Genes associated with prognosis after surgery for malignant pleural mesothelioma promote tumor cell survival in vitro.

BACKGROUND: Mesothelioma is an aggressive neoplasm with few effective treatments, one being cytoreductive surgery. We previously described a test, based on differential expression levels of four genes, to predict clinical outcome in prospectively consented mesothelioma patients after surgery. In this study, we determined whether any of these four genes could be linked to a cancer relevant phenotype. METHODS: We conducted a high-throughput RNA inhibition screen to knockdown gene expression levels of the four genes comprising the test (ARHGDIA, COBLL1, PKM2, TM4SF1) in both a human lung-derived normal and a tumor cell line using three different small inhibitory RNA molecules per gene. Successful knockdown was confirmed using quantitative RT-PCR. Detection of statistically significant changes in apoptosis and mitosis was performed using immunological assays and quantified using video-assisted microscopy at a single time-point. Changes in nuclear shape, size, and numbers were used to provide additional support of initial findings. Each experiment was conducted in triplicate. Specificity was assured by requiring that at least 2 different siRNAs produced the observed change in each cell line/time-point/gene/assay combination. RESULTS: Knockdown of ARHGDIA, COBLL1, and TM4SF1 resulted in 2- to 4-fold increased levels of apoptosis in normal cells (ARHGDIA only) and tumor cells (all three genes). No statistically significant changes were observed in apoptosis after knockdown of PKM2 or for mitosis after knockdown of any gene. CONCLUSIONS: We provide evidence that ARHGDIA, COBLL1, and TM4SF1 are negative regulators of apoptosis in cultured tumor cells. These genes, and their related intracellular signaling pathways, may represent potential therapeutic targets in mesothelioma.

Transcriptome sequencing of malignant pleural mesothelioma tumors.

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.

Differentially expressed alternatively spliced genes in malignant pleural mesothelioma identified using massively parallel transcriptome sequencing.

BACKGROUND: Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples. METHODS: We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens. RESULTS: We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively). CONCLUSION: Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.

mTOR mediates survival signals in malignant mesothelioma grown as tumor fragment spheroids.

Solid tumors such as mesothelioma exhibit a stubborn resistance to apoptosis that may derive from survival pathways, such as PI3K/Akt/mTOR, that are activated in many tumors, including mesothelioma. To address the role of PI3K/Akt/mTOR, we used a novel approach to study mesothelioma ex vivo as tumor fragment spheroids. Freshly resected mesothelioma tissue from 15 different patients was grown in vitro as 1- to 2-mm-diameter fragments, exposed to apoptotic agents for 48 hours with or without PI3K/Akt/mTOR inhibitors, and doubly stained for cytokeratin and cleaved caspase 3 to identify apoptotic mesothelioma cells. Mesothelioma cells within the tumor spheroids exhibited striking resistance to apoptotic agents such as TRAIL plus gemcitabine that were highly effective against monolayers. In a majority of tumors (67%; 10 of 15), apoptotic resistance could be reduced by more than 50% by rapamycin, an mTOR inhibitor, but not by LY294002, a PI3K inhibitor. Responsiveness to rapamycin correlated with staining for the mTOR target, p-S6K, in the original tumor, but not for p-Akt. As confirmation of the role of mTOR, siRNA knockdown of S6K reproduced the effect of rapamycin in three rapamycin-responsive tumors. Finally, in 37 mesotheliomas on tissue microarray, p-S6K correlated only weakly with p-Akt, suggesting the existence of Akt-independent regulation of mTOR. We propose that mTOR mediates survival signals in many mesothelioma tumors. Inhibition of mTOR may provide a nontoxic adjunct to therapy directed against malignant mesothelioma, especially in those with high baseline expression of p-S6K.

