Frederike C. Ling

Regulation of the multidrug resistance transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species

Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia‐inducible factor‐1 (HIF‐1) and the multidrug resistance transporter P‐glycoprotein (P‐gp) has not been investigated. Herein, we demonstrate that with increasing size of DU‐145 prostate multicellular tumor spheroids the pericellular oxygen pressure and the generation of reactive oxygen species decreased, whereas the α‐subunit of HIF‐1 (HIF‐1α) and P‐gp were up‐regulated. Furthermore, P‐gp was up‐regulated under experimental physiological hypoxia and chemical hypoxia induced by either cobalt chloride or desferrioxamine. The pro‐oxidants H2O2 and buthionine sulfoximine down‐regulated HIF‐1α and P‐gp, whereas up‐regulation was achieved with the radical scavengers dehydroascorbate, N‐acetylcysteine, and vitamin E. The correlation of HIF‐1α and P‐gp expression was validated by the use of hepatoma tumor spheroids that were either wild type (Hepa1) or mutant (Hepa1C4) for aryl hydrocarbon receptor nuclear translocator (ARNT), i.e., HIF‐1β. Chemical hypoxia robustly increased HIF‐1α as well as P‐gp expression in Hepa1 tumor spheroids, whereas no changes were observed in Hepa1C4 spheroids. Hence, our data demonstrate that expression of P‐gp in multicellular tumor spheroids is under the control of HIF‐1.

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.

ERCC1 RNA expression in peripheral blood predicts minor histopathological response to neoadjuvant radio-chemotherapy in patients with locally advanced cancer of the esophagus.

ObjectiveThe aim of this study was to determine the gene is spelled excision repair cross-complementing gene 1 (ERCC1) RNA-expression in peripheral blood as a non-invasive molecular predictor of response to neoadjuvant radio-chemotherapy in patients with locally advanced cancer of the esophagus.BackgroundOnly patients with locally advanced cancer of the esophagus with a major histopathological response to neoadjuvant radio-chemotherapy benefit from this treatment. No non-invasive molecular marker exists that can reliably predict response to neoadjuvant therapy in this disease. To improve the treatment of patients with cancer of the esophagus, molecular predictors of response are desperately needed.MethodsBlood samples were drawn from 29 patients with esophageal cancer prior to neoadjuvant radio-chemotherapy. After extraction of cellular tumor-RNA from blood samples, quantitative expression analysis of ERCC1 was done by real-time reverse transcription polymerase chain reaction.ResultsNineteen (65.5%) patients showed a minor and ten (34.5%) a major histopathological response to neoadjuvant therapy. ERCC1 expression in blood of patients was detectable in 82.8%. The median ERCC1 expression was 0.62 (minimum 0.00, maximum 2.48) in minor responders and 0.24 (minimum 0.00, maximum 0.45) in major responders (p = 0.004). No significant associations were detectable between ERCC1 levels and patients’ clinical variables. Relative ERCC1 levels above 0.452 were not associated with major histopathological response (sensitivity, 68.4; specificity, 100%), and 13 of 19 patients with minor response could be unequivocally identified.ConclusionMinor responders to the applied therapy show a significant higher ERCC1 expression level in their blood compared to major responders. ERCC1 appears to be a highly specific non-invasive predictor of response to neoadjuvant therapy in esophageal cancer.

Leukocyte Depletion in Allogeneic Blood Transfusion Does Not Change the Negative Influence on Survival Following Transthoracic Resection for Esophageal Cancer

