Mitch Raponi

Gene Expression Signatures for Predicting Prognosis of Squamous Cell and Adenocarcinomas of the Lung

Non-small-cell lung cancers (NSCLC) compose 80% of all lung carcinomas with squamous cell carcinomas (SCC) and adenocarcinoma representing the majority of these tumors. Although patients with early-stage NSCLC typically have a better outcome, 35% to 50% will relapse within 5 years after surgical treatment. We have profiled primary squamous cell lung carcinomas from 129 patients using Affymetrix U133A gene chips. Unsupervised analysis revealed two clusters of SCC that had no correlation with tumor stage but had significantly different overall patient survival (P = 0.036). The high-risk cluster was most significantly associated with down-regulation of epidermal development genes. Cox proportional hazard models identified an optimal set of 50 prognostic mRNA transcripts using a 5-fold cross-validation procedure. Quantitative reverse transcription-PCR and immunohistochemistry using tissue microarrays were used to validate individual gene candidates. This signature was tested in an independent set of 36 SCC samples and achieved 84% specificity and 41% sensitivity with an overall predictive accuracy of 68%. Kaplan-Meier analysis showed clear stratification of high-risk and low-risk patients [log-rank P = 0.04; hazard ratio (HR), 2.66; 95% confidence interval (95% CI), 1.01-7.05]. Finally, we combined the SCC classifier with our previously identified adenocarcinoma prognostic signature and showed that the combined classifier had a predictive accuracy of 71% in 72 NSCLC samples also showing significant differences in overall survival (log-rank P = 0.0002; HR, 3.54; 95% CI, 1.74-7.19). This prognostic signature could be used to identify patients with early-stage high-risk NSCLC who might benefit from adjuvant therapy following surgery.

MicroRNA Classifiers for Predicting Prognosis of Squamous Cell Lung Cancer

Non-small cell lung cancer (NSCLC), which is comprised mainly of adenocarcinoma and squamous cell carcinoma (SCC), is the cause of 80% of all lung cancer deaths in the United States. NSCLC is also associated with a high rate of relapse after clinical treatment and, therefore, requires robust prognostic markers to better manage therapy options. The aim of this study was to identify microRNA (miRNA) expression profiles in SCC of the lung that would better predict prognosis. Total RNA from 61 SCC samples and 10 matched normal lung samples was processed for small RNA species and profiled on MirVana miRNA Bioarrays (version 2, Ambion). We identified 15 miRNAs that were differentially expressed between normal lung and SCC, including members of the miR-17-92 cluster and its paralogues. We also identified miRNAs, including miR-155 and let-7, which had previously been shown to have prognostic value in adenocarcinoma. Based on cross-fold validation analyses, miR-146b alone was found to have the strongest prediction accuracy for stratifying prognostic groups at approximately 78%. The miRNA signatures were superior in predicting overall survival than a previously described 50-gene prognostic signature. Whereas there was no overlap between the mRNAs targeted by the prognostic miRNAs and the 50-gene expression signature, there was a significant overlap in the corresponding biological pathways, including fibroblast growth factor and interleukin-6 signaling. Our data indicate that miRNAs may have greater clinical utility in predicting the prognosis of patients with squamous cell lung carcinomas than mRNA-based signatures.

Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer

BackgroundMicroRNAs (MiRNAs) are short non-coding RNAs that control protein expression through various mechanisms. Their altered expression has been shown to be associated with various cancers. The aim of this study was to profile miRNA expression in colorectal cancer (CRC) and to analyze the function of specific miRNAs in CRC cells. MirVana miRNA Bioarrays were used to determine the miRNA expression profile in eight CRC cell line models, 45 human CRC samples of different stages, and four matched normal colon tissue samples. SW620 CRC cells were stably transduced with miR-143 or miR-145 expression vectors and analyzed in vitro for cell proliferation, cell differentiation and anchorage-independent growth. Signalling pathways associated with differentially expressed miRNAs were identified using a gene set enrichment analysis.ResultsThe expression analysis of clinical CRC samples identified 37 miRNAs that were differentially expressed between CRC and normal tissue. Furthermore, several of these miRNAs were associated with CRC tumor progression including loss of miR-133a and gain of miR-224. We identified 11 common miRNAs that were differentially expressed between normal colon and CRC in both the cell line models and clinical samples. In vitro functional studies indicated that miR-143 and miR-145 appear to function in opposing manners to either inhibit or augment cell proliferation in a metastatic CRC model. The pathways targeted by miR-143 and miR-145 showed no significant overlap. Furthermore, gene expression analysis of metastatic versus non-metastatic isogenic cell lines indicated that miR-145 targets involved in cell cycle and neuregulin pathways were significantly down-regulated in the metastatic context.ConclusionMiRNAs showing altered expression at different stages of CRC could be targets for CRC therapies and be further developed as potential diagnostic and prognostic analytes. The identified biological processes and signalling pathways collectively targeted by co-expressed miRNAs in CRC provide a basis for understanding the functional role of miRNAs in cancer.

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

An anti‐HIV‐1 tat ribozyme, termed Rz2, has been shown to inhibit HIV‐1 infection/replication and to decrease HIV‐1‐induced pathogenicity in T‐lymphocyte cell lines and normal peripheral blood T‐lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2.

Microarray analysis reveals genetic pathways modulated by tipifarnib in acute myeloid leukemia

BackgroundFarnesyl protein transferase inhibitors (FTIs) were originally developed to inhibit oncogenic ras, however it is now clear that there are several other potential targets for this drug class. The FTI tipifarnib (ZARNESTRA™, R115777) has recently demonstrated clinical responses in adults with refractory and relapsed acute leukemias. This study was conducted to identify genetic markers and pathways that are regulated by tipifarnib in acute myeloid leukemia (AML).MethodsTipifarnib-mediated gene expression changes in 3 AML cell lines and bone marrow samples from two patients with AML were analyzed on a cDNA microarray containing approximately 7000 human genes. Pathways associated with these expression changes were identified using the Ingenuity Pathway Analysis tool.ResultsThe expression analysis identified a common set of genes that were regulated by tipifarnib in three leukemic cell lines and in leukemic blast cells isolated from two patients who had been treated with tipifarnib. Association of modulated genes with biological functional groups identified several pathways affected by tipifarnib including cell signaling, cytoskeletal organization, immunity, and apoptosis. Gene expression changes were verified in a subset of genes using real time RT-PCR. Additionally, regulation of apoptotic genes was found to correlate with increased Annexin V staining in the THP-1 cell line but not in the HL-60 cell line.ConclusionsThe genetic networks derived from these studies illuminate some of the biological pathways affected by FTI treatment while providing a proof of principle for identifying candidate genes that might be used as surrogate biomarkers of drug activity.

Identification of Molecular Predictors of Response in a Study of Tipifarnib Treatment in Relapsed and Refractory Acute Myelogenous Leukemia

Purpose: Microarray technology was used to identify gene expression markers that predict response to the orally available farnesyltransferase inhibitor tipifarnib (Zarnestra, R115777) in acute myelogenous leukemia (AML). Experimental Design: Gene expression profiles from 58 bone marrow samples from a cohort of relapsed and refractory AML patients were analyzed on the Affymetrix U133A gene chip that contains ∼22,000 genes. Results: Supervised statistical analysis identified eight gene expression markers that could predict patient response to tipifarnib. The most robust gene was the lymphoid blast crisis oncogene (AKAP13), which predicted response with an overall accuracy of 63%. This gene provided a negative predictive value of 93% and a positive predictive value of 31% (increased from 18%). AKAP13 was overexpressed in patients who were resistant to tipifarnib. When overexpressed in the HL60 and THP1 cell lines, AKAP13 increased the resistance to tipifarnib by approximately 5- to 7-fold. Conclusion: Diagnostic gene expression signatures may be used to select a group of AML patients that might respond to tipifarnib.

