Sai Yendamuri

Evaluation of MicroRNA Expression Profiles That May Predict Recurrence of Localized Stage I Non–Small Cell Lung Cancer after Surgical Resection

Prognostic markers that can predict the relapse of localized non-small cell lung cancer (NSCLC) have yet to be defined. We surveyed expression profiles of microRNA (miRNA) in stage I NSCLC to identify patterns that might predict recurrence after surgical resection of this common deadly cancer. Small RNAs extracted from formalin-fixed and paraffin-embedded tissues were hybridized to locked nucleic acid probes against 752 human miRNAs (representing 82% of the miRNAs in the miRBase 13.0 database) to obtain expression profiles for 37 cases with recurrence and 40 cases without recurrence (with clinical follow-up for at least 32 months). Differential expression between the two case groups was detected for 49% of the miRNAs (Wilcoxon rank sum test; P<0.01). The performance of expression profiles at differentiating the two case groups was assessed by leave-one-out and Monte Carlo cross-validations. In leave-one-out cross-validation using support vector machines- or top-scoring gene pair classifier methods, which looked for six- or two-miRNA-based classifiers, the identified miRNA expression pattern predicted recurrence with an accuracy of 70% and 83%, and hazard ratio of 3.6 [95% confidence interval (95% CI), 1.8-7.1] and 9.0 (95% CI, 4.4-18.2), respectively. Mean accuracy in Monte Carlo cross-validation using 1,000 random 60-17 splits was 69% (95% CI, 68-70) and 72% (95% CI, 71-72), respectively. The specific miRNAs mir-200b*, mir-30c-1*, mir-510, mir-630, mir-657, and mir-146b-3p and mir-124*, mir-585, and mir-708, respectively, represented most commonly among the 1,000 classifiers identified in Monte Carlo cross-validation by the two methods. MiRNAs mir-488, mir-503, and mir-647 were identified as potential reference miRNAs for future studies, based on the stability of their expression patterns across the 77 cases and the two case-groups. Our findings reinforce efforts to profile miRNA expression patterns for better prognostication of stage I NSCLC.

WWOX gene restoration prevents lung cancer growth in vitro and in vivo

The WWOX (WW domain containing oxidoreductase) gene at the common fragile site, FRA16D, is altered in many types of cancer, including lung cancer. We have examined the tumor suppressor function of WWOX in preclinical lung cancer models. The WWOX gene was expressed in lung cancer cell lines through recombinant adenovirus (Ad) infection (Ad-WWOX), and through a drug [ponasterone A, (ponA)]-inducible system. After WWOX restoration in vitro, endogenous Wwox protein-negative cell lines (A549, H460, and H1299) underwent apoptosis through activation of the intrinsic apoptotic caspase cascade in A549 and H460 cells. Ectopic expression of Wwox caused dramatic suppression of tumorigenicity of A549, H460, and H1299 cells in nude mice after Ad-WWOX infection and after ponA induction of Wwox expression in H1299 lung cancer cells. Tumorigenicity and in vitro growth of U2020 (Wwox-positive) lung cancer cells was unaffected by Wwox overexpression. This study confirms that WWOX is a tumor suppressor gene and is highly effective in preventing growth of lung cancer xenografts, whether introduced through viral infection or by induction of a silent WWOX transgene.

The Tumor Suppressor Gene WWOX at FRA16D Is Involved in Pancreatic Carcinogenesis

Purpose: WWOX (WW domain containing oxidoreductase) is a tumor suppressor gene that maps to the common fragile site FRA16D. We showed previously that WWOX is frequently altered in human lung and esophageal cancers. The purpose of this study was to delineate more precisely the role of WWOX in pancreatic carcinogenesis. Experimental Design: We analyzed 15 paired pancreatic adenocarcinoma samples and 9 pancreatic cancer cell lines for WWOX alterations. Colony assay and cell cycle analysis were also performed to evaluate the role of the WWOX as a tumor suppressor gene. Results: Loss of heterozygosity at the WWOX locus was observed in 4 primary tumors (27%). Methylation analysis showed that site-specific promoter hypermethylation was detected in 2 cell lines (22%) and treatment with the demethylating agent 5-aza-2′-deoxycytidine demonstrated an increase in the expression of WWOX. In addition, 2 primary tumor samples (13%) showed promoter hypermethylation including the position of site-specific methylation. Transcripts missing WWOX exons were detected in 4 cell lines (44%) and in 2 tumor samples (13%). Real-time reverse transcription PCR revealed a significant reduction of WWOX expression in all of the cell lines and in 6 primary tumors (40%). Western blot analysis showed a significant reduction of the WWOX protein in all of the cell lines. Furthermore, transfection with WWOX inhibited colony formation of pancreatic cancer cell lines by triggering apoptosis. Conclusion: These results indicate that the WWOX gene may play an important role in pancreatic tumor development.

