Paul M. Schneider

MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer.

Early-stage non-small cell lung cancer (NSCLC) can be cured by surgical resection, but a substantial fraction of patients ultimately dies due to distant metastasis. In this study, we used subtractive hybridization to identify gene expression differences in stage I NSCLC tumors that either did or did not metastasize in the course of disease. Individual clones (n=225) were sequenced and quantitative RT–PCR verified overexpression in metastasizing samples. Several of the identified genes (eIF4A1, thymosin β4 and a novel transcript named MALAT-1) were demonstrated to be significantly associated with metastasis in NSCLC patients (n=70). The genes’ association with metastasis was stage- and histology specific. The Kaplan–Meier analyses identified MALAT-1 and thymosin β4 as prognostic parameters for patient survival in stage I NSCLC. The novel MALAT-1 transcript is a noncoding RNA of more than 8000 nt expressed from chromosome 11q13. It is highly expressed in lung, pancreas and other healthy organs as well as in NSCLC. MALAT-1 expressed sequences are conserved across several species indicating its potentially important function. Taken together, these data contribute to the identification of early-stage NSCLC patients that are at high risk to develop metastasis. The identification of MALAT-1 emphasizes the potential role of noncoding RNAs in human cancer.

Neoadjuvant Chemotherapy Compared With Surgery Alone for Locally Advanced Cancer of the Stomach and Cardia: European Organisation for Research and Treatment of Cancer Randomized Trial 40954

PURPOSE Patients with locally advanced gastric cancer benefit from combined pre- and postoperative chemotherapy, although fewer than 50% could receive postoperative chemotherapy. We examined the value of purely preoperative chemotherapy in a phase III trial with strict preoperative staging and surgical resection guidelines. PATIENTS AND METHODS Patients with locally advanced adenocarcinoma of the stomach or esophagogastric junction (AEG II and III) were randomly assigned to preoperative chemotherapy followed by surgery or to surgery alone. To detect with 80% power an improvement in median survival from 17 months with surgery alone to 24 months with neoadjuvant, 282 events were required. RESULTS This trial was stopped for poor accrual after 144 patients were randomly assigned (72:72); 52.8% patients had tumors located in the proximal third of the stomach, including AEG type II and III. The International Union Against Cancer R0 resection rate was 81.9% after neoadjuvant chemotherapy as compared with 66.7% with surgery alone (P = .036). The surgery-only group had more lymph node metastases than the neoadjuvant group (76.5% v 61.4%; P = .018). Postoperative complications were more frequent in the neoadjuvant arm (27.1% v 16.2%; P = .09). After a median follow-up of 4.4 years and 67 deaths, a survival benefit could not be shown (hazard ratio, 0.84; 95% CI, 0.52 to 1.35; P = .466). CONCLUSION This trial showed a significantly increased R0 resection rate but failed to demonstrate a survival benefit. Possible explanations are low statistical power, a high rate of proximal gastric cancer including AEG and/or a better outcome than expected after radical surgery alone due to the high quality of surgery with resections of regional lymph nodes outside the perigastic area (celiac trunc, hepatic ligament, lymph node at a. lienalis; D2).

Histomorphologic Tumor Regression and Lymph Node Metastases Determine Prognosis Following Neoadjuvant Radiochemotherapy for Esophageal Cancer: Implications for Response Classification