Preclinical studies of the proteasome inhibitor bortezomib in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly lethal neoplasm that is resistant to chemotherapy. Bortezomib is an FDA-approved proteasome inhibitor that is currently under clinical investigation in multiple neoplasms but has not been studied extensively in MPM. In this report, we determine the biological and molecular response of cultured MPM cells to bortezomib alone and in combination with cisplatin or pemetrexed. We used four MPM cell lines (MS589, H28, H2052, JMN), a normal mesothelial cell line (HM3), and a lung cancer cell line (H23) in survival studies utilizing bortezomib, cisplatin, and pemetrexed alone and in combination by administering concurrently or by varying the order of administration. We determined the effect of bortezomib on the cell cycle, apoptosis, and on the expression of cell cycle proteins p21/WAF1 and p27/KIP1 and on apoptosis-related proteins IAP-1, IAP-2, survivin, and XIAP. Bortezomib was highly cytotoxic to MPM cells and induced both G(2)/M and G(1)/S cell cycle arrest. Apoptosis increased in a concentration- and time-dependent manner in 3 of 4 MPM cell lines. Bortezomib stabilized or increased protein levels of p21/WAF1 and IAP-1 and to a lesser degree p27/KIP1, IAP-2, XIAP, and survivin. In combination studies with cisplatin, bortezomib was generally synergistic at high concentrations and antagonistic at low concentrations. Bortezomib increased the cytotoxicity of cisplatin and pemetrexed in a concentration-dependent manner when administered prior to either. Bortezomib may improve outcome in MPM patients alone or in combination with standard chemotherapy but the order of administration is likely to be important. This study justifies further evaluation of bortezomib in MPM.

Functional analysis of c-Met/hepatocyte growth factor pathway in malignant pleural mesothelioma.

c-Met receptor tyrosine kinase (RTK) has not been extensively studied in malignant pleural mesothelioma (MPM). In this study, c-Met was overexpressed and activated in most of the mesothelioma cell lines tested. Expression in MPM tissues by immunohistochemistry was increased (82%) in MPM in general compared with normal. c-Met was internalized with its ligand hepatocyte growth factor (HGF) in H28 MPM cells, with robust expression of c-Met. Serum circulating HGF was twice as high in mesothelioma patients as in healthy controls. There was a differential growth response and activation of AKT and extracellular signal-regulated kinase 1/2 in response to HGF for the various cell lines. Dose-dependent inhibition (IC50 < 2.5 micromol/L) of cell growth in mesothelioma cell lines, but not in H2052, H2452, and nonmalignant MeT-5A (IC50 > 10 micromol/L), was observed with the small-molecule c-Met inhibitor SU11274. Furthermore, migration of H28 cells was blocked with both SU11274 and c-Met small interfering RNA. Abrogation of HGF-induced c-Met and downstream signaling was seen in mesothelioma cells. Of the 43 MPM tissues and 7 cell lines, we have identified mutations within the semaphorin domain (N375S, M431V, and N454I), the juxtamembrane domain (T1010I and G1085X), and an alternative spliced product with deletion of the exon 10 of c-Met in some of the samples. Interestingly, we observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited the most dramatic reduction of cell growth with SU11274 when compared with wild-type H28 and nonmalignant MeT-5A cells. Ultimately, c-Met would be an important target for therapy against MPM.

Validation of genomics-based prognostic tests in malignant pleural mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is a highly lethal neoplasm with limited pretreatment prognostication strategies. In this report, we examine the accuracy of a previously proposed prognostic test in an independent cohort of MPM patients. This test uses simple ratios of gene expression levels to provide a novel prognostication scheme. EXPERIMENTAL DESIGN: Gene expression data using high-density oligonucleotide microarrays (approximately 22,000 genes) were obtained for a new cohort of human MPM tumors from patients undergoing similar treatments (n = 39). The relative expression levels for specific genes were also determined using real-time quantitative reverse transcription-PCR. We also used a subset of these tumors associated with widely divergent patient survival (n = 23) as a training set to identify new treatment-specific candidate prognostic molecular markers and gene ratio-based prognostic tests. The predictive nature of these newly discovered markers and gene ratio-based prognostic tests were then examined in an independent group of tumors (n = 52) using microarray data and quantitative reverse transcription-PCR. RESULTS: Previously described MPM prognostic genes and gene ratio-based prognostic tests predicted clinical outcome in 39 independent MPM tumor specimens in a statistically significant manner. Newly discovered treatment-specific prognostic genes and gene ratio-based prognostic tests were highly accurate and statistically significant when examined in an independent group of 52 tumors from patients undergoing similar treatment. CONCLUSIONS: The data support the use of gene ratios in translating gene expression data into easily reproducible, statistically validated clinical tests for the prediction of outcome in MPM.