BackgroundPerioperative transfusion of allogeneic blood has been hypothesized to have an immunomodulatory effect and influence survival in several cancer types. This study evaluates the association between receipt of leucocyte-depleted and non-depleted allogeneic blood and survival following esophagectomy for cancer.MethodsA retrospective analysis was performed including 291 patients with esophageal cancers who underwent transthoracic en bloc esophagectomy and extended mediastinal lymphadenectomy. Neoadjuvant chemoradiation was administered in 152 (52.2%) patients. Perioperative blood transfusions were quantified and the potential prognostic cutoff for transfused units was calculated according to LeBlanc.ResultsThe median number of perioperative blood transfusions was 2 (0–24), and 106 patients (36.4%) received no transfusions. Patients with one or less blood transfusion showed a significantly improved survival compared to patients receiving more than one unit (p < 0.009). In multivariate analysis, blood transfusion categories showed significance (p < 0.015) next to pT, pN, pM category, and residual tumor categories (R-categories). Separate analysis of 183 patients treated after the mandatory introduction of leukocyte-depleted blood transfusions detected a strong tendency, but no significant difference in survival for patients getting one or less or more than one transfusion (p = 0.056). Receipt of leukocyte-depleted versus non-depleted units, however, had no influence on survival (p = 0.766).ConclusionsThe need for perioperative allogeneic blood transfusions is significantly associated with poorer survival following resection for esophageal cancer by univariate and multivariate analysis. Our data suggest that the reduction of leukocytes in allogeneic transfusions is not sufficient to overcome the negative influence on survival.

Roles of thymidylate synthase and dihydropyrimidine dehydrogenase expression in blood as predictors of response to multimodal therapy in esophageal cancer

BACKGROUND Thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) RNA expression in peripheral blood was examined as a noninvasive molecular predictor of response to neoadjuvant radiochemotherapy in patients with locally advanced cancer of the esophagus. METHODS Blood samples were drawn from 29 patients with esophageal cancer (10 squamous cell carcinomas and 19 adenocarciomas) before neoadjuvant radiochemotherapy. After extraction of cellular tumor RNA from blood samples, quantitative expression analysis of TS and DPD was performed with quantitative real-time reverse-transcription polymerase chain reaction. RESULTS Twenty of 29 (68%) of patients had a minor histopathologic response, and 9 of 29 (32%) had a major response to neadjuvant radiochemotherapy. RNA expression in the blood of patients was detectable for TS in 86%, for DPD in 97%, and in 100% for β-actin. No significant associations were detected between TS and DPD expression levels and clinical variables of the patients. A high expression level for TS was associated with a minor response to neoadjuvant treatment (P = .046), while there was no significant association between DPD and response to therapy. Combined analysis of TS and DPD expression increased the specificity for the prediction of response to 100%. No major responder to therapy had high expression levels for both genes in their peripheral blood. CONCLUSION Quantitation of TS and DPD in peripheral blood may be a highly specific analysis to identify a subset of patients who do not respond to neoadjuvant radiochemotherapy and may therefore prevent expensive, noneffective, and potentially harmful therapies in a substantial number of patients with esophageal cancer.

The value of survivin gene expression in peripheral blood as a non-invasive prognostic factor in patients with esophageal cancer

Background: Although circulating tumor DNA has been detected in patients with different types of cancer, less is known about free RNA in cancer patients. Therefore, we evaluated if mRNA expression of survivin, an inhibitor of apoptosis, can be used to detect circulating tumor cells in peripheral blood of patients with esophageal cancer. Furthermore, we assessed the influence of complete surgical resection and the association of survivin levels in peripheral blood with prognosis. Methods: Sixty-two patients (53 men, 9 women; median age 60 years) with esophageal cancer (adenocarcinoma: n=37; squamous cell cancer: n=25) scheduled for surgical resection were included. A neoadjuvant radiochemotherapy was performed in 24 (39%) patients. Whole blood was drawn one day pre- and 10 days postoperatively from all study patients. After extraction of cellular tumor-RNA from blood samples, quantitative expression analysis of survivin was done by real-time reverse transcription polymerase chain reaction. Results: Survivin expression in blood was detectable in 81% of the study patients. The pre- and postoperative mRNA amount of survivin did not differ between patients with adenocarcinoma and squamous cell cancer (p=0.26). Postoperative survivin expression levels were significantly lower than preoperative levels (p=0.02). With a median overall survival of 29.4 months, patients with a preoperative survivin expression above the 60th percentile had a significant shorter overall survival than patients with a reduced expression profile (p=0.03; 23 vs. 38 months). Conclusions: Direct quantification of survivin mRNA expression in peripheral blood of patients with esophageal cancer is technically feasible and is significantly reduced by complete surgical resection. Moreover, survivin gene expression in blood might become a non-invasive prognostic molecular marker in patients with esophageal cancer.

