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 ß-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.

Evaluation of quality indicators following implementation of total mesorectal excision in primarily resected rectal cancer changed future management.

BACKGROUND AND AIMS: We evaluated the outcome of primarily resected rectal cancer patients immediately after the implementation of total meserectal excision (TME) based on potential quality indicators. PATIENTS AND METHODS: Following initial teaching of two staff surgeons (PMS and AHH) by RJ Heald, 164 consecutive patients were analyzed. The following quality indicators were evaluated: (a) frequency of local recurrence, (b) number of resected lymph nodes, (c) selection of operative technique depending on tumor localization, (d) use of a protective loop ileostomy, and (e) frequency and type of adjuvant therapy. RESULTS: Local recurrence rate was 8.5% after a minimum follow-up of 5 years. An increasing pT category (p < 0.02) and the presence of lymph node metastases (pN+, p < 0.05) were significantly associated with local recurrence rates. The number of resected lymph nodes was significantly associated with nodal metastases rate (p < 0.02). Patients with distal third rectal cancer underwent significantly more often an abdominoperineal amputation (p < 0.0001). Clinical course, but not the rate of anastomotic leakage (9.5%) itself was influenced by using a protective loop ileostomy. Forty-two (29.7%) patients received adjuvant therapy; however, local recurrence rate was higher in patients with adjuvant chemo-/radiotherapy (14.2% vs. 6.1%). CONCLUSIONS: The local recurrence rate of 8.5% demonstrates that through consequent implementation of TME excellent onclogical results can be achieved. The number of resected lymph nodes significantly influenced the pN category. The primary construction of a protective loop ileostomy after TME became standard. Neoadjuvant chemoradiation was systematically introduced in order to improve local tumor control and prevent abdominoperineal amputations. No conclusions can be drawn concerning adjuvant therapy.

Increased platelet counts after transthoracic en bloc resection for esophageal cancer is associated with significantly improved survival.

BACKGROUND: We analyzed perioperative platelet counts as a potential clinical marker for survival after transthoracic en bloc resection for esophageal cancer. Recent data described preoperative thrombocytosis in malignancies to be associated with poor prognosis. METHODS: A retrospective analysis from a prospective database (1997-2006) was performed for 291 consecutive patients with esophageal cancer who underwent transthoracic en bloc esophagectomy and extended lymphadenectomy. Squamous cell cancer was found in 47.0% and adenocarcinoma in 50.9% (2.1% had rare histologies). Neoadjuvant chemoradiation was performed in 152 (52%) patients. Platelet counts before surgery and on postoperative days (PODs) 1, 10, and 30 were evaluated. We used the published cutoff value of 293 × 10(9)/l (mean of 80 healthy controls ± standard deviation) for platelet counts. RESULTS: High platelet counts before surgery missed significance for poorer survival (p = 0.054). Following a perioperative fall in thrombocytes, a significant rise at POD 10 after surgery was evident. Platelet counts of more than 293 × 10(9)/l at this time correlated with a significantly improved survival rate (p = 0.027). Patients with no increase in thrombocytes until POD 10 had significantly poorer survival (p = 0.012). Multivariate analysis confirmed that a thrombocyte increase between the preoperative count and that on POD 10 is an independent prognostic indicator (p = 0.035) for patients with completely (R0) resected tumors. CONCLUSIONS: An increase in platelet counts measured on POD 10 following transthoracic en bloc esophagectomy and extended lymphadenectomy is an independent prognostic indicator for improved survival in patients with esophageal cancer.

The role of the homeobox genes BFT and CDX2 in the pathogenesis of non-small cell lung cancer.

BACKGROUND: The role of the homeobox genes Backfoot (BFT) and caudal-related Homeobox 2 (CDX2) in the pathogenesis of non-small cell lung cancer (NSCLC) is unclear. The goal of this study was to investigate the mRNA expression of BFT and CDX2 in NSCLC and to determine the association with the pathogenesis and the potential as a biomarker of this disease. MATERIALS AND METHODS: The mRNA expression of BFT and CDX2 was analyzed by quantitative real-time RT-PCR in the tumor and matching normal tissue from 23 patients with NSCLC. RESULTS: The mRNA expression was detectable with the following frequencies in the tumor (t) and normal (n) tissues: BFT=100% (n), 100% (t); CDX2=100% (n), 100% (t). The median CDX2 mRNA expression was 0.85 (range: 0.01-15.47) in the tumor tissue and 0.045 (range: 0-1.36) in the matching normal lung tissue (p=0.001). The median BFT mRNA expression was 0.0034 (range: 0-0.35) in the tumor tissue and 0.0001 (range: 0-0.10) in the matching normal lung tissue (p=n.s.). There were no associations between the mRNA expression levels of BFT and CDX2 and clinicopathological variables. CONCLUSION: The mRNA expression of the homeobox genes is detectable at a high frequency in the tumor and normal tissue of patients with non-small cell lung cancer. Up-regulation of CDX2 mRNA expression appears to be associated with the pathogenesis of this malignant disease. The quantification of CDX2 and BFT mRNA expression in lung tissue is a potential biomarker for the identification of patients at risk of the development of NSCLC.