Outcomes of sarcomatoid carcinoma of the lung: a Surveillance, Epidemiology, and End Results Database analysis.

BACKGROUND Sarcomatoid lung carcinomas are unusual, and reports from small single institution case series suggest poor survival rates. We sought to study the clinical characteristics of this form of non-small cell lung cancer using the Surveillance Epidemiology, End Results database. METHODS The Surveillance, Epidemiology, and End Results database was queried for respiratory tract malignancies of sarcomatoid histology. The demographic information and oncologic characteristics of this population were examined. A propensity score-matched analysis of patients was performed to test the hypothesis that patients with sarcomatoid cancers undergoing lobectomies perform worse that those with other non-small cell lung cancers. RESULTS Of 878,810 patients with lung cancer, only 3,647 patients had a diagnosis of sarcomatoid cancer (0.4%). For the additional analyses of outcomes, only patients with lifetime incidence of a single cancer, known Surveillance, Epidemiology, and End Results historic stage and inpatient reporting were selected (n = 1,921). Demographics, tumor characteristics, and outcomes of these patients were described. Non-small cell lung cancer cohorts (with and without sarcomatoid cancer propensity-matched on age, gender, race, year of diagnosis, grade, and Surveillance, Epidemiology, and End Results historic stage) that underwent lobectomies or pneumonectomies were selected (n = 758). Univariate (hazard ratio, 1.60; 95% confidence interval, 1.31-1.97) and multivariate analysis (hazard ratio, 1.67; 95% confidence interval, 1.36-2.05) revealed a significantly worse overall survival for patients with sarcomatoid cancer compared to matched nonsarcomatoid lung cancer controls. CONCLUSION Sarcomatoid cancer is a rare form of lung malignancy with outcomes significantly worse than other forms of non-small cell lung cancer. Novel multimodality treatment strategies are necessary to improve outcomes of this disease.

MicroRNA Expression Profiles of Whole Blood in Lung Adenocarcinoma

The association of lung cancer with changes in microRNAs in plasma shown in multiple studies suggests a utility for circulating microRNA biomarkers in non-invasive detection of the disease. We examined if presence of lung cancer is reflected in whole blood microRNA expression as well, possibly because of a systemic response. Locked nucleic acid microarrays were used to quantify the global expression of microRNAs in whole blood of 22 patients with lung adenocarcinoma and 23 controls, ten of whom had a radiographically detected non-cancerous lung nodule and the other 13 were at high risk for developing lung cancer because of a smoking history of >20 pack-years. Cases and controls differed significantly for age with a mean difference of 10.7 years, but not for gender, race, smoking history, blood hemoglobin, platelet count, or white blood cell count. Of 1282 quantified human microRNAs, 395 (31%) were identified as expressed in the study’s subjects, with 96 (24%) differentially expressed between cases and controls. Classification analyses of microRNA expression data were performed using linear kernel support vector machines (SVM) and top-scoring pairs (TSP) methods, and classifiers to identify presence of lung adenocarcinoma were internally cross-validated. In leave-one-out cross-validation, the TSP classifiers had sensitivity and specificity of 91% and 100%, respectively. The values with SVM were both 91%. In a Monte Carlo cross-validation, average sensitivity and specificity values were 86% and 97%, respectively, with TSP, and 88% and 89%, respectively, with SVM. MicroRNAs miR-190b, miR-630, miR-942, and miR-1284 were the most frequent constituents of the classifiers generated during the analyses. These results suggest that whole blood microRNA expression profiles can be used to distinguish lung cancer cases from clinically relevant controls. Further studies are needed to validate this observation, including in non-adenocarcinomatous lung cancers, and to clarify upon the confounding effect of age.

Regression of upper gastric cancer in mice by FHIT gene delivery

Fhit expression is reduced in most cancers, and Fhit replacement by FHIT expression viruses in lung, esophageal, pancreatic, and cervical cancers induces apoptosis in the cancer cells. Mice carrying one or two inactivated Fhit alleles are hypersensitive to development of N‐nitrosomethylbenzylamine (NMBA)‐induced forestomach tumors. In the present study, we investigated the kinetics and mechanism of tumor reversal and intervention by oral delivery of FHIT expression viruses. Tumor analysis showed that: a) by 37 days post‐NMBA, control mice showed ∼7 tumors and by 84 days ∼10 tumors/forestomach; b) mice receiving FHIT virus at 2 or 42 days post‐NMBA showed significantly reduced tumor burdens; c) Fhit was still expressed at 82 days postinfection; d) control viral infection had no effect on tumor development; and e) reduced Bcl2, increased Bax expression, and increased TUNEL‐positive apoptotic nuclei characterized the restored epithelia of FHIT transduced forestomachs. Thus, FHIT viral gene delivery prevents or retards development of carcinogen‐induced forestomach tumors and reverses development of established tumors by 60–70% through an apoptotic pathway. This dramatic reduction in tumor burden emphasizes the efficacy of targeting the FHIT apoptotic pathway for tumor eradication.

