Multiregion gene expression profiling reveals heterogeneity in molecular subtypes and immunotherapy response signatures in lung cancer.

Intra-tumor heterogeneity may be present at all molecular levels. Genomic intra-tumor heterogeneity at the exome level has been reported in many cancer types, but comprehensive gene expression intra-tumor heterogeneity has not been well studied. Here, we delineated the gene expression intra-tumor heterogeneity by exploring gene expression profiles of 35 tumor regions from 10 non-small cell lung cancer tumors (three or four regions/tumor), including adenocarcinoma, squamous cell carcinoma, large-cell carcinoma, and pleomorphic carcinoma of the lung. Using Affymetrix Gene 1.0 ST arrays, we generated the gene expression data for every sample. Inter-tumor heterogeneity was generally higher than intra-tumor heterogeneity, but some tumors showed a substantial level of intra-tumor heterogeneity. The analysis of various clinically relevant gene expression signatures including molecular subtype, epithelial-to-mesenchymal transition, and anti-PD-1 resistance signatures also revealed heterogeneity between different regions of the same tumor. The gene expression intra-tumor heterogeneity we observed was associated with heterogeneous tumor microenvironments represented by stromal and immune cells infiltrated. Our data suggest that RNA-based prognostic or predictive molecular tests should be carefully conducted in consideration of the gene expression intra-tumor heterogeneity.

Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer.

Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

Elevated PDGFRB gene copy number gain is prognostic for improved survival outcomes in resected malignant pleural mesothelioma.

PDGF/PDGFR pathway has been implicated in malignant pleural mesothelioma (MPM) carcinogenesis, and evidence suggests autocrine mechanisms of proliferation. We sought to evaluate the incidence of PDGFRB gene copy number gain (CNG) by fluorescence in situ hybridization and PDGFR pathway protein expression by immunohistochemistry (IHC) and correlate it to patient clinical outcome. Eighty-eight archived tumor blocks from resected MPM with full clinical information were used to perform IHC biomarkers (PDGFRα, PDGFRß, p-PDGFRß) and fluorescence in situ hybridization analysis of PDGFRB gene CNG. Spearman rank correlation, Wilcoxon rank-sum test, Kruskal-Wallis test, BLiP plots, and Kaplan-Meier method were used to analyze the biomarkers and correlation to clinical outcome. Several correlations between the IHC biomarkers were seen; however, none correlated to clinically relevant patient demographics or histology. In the CNG analysis, PDGFRB gene CNG in >10% of tumor cells had lower cytoplasmic p-PDGFRß (P=.029), while PDGFRB gene CNG in >40% of tumor cells had a higher cytoplasmic PDGFRß (P=.04). PDGFRB gene CNG status did not associate with patient demographics or tumor characteristics. PDGFR pathway IHC biomarkers did not associate with survival outcomes. However, patients with PDGFRB CNG >40% of tumor cells had improved relapse-free survival (HR 0.25 [95% CI 0.09-0.72], P=.0096) and improved overall survival (HR 0.32 [95% CI 0.11-0.89], P=.029). PDGFRB CNG >40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients. Future validation of this biomarker in prospective trials is needed. From a retrospective review of archived tissue specimens from patients with resected malignant pleural mesothelioma tumors, we show that patients with PDGFRB CNG >40% of tumor cells had improved relapse-free survival (HR 0.25 [95% CI 0.09-0.72], P=.0096) and improved overall survival (HR 0.32 [95% CI 0.11-0.89], P=.029). PDGFRB CNG >40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients.

Prognostic impact of insulin receptor expression on survival of patients with nonsmall cell lung cancer.

BACKGROUND: The purpose of this study was to characterize insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) expression in patients with nonsmall cell lung cancer (NSCLC). METHODS: A total of 459 patients who underwent curative resection of NSCLC were studied (median follow-up duration, 4.01 years). Expression of the IR and IGF-1R protein in tumor specimens was assessed immunohistochemically using tissue microarrays. RESULTS: The cytoplasmic IR score was higher in patients with adenocarcinoma (ADC) than in those with squamous cell carcinoma (SCC), whereas cytoplasmic IGF-1R score was higher in patients with SCC than those with ADC. Neither IR nor IGF-1R expression was associated with sex, smoking history, or clinical stage. Patients with positive IR or IGF-1R expression levels had poor recurrence-free (RFS) (3.8 vs 3.3 years; 3.8 vs 2.0 years, respectively), but similar overall survival (OS). Patients with high expression levels of IR and IGF-1R had shorter RFS and OS compared with those with low levels of IR and/or IGF-1R expression. Finally, a multivariate analysis revealed the impact of IR, but not of IGF-1R, as an independent predictive marker of NSCLC survival: hazard ratio (HR) for OS, 1.005 (95% confidence interval [CI], 1.001-1.010], HR for RFS, 1.005 (95% CI, 1.001-1.009), when IR score was tested as a continuous variable. CONCLUSIONS: Overexpression of IR predicts a poor survival among patients with NSCLC, especially those with SCC. These results might serve as future guidance to the clinical trials involving IR or IGR-1R targeting agents.