Using gene expression ratios to predict outcome among patients with mesothelioma.

BACKGROUND: We have recently demonstrated that simple ratios of the expression levels of selected genes in tumor samples can be used to distinguish among types of thoracic malignancies. We examined whether this technique could predict treatment-related outcome for patients with mesothelioma. METHODS: We used gene expression profiling data previously collected from 17 mesothelioma patients with different overall survival times to define two outcome-related groups of patients and to train an expression ratio-based outcome predictor model. A Student's t test was used to identify genes among the two outcome groups that had statistically significant, inversely correlated expression levels; those genes were used to form prognostic expression ratios. We used a combination of several highly accurate expression ratios and cross-validation techniques to assess the internal consistency of this predictor model, quantitative reverse transcription-polymerase chain reaction of tumor RNA to confirm the microarray data, and Kaplan-Meier survival analysis to validate the model among an independent set of 29 mesothelioma tumors. All statistical tests were two-sided. RESULTS: We developed an expression ratio-based test capable of identifying 100% (17/17) of the samples used to train the model. This test remained highly accurate (88%, 15/17) after cross-validation. A four-gene expression ratio test statistically significantly (P =.0035) predicted treatment-related patient outcome in mesothelioma independent of the histologic subtype of the tumor. CONCLUSIONS: Gene expression ratio-based analysis accurately predicts treatment-related outcome in mesothelioma samples. This technique could impact the clinical treatment of mesothelioma by allowing the preoperative identification of patients with widely divergent prognoses.

Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma.

The pathological distinction between malignant pleural mesothelioma (MPM)and adenocarcinoma (ADCA) of the lung can be cumbersome using established methods. We propose that a simple technique, based on the expression levels of a small number of genes, can be useful in the early and accurate diagnosis of MPM and lung cancer. This method is designed to accurately distinguish between genetically disparate tissues using gene expression ratios and rationally chosen thresholds. Here we have tested the fidelity of ratio-based diagnosis in differentiating between MPM and lung cancer in 181 tissue samples (31 MPM and 150 ADCA). A training set of 32 samples (16 MPM and 16 ADCA) was used to identify pairs of genes with highly significant, inversely correlated expression levels to form a total of 15 diagnostic ratios using expression profiling data. Any single ratio of the 15 examined was at least 90% accurate in predicting diagnosis for the remaining 149 samples (e.g., test set). We then examined (in the test set) the accuracy of multiple ratios combined to form a simple diagnostic tool. Using two and three expression ratios, we found that the differential diagnoses of MPM and lung ADCA were 95% and 99% accurate, respectively. We propose that using gene expression ratios is an accurate and inexpensive technique with direct clinical applicability for distinguishing between MPM and lung cancer. Furthermore, we provide evidence suggesting that this technique can be equally accurate in other clinical scenarios.

Transcriptional profiling of mesothelioma using microarrays.

Mesothelioma is an asbestos-related neoplasm of the thoracic pleura about which little is known and for which effective therapy is lacking. Large-scale transcriptional profiling using microarrays is frequently a part of studies to explore gene expression patterns in cancer and other diseases. In general, microarray based experiments can facilitate the identification of tumor molecular markers, provide clues relating to mechanisms carcinogenesis, as well as aid in the discovery of candidate targets for therapy. Relatively few studies of this sort have been attempted for mesothelioma, likely due to its relatively rare incidence and by extension the difficulty in acquiring suitable tissues for analysis. Microarray analysis of mesothelioma will likely lead to a better understanding of a highly lethal malignancy and result in the identification of potential therapeutic targets to ultimately affect better treatment options and patient clinical outcome. This mini-review will address general issues pertaining to all expression profiling experiments (e.g., data interpretation) and summarize similar studies that have been attempted for mesothelioma.

A prognostic test for adenocarcinoma of the lung from gene expression profiling data.