Number of Lymph Nodes and Metastatic Lymph Node Ratio Are Associated With Survival in Lung Cancer

BACKGROUND The non-small cell lung cancer TNM classification system uses only the anatomic extent of lymph node (LN) metastases to define the N category. The number of LNs resected and the ratio of positive LNs to total examined LNs are prognostic in other solid tumors. We used the Surveillance, Epidemiology and End Results database to investigate the effect of these factors on the overall survival of non-small cell lung cancer. METHODS All patients with non-small cell lung cancer in the Surveillance, Epidemiology and End Results database from 1988 through 2007 who had curative resections and had at least one LN examined were included. The prognostic value of age, race, sex, tumor size, histologic grade, number of examined LNs, and ratio of positive LNs to total examined LNs was assessed using a multivariate Cox proportional hazards model for overall survival. The number of LNs examined was categorized into four levels. The percentage of positive LNs was stratified into three levels. RESULTS Among patients with localized disease, fewer LNs examined corresponded with a worse prognosis. Prognosis improved as more LNs were examined. For patients with regional disease, the differences were significant only at the extremes. Older patients, males, and those with higher grade or larger tumors did worse. Patients with low or moderate ratios of positive to total LNs had better prognoses than those with high ratios. CONCLUSIONS More LNs resected and lower ratios of positive LNs to total examined LNs are associated with better patient survival after non-small cell lung cancer resection independent of age, sex, grade, tumor size, and stage of disease.

Detection of microRNAs in dried serum blots

We observed the preservation of microRNAs in unrefrigerated dried serum blots. Preservation was not adversely affected by drying or storing at 37, 45, or 60°C instead of room temperature, but it was harmed when blots were dried incompletely before storage. Preservation of microRNAs in serum was not diminished if, instead of being kept frozen at -80°C, it was stored as dried blots at room temperature for 5 months or at 37°C for 4 weeks. Thus, dried blots can be a convenient and safer way to save, transport, and store serum for microRNA assays.

MicroRNAs and lung cancer: Biology and applications in diagnosis and prognosis.

MicroRNAs are tiny non-coding RNA molecules which play important roles in the epigenetic control of cellular processes by preventing the translation of proteins from messenger RNAs (mRNAs). A single microRNA can target different mRNAs, and an mRNA can be targeted by multiple microRNAs. Such complex interplays underlie many molecular pathways in cells, and specific roles for many microRNAs in physiological as well as pathological phenomena have been identified. Changes in expression of microRNAs have been associated with a wide variety of disease conditions, and microRNA-based biomarkers are being developed for the identification and monitoring of such states. This review provides a general overview of the current state of knowledge about the biology of microRNAs, and specific information about microRNAs with regard to the diagnosis and prognosis of lung cancer.

Is Sublobar Resection Sufficient for Carcinoid Tumors

BACKGROUND The existing guidelines for extent of resection of carcinoid tumors are based on other, more malignant non-small cell lung cancers. Because of the small number of patients in any single institution, we analyzed the Surveillance Epidemiology and End Results (SEER) database to study the effect of the extent of resection of these tumors on overall survival. METHODS All patients with lung cancer in the SEER database from 1973 to 2006 with carcinoid tumors as their only cancer were included. Variables examined included age, race (white, black, others), gender, histologic type (atypical versus typical carcinoid), stage (localized, regional, and distant), extent of resection (sublobar resection, lobectomy, or more extensive) and survival. Univariate analyses (Kaplan-Meier method) were used to select variables for multivariate analysis (Cox regression analysis). Associations were considered significant with an alpha error < 5%. In addition, propensity score-matched Cox regression analysis was performed for patients with typical carcinoid disease. RESULTS Most patients with carcinoid tumors did not acquire any other cancers (4,785/6,819; 70.2%). Of these, 797 patients had sublobar resection and 2,681 patients had lobectomy or more extensive resections. On univariate analysis, gender (p = 0.014), race (p < 0.001), stage (p < 0.001), histologic type (p < 0.001) and extent of resection (p = 0.04) were associated with overall survival. Multivariate analysis demonstrated that age, gender, race, stage, and histologic type remain statistically associated with overall survival and disease-specific survival, whereas extent of resection is not. Propensity score-matched analysis demonstrates that for typical carcinoid, extent of resection is not associated with overall survival when adjusted for age, gender, race, and stage. CONCLUSIONS Sublobar resection of carcinoid tumors did not compromise oncologic outcomes in a large population-based database. Lobectomy for typical carcinoid tumors is not mandatory as long as complete resection and adequate mediastinal staging are performed.