Objective:We sought to quantitatively and objectively evaluate histomorphologic tumor regression and establish a relevant prognostic regression classification system for esophageal cancer patients receiving neoadjuvant radiochemotherapy. Patients and Methods:Eighty-five consecutive patients with localized esophageal cancers (cT2-4, Nx, M0) received standardized neoadjuvant radiochemotherapy (cisplatin, 5-fluorouracil, 36 Gy). Seventy-four (87%) patients were resected by transthoracic en bloc esophagectomy and 2-field lymphadenectomy. The entire tumor beds of the resected specimens were evaluated histomorphologically, and regression was categorized into grades I to IV based on the percentage of vital residual tumor cells (VRTCs). A major response was achieved when specimens contained either less than 10% VRTCs (grade III) or a pathologic complete remission (grade IV). Results:Complete resections (R0) were performed in 66 of 74 (89%) patients with 3-year survival rates of 54% ± 7.05% for R0-resected cases and 0% for patients with incomplete resections ortumor progression during neoadjuvant therapy (P < 0.01). Minor histopathologic response was present in 44 (59.5%) and major histopathologic response in 30 (40.5%) tumors. Significantly different 3-year survival rates (38.8% ± 8.1% for minor versus 70.7 ± 10.1% for major response) were observed. Univariate survival analysis identified histomorphologic tumor regression (P < 0.004) and lymph node category (P < 0.01) as significant prognostic factors. Pathologic T category (P < 0.08), histologic type (P = 0.15), or grading (P = 0.33) had no significant impact on survival. Cox regression analysis identified dichotomized regression grades (minor and major histomorphologic regression, P < 0.028) and lymph node status (ypN0 and ypN1, P < 0.036) as significant independent prognostic parameters. A 2-parameter regression classification system that includes histomorphologic regression (major versus minor) and nodal status (ypN0 versus ypN1) was established (P < 0.001). Conclusions:Histomorphologic tumor regression and lymph node status (ypN) were significant prognostic parameters for patients with complete resections (R0) following neoadjuvant radiochemotherapy for esophageal cancer. A regression classification based on 2 parameters could lead to improved objective evaluation of the effectiveness of treatment protocols, accuracy of staging and restaging modalities, and molecular response prediction.

S100 Family Members and Trypsinogens Are Predictors of Distant Metastasis and Survival in Early-Stage Non-Small Cell Lung Cancer

Distant metastasis is the predominant cause of death in early-stage non-small cell lung cancer (NSCLC). Currently, it is impossible to predict the occurrence of metastasis at early stages and thereby separate patients who could be cured by surgical resection alone from patients who would benefit from additional chemotherapy. In this study, we applied a comparative microarray approach to identify gene expression differences between early-stage NSCLC patients whose cancer ultimately did or did not metastasize during the course of their disease. Transcriptional profiling of 82 microarrays from two patient groups revealed differential expression of several gene families including known predictors of metastasis (e.g., matrix metalloproteinases). In addition, we found S100P, S100A2, trypsinogen C (TRY6), and trypsinogen IVb (PRSS3) to be overexpressed in tumors that metastasized during the course of the disease. In a third group of 42 patients, we confirmed the induction of S100 proteins and trypsinogens in metastasizing tumors and its significant correlation with survival by real-time quantitative reverse transcription-PCR. Overexpression of S100A2, S100P, or PRSS3 in NSCLC cell cultures led to increased transendothelial migration, corroborating the role of S100A2, S100P, and PRSS3 in the metastatic process. Taken together, we provide evidence that expression of S100 proteins and trypsinogens is associated with metastasis and predicts survival in early stages of NSCLC. For the first time, this implicates a role of S100 proteins and trypsinogens in the metastatic process of early-stage NSCLC.

Laparoscopic Ischemic Conditioning of the Stomach for Esophageal Replacement

Objectives:A considerable percentage of morbidity and mortality after esophagectomy and gastric pull-up is due to leakage of the esophagogastrostomy, which is mainly caused by ischemia of the gastric fundus. Previous clinical studies demonstrated that impaired microcirculation of the gastric conduit almost recovers within the first 5 postoperative days. Therefore, this study was designed to improve gastric perfusion by laparoscopic ischemic conditioning of the stomach. Methods:The study group consisted of 83 patients with 44 esophageal adenocarcinomas and 39 squamous cell carcinomas. A total of 51% received neoadjuvant radiochemotherapy. First, all patients underwent laparoscopic mobilization of the stomach including the cardia and preparation of the gastric conduit. After a mean delay of 4.3 days (range, 3–7 days), a conventional right-sided transthoracic en bloc esophagectomy was performed. Reconstruction was done by gastric pull-up and high intrathoracic esophagogastrostomy. Results:Three conversions (3.6%) to open surgery were necessary during laparoscopic mobilization of the stomach. The reoperation rate was 2.4% (one relaparoscopy for control of a bleeding of the stapler line, one rethoracotomy for chylothorax). Two patients showed circumscribed necroses of the upper part of the fundus after gastric pull-up into the chest. These necroses were resected for reconstruction by esophagogastrostomy. Five patients (6.0%) developed small anastomotic leakages with minor clinical symptoms; however, the gastric conduits were well vascularized. All leakages healed after endoscopic stenting. Major postoperative complications were observed in 13.3% of the patients and the 90-day mortality was 0%. Conclusion:Laparoscopic ischemic conditioning of the gastric conduit is feasible and safe and may contribute to the reduction of postoperative morbidity and mortality after esophagectomy and gastric pull-up.