In vitro and In vivo Characterization of Irreversible Mutant-Selective EGFR Inhibitors that are Wild-type Sparing

Patients with non–small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFRWT). These inhibitors have not provided compelling clinical benefit in T790M-positive patients, apparently because of dose-limiting toxicities associated with inhibition of EGFRWT. Thus, there is an urgent clinical need for therapeutics that overcome T790M drug resistance while sparing EGFRWT. Here, we describe a lead optimization program that led to the discovery of four potent irreversible 2,4-diaminopyrimidine compounds that are EGFR mutant (EGFRmut) selective and have been designed to have low affinity for EGFRWT. Pharmacokinetic and pharmacodynamic studies in H1975 tumor–bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. Importantly, evaluation of normal lung tissue from the same animals showed no inhibition of EGFRWT. Of all the compounds tested, compound 3 displayed the best efficacy in EGFRL858R/T790M-driven tumors. Compound 3, now renamed CO-1686, is currently in a phase I/II clinical trial in patients with EGFRmut-advanced NSCLC that have received prior EGFR-directed therapy. Mol Cancer Ther; 13(6); 1468–79. ©2014 AACR.

RNA-mediated gene silencing in non-pathogenic and pathogenic fungi

Many fungal genomes have now been sequenced and thousands of genes are being discovered. Gene disruption or inactivation technology offers an important tool not only for elucidating the function of the many unknown genes but also for the identification of genes essential for fungal growth and pathogenesis. A variety of gene-silencing methods that inhibit genes at the post-transcriptional level are now being used in both non-pathogenic and human pathogenic fungi. We focus on the recent advances in RNA-mediated gene silencing technologies and their potential for functional genomics studies in fungi.

Control of specific gene expression in mammalian cells by co‐expression of long complementary RNAs

The use of long double‐stranded RNA (dsRNA) for gene silencing in mammalian cells has generally been restricted to embryonic cell types and proposed to induce non‐specific effects on gene expression in differentiated cells. In this study, we report that foreign and endogenous gene expression can be regulated in immortalised human cell lines by co‐expression of long complementary RNAs with the potential to form dsRNA. The observed gene silencing effect was transferable to recipient control cells, occurred independently of cytoplasmic Dicer and produced an epi‐allelic series of clones suitable for gene function studies. This complementary RNA co‐expression approach permits the use of long complementary RNAs for regulating specific gene expression in mammalian cells.

Multi-institutional phase 2 clinical and pharmacogenomic trial of tipifarnib plus etoposide for elderly adults with newly diagnosed acute myelogenous leukemia

Tipifarnib (T) exhibits modest activity in elderly adults with newly diagnosed acute myelogenous leukemia (AML). Based on preclinical synergy, a phase 1 trial of T plus etoposide (E) yielded 25% complete remission (CR). We selected 2 comparable dose levels for a randomized phase 2 trial in 84 adults (age range, 70-90 years; median, 76 years) who were not candidates for conventional chemotherapy. Arm A (T 600 mg twice a day × 14 days, E 100 mg days 1-3 and 8-10) and arm B (T 400 mg twice a day × 14 days, E 200 mg days 1-3 and 8-10) yielded similar CR, but arm B had greater toxicity. Total CR was 25%, day 30 death rate 7%. A 2-gene signature of high RASGRP1 and low aprataxin (APTX) expression previously predicted for T response. Assays using blasts from a subset of 40 patients treated with T plus E on this study showed that AMLs with a RASGRP1/APTX ratio of more than 5.2 had a 78% CR rate and negative predictive value 87%. This ratio did not correlate with outcome in 41 patients treated with conventional chemotherapies. The next T-based clinical trials will test the ability of the 2-gene signature to enrich for T responders prospectively. This study is registered at www.clinicaltrials.gov as #NCT00602771.