Wertigkeit der ERCC1-RNA Expression im Serum als Responsemarker auf die neoadjuvante Radio-Chemotherapie beim Ösophaguscarcinom

Background: Only patients with locally advanced cancer of the esophagus with a major histopathological response to neadjuvant radio-chemotherapy benefit from this treatment. Unfortunately, no non-invasive molecular marker exists that can reliably predict response or non-response to neadjuvant therapy in this disease. To further improve the treatment of patients with locally advanced cancer of the esophagus, molecular predictors of response are desperately needed. The aim of this study was to determine the value of ERCC1 RNA-Expression in sera of patients with locally advanced cancer of the esophagus as a non-invasive molecular predictor of response to neoadjuvant radio-chemotherapy. Material and methods: A total of 29 patients with locally advanced cancer (cT3-T4, Nx, M0) of the esophagus were included in this study. Blood samples from each patient were drawn prior to neoadjuvant radio-chemotherapy (cis-Platin, 5-FU, 36Gy). Transthoracic en-bloc esophagectomy was performed in all patients following completion of neadjuvant therapy. After extraction of cellular tumor-RNA from sera samples, quantitative expression analysis of ERCC1 and the internal reference gene beta-Actin was done by real-time RT-PCR (Taqman®). Histomorphological regression was defined as major response when resected specimen contained 10 % of vital residual tumor cells. Results: Nineteen of 29 (65.5 %) of patients showed a minor histopathological response and 10 of 29 (34.5 %) showed a major-response to neadjuvant radio-chemotherapy. ERCC1 RNA expression in sera of patients was detectable for ERCC1 in 82.8 % (24 of 29) and 100 % (29 of 29) for beta-Actin. The median ERCC1 expression was 0.62 (min.: 0.00, max.: 2.48) in minor-responders and 0.24 (min.: 0.00, max.: 0.45) in major-responders and was significantly different (p = 0.004 Mann-Whitney test). No significant associations were detected between ERCC1 expression levels and patients clinical variables (histology, tumor stage, gender etc.). Relative ERCC1 expression levels above 0.452 were not associated with major histopathological response (sensitivity: 68.4; specificity: 100 %) and 13 of 19 patients with minor histopathological response to the delivered neadjuvant therapy could be unequivocally identified. This association of dichotomized relative ERCC1 expression and histopathological response was statistically significant (P < 0.001). Conclusion: The applied method is technically feasible for the analysis of cellular ERCC1 RNA expression in sera of patients with locally advanced cancer of the esophagus. Minor-responders to the applied radiochemotherapy show a significant higher ERCC1 expression level in their sera compared to major-responders prior to therapy. ERCC1 expression levels in sera appear to be highly specific to predict minor-response to neoadjuvant radiochemotherapy in patients with locally advanced esophageal cancer and could be applied to prevent expensive, noneffective, and potentially harmful therapies in a substantial number (45 %) of patients.

948 The Role of ERCC1 RNA Expression in Blood As a Non-Invasive Predictor of Response to Neadjuvant Radio-Chemotherapy in Patients with Locally Advanced Cancer of the Esophagus

