining of tumor cells. Results: Median COX-2 mRNA expression levels were significantly (P < 0.0001) different between paired tumor (median, 2.2) and normal tissues (median, 0.159). Comparison of pre-therapeutic and posttherapeutic specimens showed a significant difference (P < 0.006) in COX-2 protein expression. Twelve of 52 tumors showed down-regulation and 3 of 52 showed up-regulation of COX-2 protein expression during neoadjuvant radiochemotherapy. High COX-2 protein expression in post-therapeutic resection specimens was significantly associated with minor histopathologic response (P < 0.04) and poor prognosis (5-year survival probabilities: 26.3 ± 8.2% for minor and 58.6% ± 12.9% for major histopathologic response; P < 0.01). Conclusion: High COX-2 protein expression following neoadjuvant radiochemotherapy in resection specimens is significantly associated with minor histopathologic response to neoadjuvant therapy and very poor prognosis.