Death-associated protein kinase (DAPK) promoter methylation and response to neoadjuvant radiochemotherapy in esophageal cancer.

BACKGROUND: Multiple studies have shown that promoter methylation of tumor suppressor genes underlies esophageal carcinogenesis. Hypothetically, methylation resulting in tumor suppressor gene inactivation might result in tumors that are unresponsive to chemotherapy and radiation. Accordingly, our aim was to investigate if aberrant methylation of the apoptosis-related gene Death-Associated Protein Kinase (DAPK) could be used as a predictor of response to neoadjuvant therapy in locally advanced cancer of the esophagus. METHODS: Tumor and normal esophageal tissues were obtained from 50 patients with locally advanced cancer of the esophagus prior to neoadjuvant radiochemotherapy. DAPK methylation analysis was performed on all samples by methylation-specific real-time polymerase chain reaction (PCR). RESULTS: Seventeen (34%) patients showed a major and 33 (66%) a minor histomorphological response to neoadjuvant therapy. DAPK methylation was detectable in normal esophageal tissues with a frequency of 10% and in tumor tissue with a frequency of 78%. The median methylation level for DAPK was 2.7 x 10(-3) in tumor compared with 0.1 x 10(-3) in normal tissues (p < 0.001). DAPK methylation was not associated with response to neoadjuvant therapy or prognosis after esophagectomy. CONCLUSION: Aberrant DAPK methylation in tumor tissues is significantly higher compared with matching normal esophageal tissues, suggesting a fundamental role of this epigenetic alteration in the pathogenesis of this disease. The level of DAPK methylation in pretreatment biopsies of patients with locally advanced cancer of the esophagus is no marker for the prediction of histomorphological regression or prognosis following neoadjuvant chemoradiation in this disease.

Leukocyte depletion in allogeneic blood transfusion does not change the negative influence on survival following transthoracic resection for esophageal cancer.

BACKGROUND: Perioperative 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. METHODS: A 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. RESULTS: The 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). CONCLUSIONS: The 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.

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

OBJECTIVE: The 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. BACKGROUND: Only 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. METHODS: Blood 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. RESULTS: Nineteen (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. CONCLUSION: Minor 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.

HIF-1alpha mRNA is not associated with histopathological regression following neoadjuvant chemoradiation in esophageal cancer.

BACKGROUND: Hypoxia-inducible factor-1alpha (HIF-1alpha) expression was reported to be associated with tumor growth, progression and resistance to radio-/chemotherapy. Whether HIF-1alpha mRNA or protein expression is associated with histomorphological response or prognosis following neoadjuvant chemoradiation and surgery in resectable, locally-advanced esophageal cancer was analyzed. PATIENTS AND METHODS: Fifty-three patients received neoadjuvant chemoradiation (cisplatin, 5-fluorouracil, 36 Gy) followed by transthoracic en bloc esophagectomy. HIF-1alpha mRNA and protein expressions were analyzed by quantitative RT-PCR and immunostaining. RESULTS: In squamous cell carcinoma, HIF-1alpha mRNA expression was significantly higher than in paired normal epithelium (p < 0.001). Normal squamous epithelium showed significant elevated expression in adenocarcinomas, suggesting a field effect (p < 0.04). HIF-1alpha protein expression showed a significant regulation following chemoradiation. Neither HIF-1alpha mRNA nor protein expression was associated with histomorphological regression or prognosis. CONCLUSION: HIF-1alpha mRNA expression is differentially upregulated in esophageal squamous cell carcinoma compared to adenocarcinomas, but does not predict tumor regression or prognosis.

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 beta-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.

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 alpha-subunit of HIF-1 (HIF-1alpha) 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-1alpha and P-gp, whereas up-regulation was achieved with the radical scavengers dehydroascorbate, N-acetylcysteine, and vitamin E. The correlation of HIF-1alpha 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-1beta. Chemical hypoxia robustly increased HIF-1alpha 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.