Background: Only patients with locally advanced cancer of the esophagus with a major histopathological response to neadjuvant radio-chemotherapy benefit from this treatment. Unfortunately, no non-invasive molecular marker exists that can reliably predict response or non-response to neadjuvant therapy in this disease. To further improve the treatment of patients with locally advanced cancer of the esophagus, molecular predictors of response are desperately needed. The aim of this study was to determine the value of ERCC1 RNA-Expression in sera of patients with locally advanced cancer of the esophagus as a non-invasive molecular predictor of response to neoadjuvant radio-chemotherapy. Material and methods: A total of 29 patients with locally advanced cancer (cT3-T4, Nx, M0) of the esophagus were included in this study. Blood samples from each patient were drawn prior to neoadjuvant radio-chemotherapy (cis-Platin, 5-FU, 36Gy). Transthoracic en-bloc esophagectomy was performed in all patients following completion of neadjuvant therapy. After extraction of cellular tumorRNA from sera samples, quantitative expression analysis of ERCC1 and the internal reference gene beta-Actin was done by real-time RT-PCR (Taqman©). Histomorphological regression was defined as major response when resected specimen contained 10 % of vital residual tumor cells. Results: Nineteen of 29 (65.5 %) of patients showed a minor histopathological response and 10 of 29 (34.5 %) showed a major-response to neadjuvant radio-chemotherapy. ERCC1 RNA expression in sera of patients was detectable for ERCC1 in 82.8 % (24 of 29) and 100 % (29 of 29) for beta-Actin. The median ERCC1 expression was 0.62 (min.: 0.00, max.: 2.48) in minor-responders and 0.24 (min.: 0.00, max.: 0.45) in major-responders and was significantly different (p = 0.004 Mann-Whitney test). No significant associations were detected between ERCC1 expression levels and patients clinical variables (histology, tumor stage, gender etc.). Relative ERCC1 expression levels above 0.452 were not associated with major histopathological response (sensitivity: 68.4; specificity: 100 %) and 13 of 19 patients with minor histopathological response to the delivered neadjuvant therapy could be unequivocally identified. This association of dichotomized relative ERCC1 expression and histopathological response was statistically significant (P < 0.001). Conclusion: The applied method is technically feasible for the analysis of cellular ERCC1 RNA expression in sera of patients with locally advanced cancer of the esophagus. Minor-responders to the applied radiochemotherapy show a significant higher ERCC1 expression level in their sera compared to major-responders prior to therapy. ERCC1 expression levels in sera appear to be highly specific to predict minor-response to neoadjuvant radiochemotherapy in patients with locally advanced esophageal cancer and could be applied to prevent expensive, noneffective, and potentially harmful therapies in a substantial number (45 %) of patients. 92 VI. Molekulare Onkologie: Pradiktion und Therapie

Die ansteigende Cyclooxygenase-2 Expression in der Metaplasie-Dysplasie-Karzinom Sequenz beim Barrett-Karzinom ist nicht assoziiert mit der inflammatorischen Umgebungsreaktion

Background: Epidemiological data assume a reduction of risk for developing an adenocarcinoma of the esophagus for individuals taking NSAIDs. One of the inhibited enzymes, cyclooxygenase-2, is supposed to be of major influence in the pathogenesis of Barrett’s cancer. We examined a possible association between COX-2 protein expression and the progressive severity within Barrett’s metaplasia-dysplasia-carcinoma sequence and the type and extent of the corresponding inflammatory reaction. Methods: COX-2 protein expression was examined in precurser lesions (metaplasia, low-grade-and high-grade-dysplasia) as well as tumor tissue of 49 resection specimens. Thereby interindividual variability was minimized. Immunohistochemical staining was graded on a scale 0–3 based on the percentage of specific tumor cell staining. Active and chronic inflammatory reaction was scored on a scale 0–3 as defined by the Updated Sydney Classification. Results: COX-2 expression is significant lower in squamous epithelium than in Barrett’s metaplasia (p < 0.001). Within Barrett’s metaplasia-dysplasia-carcinoma sequence a significant progressive increase in protein expression was noticed (Friedman test: p < 0.0001). Individual statistical testing detected the most significant differences between squamous epithelium and Barrett’s metaplasia (Wilcoxon test: p < 0.001) as well as between low-grade and high-grade dysplasia (Wilcoxon test: p < 0.0001). No association was present between COX-2 expression and pT-categories or grading of the tumor. Active and chronic inflammation were significantly different between squamous epithelium and Barrett’s metaplasia (Wilcoxon test: p < 0.0001) but not between Barrett’s metaplasia and carcinoma. Conclusions: There is a significant association between COX-2 protein expression and progression of the Barrett’s metaplasia-dysplasia-carcinoma sequence not explicable through changes in kind or intensity of the inflammatory reaction of the environment. Therefore, selective COX-2 inhibitors might be used as chemoprevention strategy independently of the inflammatory reaction.