Epidermal growth factor receptor and K-Ras mutations and resistance of lung cancer to insulin-like growth factor 1 receptor tyrosine kinase inhibitors.

BACKGROUND: Most patients with nonsmall cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The authors investigated the involvement of insulinlike growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and the molecular determinants of resistance to IGF-1R TKIs. METHODS: Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in an NSCLC tissue microarray. The authors analyzed the antitumor effects of an IGF-1R TKI (PQIP or OSI-906), either alone or in combination with a small-molecular inhibitor (PD98059 or U0126) or with siRNA targeting K-Ras or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), in vitro and in vivo in NSCLC cells with variable histologic features and EGFR or K-Ras mutations. RESULTS: pIGF-1R/IR expression in NSCLC specimens was associated with a history of tobacco smoking, squamous cell carcinoma histology, mutant K-Ras, and wild-type (WT) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF-1R TKIs exhibited significant antitumor activity in NSCLC cells with WT EGFR and WT K-Ras but not in those with mutations in these genes. Introduction of mutant K-Ras attenuated the effects of IGF-1R TKIs on NSCLC cells expressing WT K-Ras. Conversely, inactivation of MEK restored sensitivity to IGF-TKIs in cells carrying mutant K-Ras. CONCLUSIONS: The mutation status of both EGFR and K-Ras could be a predictive marker of response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF-1R TKIs.

Assessing environmental and occupational risk factors for lung cancer in Mexican-Americans.

BACKGROUND: We investigated environmental and occupational exposures and smoking history (while controlling for demographics) in a population of Mexican-American lung cancer cases and controls from the Houston metropolitan area. METHODS: Data were collected between 1991 and 2005 as part of an on-going multi-racial/ethnic, lung cancer case-control study. Cases included 212 Mexican-American lung cancer cases from UT MD Anderson Cancer Center. Controls (n = 328) were recruited from Houston's largest multispecialty group practice and frequency matched to the cases by age (± 5 years), sex, and ethnicity. Environmental and occupational factors were analyzed and odds ratios and 95% confidence intervals were calculated using logistic regression. RESULTS: We detected elevated risks of lung cancer associated with pesticide exposure and found conventional and antimicrobial (e.g., sterilizers, disinfectants, antiseptics) pesticides were associated with an increased risk of lung cancer in Mexican-Americans (conventional pesticides and antimicrobial pesticides combined: OR = 1.80, 95% CI 1.13-2.86; conventional pesticides: OR = 2.05, 95% CI 1.23-2.39; antimicrobial pesticides: OR = 2.48, 95% CI 1.46-4.21). CONCLUSIONS: Although we found over a two-fold increased risk of lung cancer among Mexican-Americans for pesticides, we could not identify individual pesticides. Our findings are an important preliminary step in identifying factors that are specifically associated with lung cancer risk among Mexican Americans.

Immunohistochemical expression of basic fibroblast growth factor and fibroblast growth factor receptors 1 and 2 in the pathogenesis of lung cancer.