Until recently, it has been impossible to determine which patients with resected stage I lung cancer are among the 30% who will die of metastatic cancer within 5 years of surgery. Bioinformatics tools applied to lung cancer expression profiling data have identified prognostic genes that have been used to develop predictor models, but thus far, these models have not been incorporated into routine clinical use because of their inherent complexity and requirement for relatively large numbers of genes. We have used ratios of gene expression to overcome these limitations. Here, we evaluate the ability of this technique to identify patients with stage I lung adenocarcinoma at risk for recurrence. We derived candidate ratio-based tests from analysis of 36 stage I lung adenocarcinoma samples using previously published expression profiling data. Eleven of these tests were identified for additional study and assessed for classification accuracy in an independent set of 60 stage I lung adenocarcinoma samples. We then evaluated the optimal ratio-based test in the independent samples using Kaplan-Meier survival analysis. Finally, we examined the ability of this test to predict outcome in a set of 97 stage I breast adenocarcinoma. We found that subsets of the independent lung cancer samples predicted to be associated with either good or poor outcome using the optimal ratio-based test differed significantly (P=0.0056) in terms of survival with a classification accuracy of 74% (P=0.0043, Fisher's exact test). When this test was applied to stage II and III lung cancers, most specimens were classified as poor outcome cancers. Interestingly, we found that the same test significantly (P=0.0417) predicted recurrence of stage I breast tumors, suggesting that at least some of the marker genes we identified may have generalized prognostic significance for adenocarcinoma. Our results provide additional evidence that expression ratios are highly accurate in predicting cancer recurrence and may be used in a simple test to predict response to surgical therapy in early-stage lung adenocarcinoma.

Isolation, short-term culture, and transplantation of small hepatocyte-like progenitor cells from retrorsine-exposed rats.

BACKGROUND: Complete liver regeneration after partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine can be accomplished through the activation, expansion, and differentiation of a novel population of small hepatocyte-like progenitor cells (SHPCs). These cells have not been isolated in pure form, established in primary culture, or transplanted into syngeneic rats to examine their differentiation potential. METHODS: Primary liver cells enriched for SHPCs were prepared by differential centrifugation of primary liver cell dispersions from retrorsine-exposed rats 6-8 days and 13-15 days after PH. Isolated SHPCs were characterized for cell size, morphology, and expression of cell type-specific markers (including hepatocyte and bile duct-oval cell markers), and established in short-term primary culture. Isolated SHPCs were transplanted into the livers of syngeneic rats to evaluate their ability to engraft and differentiate into mature hepatocytes. RESULTS: SHPCs obtained from retrorsine-exposed rats 6-8 days and 13-15 days after PH were small (10-12 microm in diameter), morphologically resembled hepatocytes, and were predominantly H.4 antigen-positive, alpha-fetoprotein-positive, and OV6-negative. SHPCs did not proliferate in culture and could not be passaged, but short-term cultures were established using protein substrates (collagen or laminin) and defined medium containing epidermal growth factor and nicotinamide. After transplantation into the livers of syngeneic hosts, SHPCs insert into hepatic plates and give rise to differentiated hepatocyte progeny. The SHPC-derived hepatocyte progeny express a differentiated phenotype (albumin-positive, transferrin-positive, alpha-fetoprotein-negative) and are able to proliferate in vivo in response to the growth stimulus provided by PH. CONCLUSIONS: The results demonstrate that enriched SHPC populations can be isolated from retrorsine-exposed rats and established in short-term culture, and they can engraft and differentiate after transplantation into the livers of syngeneic rats.

Detection and quantification of SV40 large T-antigen DNA in mesothelioma tissues and cell lines.

SV40 viral DNA sequences, particularly large T-antigen (T-ag) DNA, have been reported in malignant pleural mesothelioma (MPM) and suggested to play a role in the tumorigenesis of this cancer. These results remain somewhat controversial owing to variability among a number of laboratories in reported SV40 DNA and protein detection in MPM tissues. This could be explained in part if SV40 DNA is present in relatively low abundance in many MPM tissues determined to contain viral sequences and is therefore difficult to detect. To this end, we investigated the efficacy of real time quantitative PCR in detecting low copy number SV40 DNA sequences, then we quantified SV40 copy number in MPM tissues and cell lines at our institution. Quantitative PCR demonstrated consistent precision and accuracy in detecting SV40 DNA sequences over a large logarithmic range of viral copy number. In addition, SV40 sequences were found in 2/35 tissues and 3/7 cell lines in relatively low abundance. We conclude that SV40 is not a contributing factor in the pathobiology of the majority of MPM tumors from patients at our institution.

