The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round‐table discussions were held to provide an evidence‐based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well‐controlled experiments.

Background: Gefitinib is a selective inhibitor of the epidermal growth factor (EGFR) tyrosine kinase, which is overexpressed in many cancers, including non–small-cell lung cancer (NSCLC). We carried out a clinical study to compare the relationship between EGFR gene copy number, EGFR protein expression, EGFR mutations, and Akt activation status as predictive markers for gefitinib therapy in advanced NSCLC. Methods: Tumors from 102 NSCLC patients treated daily with 250 mg of gefitinib were evaluated for EGFR status by fluorescence in situ hybridization (FISH), DNA sequencing, and immunohistochemistry and for Akt activation status (phospho-Akt [P-Akt]) by immunohistochemistry. Time to progression, overall survival, and 95% confidence intervals (CIs) were calculated and evaluated by the Kaplan–Meier method; groups were compared using the log-rank test. Risk factors associated with survival were evaluated using Cox proportional hazards regression modeling and multivariable analysis. All statistical tests were two-sided. Results: Amplification or high polysomy of the EGFR gene (seen in 33 of 102 patients) and high protein expression (seen in 58 of 98 patients) were statistically significantly associated with better response (36% versus 3%, mean difference = 34%, 95% CI = 16.6 to 50.3; P <.001), disease control rate (67% versus 26%, mean difference = 40.6%, 95% CI = 21.5 to 59.7; P <.001), time to progression (9.0 versus 2.5 months, mean difference = 6.5 months, 95% CI = 2.8 to 10.3; P <.001), and survival (18.7 versus 7.0 months, mean difference = 11.7 months, 95% CI = 2.1 to 21.4; P = .03). EGFR mutations (seen in 15 of 89 patients) were also statistically significantly related to response and time to progression, but the association with survival was not statistically significant, and 40% of the patients with mutation had progressive disease. In multivariable analysis, only high EGFR gene copy number remained statistically significantly associated with better survival (hazard ratio = 0.44, 95% CI = 0.23 to 0.82). Independent of EGFR assessment method, EGFR ⁺ /P-Akt ⁺ patients had a statistically significantly better outcome than EGFR ⁻ , P-Akt ⁻ , or EGFR ⁺ /P-Akt ⁻ patients. Conclusions: High EGFR gene copy number identified by FISH may be an effective molecular predictor for gefitinib efficacy in advanced NSCLC.

MicroRNAs (miRNAs) regulate expression by promoting degradation or repressing translation of target transcripts. miRNA target sites have been catalogued in databases based on experimental validation and computational prediction using various algorithms. Several online resources provide collections of multiple databases but need to be imported into other software, such as R, for processing, tabulation, graphing and computation. Currently available miRNA target site packages in R are limited in the number of databases, types of databases and flexibility. We present multiMiR, a new miRNA–target interaction R package and database, which includes several novel features not available in existing R packages: (i) compilation of nearly 50 million records in human and mouse from 14 different databases, more than any other collection; (ii) expansion of databases to those based on disease annotation and drug microRNAresponse, in addition to many experimental and computational databases; and (iii) user-defined cutoffs for predicted binding strength to provide the most confident selection. Case studies are reported on various biomedical applications including mouse models of alcohol consumption, studies of chronic obstructive pulmonary disease in human subjects, and human cell line models of bladder cancer metastasis. We also demonstrate how multiMiR was used to generate testable hypotheses that were pursued experimentally.

PURPOSE: E-cadherin (E-cad) and its associated intracellular molecules, catenins, are critical for intercellular epithelial adhesion and are often expressed in non–small-cell lung carcinomas (NSCLCs). We constructed tissue microarrays (TMAs) to investigate the expression of cadherins and catenins and their prognostic significance in NSCLC. PATIENTS AND METHODS: Tumor tissue samples from 193 patients with stages I to III NSCLC were obtained from the University of Colorado Cancer Center and Johns Hopkins Medical Institutions. Viable tumor was sampled in triplicate for the TMAs, and slides were stained by immunohistochemistry with antibodies against E-cad, N-cadherin, alpha (α)-, beta (β)-, and gamma (γ)-catenin, p120, p27, and adenomatous polyposis coli (APC) gene product. Clinical data were collected by the tumor registries. Patients were followed for a median period of 51 months (range, 18 to 100 months). RESULTS: Absent or severely reduced membranous expression for E-cad, α-, β-, and γ-catenin, and p120 were observed in 10%, 17%, 8%, 31%, and 61% of the cases, respectively. Tumor cell dedifferentiation correlated with reduced expression for E-cad, β-catenin, γ-catenin, and p120 in squamous cell carcinomas but not in adenocarcinomas. There was an inverse correlation between nodal metastasis and expression of E-cad and γ-catenin. Besides the traditional clinical prognostic variables, E-cad and α-, β-, and γ-catenin expression were of positive prognostic value in univariate survival analyses. In multivariate analysis, E-cad expression was the only independent prognostic factor for survival in addition to age, node status, tumor status, and pathologic surgical margins. CONCLUSION: Reduced expression of E-cad and catenins is associated with tumor cell dedifferentiation, local invasion, regional metastasis, and reduced survival in NSCLC. E-cad is an independent prognostic factor for NSCLC survival.

Micropthalmia-associated transcription factor (MITF) is the master regulator of melanocyte development, survival, and function. Frequent alteration in the expression of MITF is detected in melanoma, but the mechanism(s) underlying the alteration in expression have not been completely determined. In these studies, we have identified microRNA-137 (miR-137) as a regulator of MITF expression. The genomic locus of miR-137 at chromosome 1p22 places it in a region of the human genome previously determined to harbor an allele for melanoma susceptibility. Here, we show that expression of mature miR-137 in melanoma cell lines down-regulates MITF expression. Further, we have identified a 15-bp variable nucleotide tandem repeat located just 5′ to the pre-miR-137 sequence, which alters the processing and function of miR-137 in melanoma cell lines. [Cancer Res 2008;68(5):1362–8]

Background: Biological markers for optimal selection of patient to epidermal growth factor receptor (EGFR)-targeted therapies are not established in advanced non-small-cell lung cancer (NSCLC). Patients and methods:EGFR/HER2 gene copy number by FISH, EGFR protein and pAKT expression by immunohistochemistry (IHC) and EGFR and KRAS mutations were tested in 204 gefitinib-treated NSCLC patients. Results: Increased EGFR and HER2 gene copy number (FISH+), EGFR protein overexpression (IHC+), EGFR mutations and pAKT overexpression were all associated with significantly higher response rates (33%, 29%, 22%, 39% and 20% respectively). EGFR FISH+ (32%) and IHC+ (61%) correlated with improved survival, while EGFR mutations (27%), KRAS mutations (26%) and pAKT expression (69%) did not. In multivariate survival analysis EGFR FISH and IHC were independent predictive markers. EGFR FISH+/IHC+ patients (23%) had a median survival of 21 months versus 6 months for double-negative patients (30%). Conclusion: Combination of EGFR FISH and IHC is effective predictor for benefit from gefitinib. Patients with double-negative results are unlikely to benefit in western NSCLC populations.