PURPOSE: To identify the patterns of protein expression of basic fibroblast growth factor (bFGF) and FGF receptors 1 and 2 in non-small cell lung carcinoma (NSCLC) and their role in the early pathogenesis of squamous cell carcinoma (SCC) of the lung. EXPERIMENTAL DESIGN: Archived tissue from NSCLC (adenocarcinoma and SCC; n = 321) and adjacent bronchial epithelial specimens (n = 426) were analyzed for the immunohistochemical expression of bFGF, FGFR1, and FGFR2, and the findings were correlated with clinicopathologic features of the patients. RESULTS: High expression of bFGF, FGFR1, and FGFR2 was shown in most NSCLC tumors. The pattern of expression for all markers varied according to tumor histologic type and cellular localization. Cytoplasmic expression scores were significantly higher in tumors than in normal epithelia. Nuclear bFGF (P = 0.03) and FGFR1 (P = 0.02) levels were significantly higher in women than in men. Although cytoplasmic FGFR1 expression was significantly higher (P = 0.002) in ever smokers than in never smokers, nuclear FGFR1 (P = 0.0001) and FGFR2 (P = 0.003) expression was significantly higher in never smokers. Different prognostic patterns for the expression of these markers were detected for both NSCLC histologic types. Dysplastic changes showed significantly higher expression of all markers compared with squamous metaplasia. CONCLUSIONS: bFGF, FGFR1, and FGFR2 are frequently overexpressed in SCC and adenocarcinoma of the lung. bFGF signaling pathway activation may be an early phenomenon in the pathogenesis of SCC and thus an attractive novel target for lung cancer chemopreventive and therapeutic strategies.

Proteomics-based identification of proteins secreted in apical surface fluid of squamous metaplastic human tracheobronchial epithelial cells cultured by three-dimensional organotypic air-liquid interface method.

Squamous cell carcinoma in the lung originates from bronchial epithelial cells that acquire increasingly abnormal phenotypes. Currently, no known biomarkers are clinically efficient for the early detection of premalignant lesions and lung cancer. We sought to identify secreted molecules produced from squamous bronchial epithelial cells cultured with organotypic culture methods. We analyzed protein expression patterns in the apical surface fluid (ASF) from aberrantly differentiated squamous metaplastic normal human tracheobronchial epithelial (NHTBE) and mucous NHTBE cells. Comparative two-dimensional PAGE analysis revealed 174 unique proteins in the ASF of squamous NHTBE cells compared with normal mucociliary differentiated NHTBE cells. Among them, 64 well-separated protein spots were identified by liquid chromatography-tandem mass spectrometry, revealing 22 different proteins in the ASF from squamous NHTBE cells. Expression of six of these proteins [SCC antigen 1 (SCCA1), SCC antigen 2 (SCCA2), S100A8, S100A9, Annexin I, and Annexin II] in the squamous NHTBE cells was further confirmed with immunoblot analysis. Notably, SCCA1 and SCCA2 were verified as being expressed in squamous metaplastic NHTBE cells but not in normal mucous NHTBE or normal bronchial epithelium. Moreover, SCCA1 and SCCA2 expression increased in in vitro lung carcinogenesis model cell lines with increasing malignancy. In summary, we identified proteins that are uniquely secreted from squamous metaplastic primary human bronchial epithelial cells cultured by the organotypic air-liquid interface method. These ASF proteins may be used to detect abnormal lesions in the lung without collecting invasive biopsy specimens.

Multi-region exome sequencing reveals genomic evolution from preneoplasia to lung adenocarcinoma.

There has been a dramatic increase in the detection of lung nodules, many of which are preneoplasia atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) or invasive adenocarcinoma (ADC). The molecular landscape and the evolutionary trajectory of lung preneoplasia have not been well defined. Here, we perform multi-region exome sequencing of 116 resected lung nodules including AAH (n = 22), AIS (n = 27), MIA (n = 54) and synchronous ADC (n = 13). Comparing AAH to AIS, MIA and ADC, we observe progressive genomic evolution at the single nucleotide level and demarcated evolution at the chromosomal level supporting the early lung carcinogenesis model from AAH to AIS, MIA and ADC. Subclonal analyses reveal a higher proportion of clonal mutations in AIS/MIA/ADC than AAH suggesting neoplastic transformation of lung preneoplasia is predominantly associated with a selective sweep of unfit subclones. Analysis of multifocal pulmonary nodules from the same patients reveal evidence of convergent evolution.

Survivin expression in normal human bronchial epithelial cells: an early and critical step in tumorigenesis induced by tobacco exposure.