Genetics of malignant pleural mesothelioma: molecular markers and biologic targets.

MPM is a poorly understood lethal malignancy. Although the pathobiology of MPM is not completely elucidated, new genomic technology is likely to help shed light on the mechanisms of carcinogenesis through genome-wide screening of tumor-specific gene expression. Related efforts to identify the molecular markers of mesothelioma are pursued with the aim of refining current diagnostic capabilities, predicting prognosis, and designing appropriate trimodality programs. These new genomic tools also will assist efforts to tailor current adjuvant and neoadjuvant therapies, optimizing their effect and furthering research that may lead to new therapeutic options.

Fine-needle aspiration biopsies for gene expression ratio-based diagnostic and prognostic tests in malignant pleural mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is an aggressive disease associated with median survival between 9 and 12 months. The correct diagnosis of MPM is sometimes challenging and usually requires solid tissue biopsies rather than fine-needle aspiration biopsies (FNA). We postulated that the accuracy of FNA-based diagnosis might be improved by the addition of molecular tests using a gene expression ratio-based algorithm and that prognostic tests could be similarly performed. EXPERIMENTAL DESIGN: Two MPM and 2 lung cancer cell lines were used to establish the minimal quantity of RNA required to perform the gene ratio test. On the basis of these results, 276 ex vivo FNA biopsies from 63 MPM patients and 250 ex vivo FNA samples from 92 lung cancer patients were analyzed using previously described diagnostic and prognostic tests based on gene expression ratios. RESULTS: We found that the sensitivity of the diagnostic test for MPM was 100% [95% confidence interval (CI): 95%-100%] and the specificity in primary lung adenocarcinoma was 90% (95% CI: 81%-95%). The FNA-based prognostic classification was concordant among 76% (95% CI: 65%-87%) of patients with the risk assignment in a subset of the matched surgical specimens previously analyzed by the prognostic test. CONCLUSIONS: Sufficient RNA can be extracted from most FNA biopsies to perform gene expression molecular tests. In particular, we show that the gene expression ratio algorithms performed well when applied to diagnosis and prognosis in MPM. This study provides support for the development of additional RNA molecular tests that may enhance the utility of FNA in the management of other solid cancers.

Second generation sequencing of the mesothelioma tumor genome.

The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.

A gene expression ratio-based diagnostic test for bladder cancer.

PURPOSE: Bladder cancer is relatively common but early detection techniques such as cystoscopy and cytology are somewhat limited. We developed a broadly applicable, platform-independent and clinically relevant method based on simple ratios of gene expression to diagnose human cancers. In this study, we sought to determine whether this technique could be applied to the diagnosis of bladder cancer. EXPERIMENTAL DESIGN: We developed a model for the diagnosis of bladder cancer using expression profiling data from 80 normal and tumor bladder tissues to identify statistically significant discriminating genes with reciprocal average expression levels in each tissue type. The expression levels of select genes were used to calculate individual gene pair expression ratios in order to assign diagnosis. The optimal model was examined in two additional published microarray data sets and using quantitative RT-PCR in a cohort of 13 frozen benign bladder urothelium samples and 13 bladder cancer samples from our institution. RESULTS: A five-ratio test utilizing six genes proved to be 100% accurate (26 of 26 samples) for distinguishing benign from malignant bladder tissue samples (P < 10(-6)). CONCLUSIONS: : We have provided a proof of principle study for the use of gene expression ratios in the diagnosis of bladder cancer. This technique may ultimately prove to be a useful adjunct to cytopathology in screening urine specimens for bladder cancer.

Four-gene expression ratio test for survival in patients undergoing surgery for mesothelioma.