MUC1 and Nuclear β-Catenin Are Coexpressed at the Invasion Front of Colorectal Carcinomas and Are Both Correlated with Tumor Prognosis

Purpose: Overexpression of MUC1 and cytosolic interaction of the mucin with β-catenin are claimed to be involved in colorectal carcinogenesis. In vitro data published recently suggest that MUC1 overexpression results in an increase of steady state levels of nuclear β-catenin. We tried to elucidate the coexpression of both molecules in colorectal cancer to demonstrate possible correlations with clinical, pathological, and prognostic data. Experimental Design: An immunohistochemical double staining study was performed to characterize the expression and subcellular distribution of MUC1 and β-catenin in a series of 205 patients with colorectal carcinoma. The results were correlated with clinicopathological variables as well as overall survival. Results: MUC1 was strongly expressed in the tumor center and at the invasion front in ∼50% of the cases. Similar results were obtained with regard to nuclear accumulation of β-catenin at the invasive tumor parts. MUC1 protein expression in the tumor center correlated significantly with a low grade of differentiation, and nuclear β-catenin in the tumor periphery was more frequent in carcinomas of the left colon and rectum. Overexpression of MUC1 and β-catenin, as well as their nuclear coexpression at the invasion front correlated with a worse overall survival in an univariate analysis. However, only pathological tumor-node-metastasis staging and MUC1 at the invasion front revealed as independent prognostic factors. Conclusions: These results suggest that MUC1 and β-catenin are coexpressed at the invasion front of colorectal carcinomas and that this feature is associated with an accelerated course of disease and worse prognosis.

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.

Mutations of p53 in barrett's esophagus and barrett's cancer: A prospective study of ninety-eight cases

We had previously identified p53 mutations in Barretts esophagus and therefore began a multiinstitutional study to determine their significance as a marker for malignancy. Ninety-eight patients from four institutions were studied. Forty-eight patients (37 men and 11 women, mean age 56.2 years) had Barretts esophagus with metaplasia or dysplasia but no evidence of malignancy at a mean follow-up of 2.2 years. Barretts esophagus was classified as metaplasia with no evidence of dysplasia in 32 patients, as low-grade dysplasia in 13, and as high-grade dysplasia in three. The other 50 patients (46 men and four women, mean age 60.2 years) had adenocarcinoma arising in Barretts esophagus. Tissues from normal stomach or esophagus, tumor, and Barretts esophagus were obtained for deoxyribonucleic acid analysis by endoscopic biopsy from patients with Barretts esophagus or cancer or during operations on some patients with Barretts cancer. Exons 5 through 9 of the p53 gene were studied for mutations by single-strand conformational polymorphism analysis after polymerase chain reaction amplification. Mutations detected by single-strand conformational polymorphism analysis were confirmed by deoxyribonucleic acid sequencing. None of the tissue samples from patients with Barretts esophagus alone and no dysplasia or low-grade dysplasia had any p53 mutations, but one of the three patients with high-grade dysplasia and no evidence of invasive malignancy did have a p53 mutation. Of the 50 patients with Barretts cancer, however, 23 (46%) had p53 mutations in Barretts epithelium, tumors, or both. Twenty of these patients had p53 mutations in the tumor only (n = 16) or in both tumor and Barretts epithelium (n = 4), suggesting that the mutation plays a direct role in carcinogenesis. Mutations in Barretts epithelium were found in one patient in the group without malignancy and in seven patients with cancer (one with no dysplasia, two with low-grade dysplasia, and five with high-grade dysplasia). In three patients with cancer, mutations occurred only in Barretts epithelium, suggesting that such mutations may also be a marker for genomic instability. Mutations were predominantly found in exons 5, 7, and 8, and transitions from guanine to adenine were the most frequent changes. Mutations of p53 are clearly involved in the pathogenesis of Barretts cancer for a subset of patients (46%), and the fact that we could detect mutations in premalignant Barretts epithelium supports the hypothesis that p53 mutations may be a useful marker for patients at increased risk for development of invasive cancer.