BACKGROUND: Malignant pleural mesothelioma has few effective treatments, one being cytoreductive surgery. We previously developed a gene ratio test to predict outcome of malignant pleural mesothelioma patients undergoing surgery. In this study, we investigated the predictive value and technical assay performance of this test in patients with malignant pleural mesothelioma. METHODS: Clinical data were obtained prospectively from 120 consecutive patients with malignant pleural mesothelioma who were scheduled for debulking surgery at one institution. Specimens were obtained at surgery or by pleural biopsy examination. Expression data for four genes were collected from tumor specimens, and three ratios of gene expression (TM4SF1/PKM2, TM4SF1/ARHGDIA, and COBLL1/ARHGDIA) were determined by quantitative reverse transcriptase-polymerase chain reaction. Patients were assigned to good or poor outcome groups by the gene ratio test. Survival was estimated by the Kaplan-Meier method and the log-rank test in univariate analyses. A multivariable Cox proportional hazards model was used to control for prognostic factors. Technical robustness was determined by using up to 30 specimens per patient, two biopsy techniques, and two performance sites. All statistical tests were two-sided. RESULTS: The test predicted overall survival (P < .001) and cancer-specific survival (P = .007) in univariate analysis and overall survival in multivariable analysis (hazard ratio for death = 2.09, 95% confidence interval [CI] = 1.27 to 3.45, P = .004). The test was reproducible within patients and repeatable between two determinations for specimens with widely varying tumor cell contents. Repeatability between two determinations was 88.5% (95% CI = 84.0% to 92.2%) or, when technically unacceptable test values were excluded, 91.9% (95% CI = 87.4% to 95.1%). Reproducibility between two determinations was 96.1% (95% CI = 86.5% to 99.5%). Combining the gene ratio test and other prognostic factors allowed prospective discrimination between patients at high risk (median survival = 6.9 months, 95% CI = 2.6 to 8.9 months; 3-year survival = 0%) and low risk (median survival = 31.9 months, 95% CI = 21.9 to 41.7 months; 3-year survival = 42%). CONCLUSION: The gene ratio test for survival of patients with malignant pleural mesothelioma has robust predictive value and technical assay performance.

Paxillin is a target for somatic mutations in lung cancer: implications for cell growth and invasion.

Lung cancer is characterized by abnormal cell growth and invasion, and the actin cytoskeleton plays a major role in these processes. The focal adhesion protein paxillin is a target of a number of oncogenes involved in key signal transduction and important in cell motility and migration. In lung cancer tissues, we have found that paxillin was highly expressed (compared with normal lung), amplified (12.1%, 8 of 66) and correlated with increased MET and epidermal growth factor receptor (EGFR) gene copy numbers, or mutated (somatic mutation rate of 9.4%, 18 of 191). Paxillin mutations (19 of 21) were clustered between LD motifs 1 and 2 and the LIM domains. The most frequent point mutation (A127T) enhanced lung cancer cell growth, colony formation, focal adhesion formation, and colocalized with Bcl-2 in vitro. Gene silencing from RNA interference of mutant paxillin led to reduction of cell viability. A murine in vivo xenograft model of A127T paxillin showed an increase in tumor growth, cell proliferation, and invasion. These results establish an important role for paxillin in lung cancer.

Differential diagnosis of solitary lung nodules with gene expression ratios.

OBJECTIVE: We have developed a new technique that uses the ratios of select gene expression levels to translate complex genomic data into simple clinically relevant tests for the diagnosis and prognosis of cancer. We determined whether select gene pair ratio combinations can be used to detect and diagnose lung cancer with high accuracy and sensitivity. METHODS: We used gene expression profiling data to train a ratio-based predictor model to discriminate among a set of samples (n = 145 total) composed of normal lung, small cell lung cancer, adenocarcinoma, squamous cell carcinoma, and pulmonary carcinoid (the training set). We then examined the optimal test in an independent set of samples (the test set, n = 122). Finally, we used one aspect of the test to determine whether the gene ratio technique was capable of detecting cancer in specimens from fine-needle aspirations performed ex vivo with normal lung (n = 14) and suspected tumor nodules (n = 15) acquired at our institution. RESULTS: We found that a ratio-based test with 23 genes could be used to classify training set samples with 90% accuracy. This same test was similarly accurate (88%) when applied to the test set of samples. We also found that this test was 87% and 100% accurate at detecting cancer in normal and tumorous fine-needle aspiration specimens, respectively. CONCLUSION: The gene expression ratio diagnostic technique is likely to aid in the differential diagnosis of solitary lung nodules in patients with suspected cancer and may also prove useful in developing lung cancer screening strategies that incorporate analysis of fine-needle aspiration specimens.