The inhibitor of apoptosis protein survivin is selectively expressed in tumor cells. The tobacco component nicotine increases the transcription of the survivin gene in non-small cell lung cancer cells. However, the role of survivin expression induced by tobacco component is not clear during lung carcinogenesis. We investigated the effects of the tobacco components nicotine and its related carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on survivin expression in normal human bronchial epithelial (NHBE) cells and examined the role of survivin in the malignant transformation of normal human bronchial epithelial (HBE) cells induced by these components. We found that survivin messenger RNA (mRNA) expression was detected in 41% (7 of 17) of bronchial brush specimens from heavy smokers. Nicotine and NNK increased survivin mRNA and protein expression levels in primary cultured NHBE cells and immortalized HBE cells. Bronchial epithelium in mice administered NNK also showed increased staining for survivin. Nicotine and NNK stimulated the Akt-mammalian target of rapamycin (mTOR) pathway in NHBE cells, leading to increased de novo synthesis of survivin protein. Induced survivin expression increased the survival potential of the cells, which was blocked by transfection with survivin-specific small interfering RNA (siRNA). siRNA-induced down-regulation of survivin expression also suppressed the tumorigenic potential of premalignant and malignant HBE cells exposed to the tobacco components. These findings suggest that NNK and nicotine induce survivin protein synthesis in NHBE cells by activating the Akt-mTOR pathway and thus blockade of the pathway effectively inhibits the tobacco-induced malignant transformation of HBE cells.

Identification of novel antiangiogenic anticancer activities of deguelin targeting hypoxia-inducible factor-1 alpha.

Hypoxia-inducible factor 1 (HIF-1) plays an essential role in tumor angiogenesis and growth by regulating the transcription of several genes in response to hypoxic stress and changes in growth factors. This study was designed to investigate the effects of deguelin on tumor growth and angiogenesis, and the mechanisms underlying the antitumor activities of deguelin. We show here that orally administered deguelin inhibits tumor growth and blocks tumor angiogenesis in mice. Deguelin decreased expression of HIF-1alpha protein and its target genes, such as VEGF, in a subset of cancer cell lines, including H1299 lung cancer cells, and vascular endothelial cells in normoxic and hypoxic conditions. Overexpression of vascular endothelial growth factor by adenoviral vector infection abolished the antiangiogenic effects of deguelin on H1299 nonsmall cell lung cancer cells. Deguelin inhibited de novo synthesis of HIF-1alpha protein and reduced the half-life of the synthesized protein. MG132, a proteasome inhibitor, protected the hypoxia- or IGF-induced HIF-1alpha protein from deguelin-mediated degradation. Our findings suggest that deguelin is a promising antiangiogenic therapeutic agent in cancer targeting HIF-1alpha. Considering that HIF-1alpha is overexpressed in a majority of human cancers, deguelin could offer a potent therapeutic agent for cancer.

Concomitant DFMO and sulindac chemoprevention of colorectal adenomas: a major clinical advance.

In a randomized, placebo-controlled, double-blind clinical trial by Meyskens et al. the combination of difluoromethylornithine and sulindac has been shown to be strikingly effective for prevention of sporadic colorectal adenomas. This concomitant use of two drugs to suppress the progression of preneoplastic lesions represents the first major clinical success of the application of the principle of 'combination chemoprevention'. Neither drug alone has previously had clinical utility at the low doses used in this trial. The combination of the two agents has provided synergistic efficacy in suppression of carcinogenesis, while minimizing any undesirable adverse effects. This study should be the impetus for further clinical investigation of the use of multiple drugs for chemoprevention of cancer.

Implication of the insulin-like growth factor-IR pathway in the resistance of non-small cell lung cancer cells to treatment with gefitinib.

PURPOSE: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been found to be effective against lung cancer in vitro, but clinical resistance to these agents has developed as their usage has increased. In this study, we determined whether the insulin-like growth factor I (IGF-I) signaling pathway induces resistance of non-small cell lung cancer (NSCLC) cells to the EGFR tyrosine kinase inhibitor gefitinib. EXPERIMENTAL DESIGN: The effects of gefitinib and cetuximab on NSCLC cells, alone or with an IGF-I receptor (IGF-IR) inhibitor, were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the flow cytometry-based terminal nucleotidyl transferase-mediated nick end labeling assay, coimmunoprecipitation, and Western blot analysis. EGFR and IGFR expression in NSCLC tissues were examined by Western blot analysis. RESULTS: Gefitinib inhibited NSCLC cell proliferation by inducing apoptosis when IGF-IR signaling was suppressed. Treatment with gefitinib, but not cetuximab, induced EGFR:IGF-IR heterodimerization and activation of IGF-IR and its downstream signaling mediators, resulting in increased survivin expression in NSCLC cell lines with high levels of IGF-IR expression. Inhibition of IGF-IR activation and knockdown of survivin expression led to increased apoptosis. In contrast, overexpression of survivin protected cells with low IGF-IR expression from gefitinib-induced apoptosis. Most NSCLC tissues with EGFR overexpression had associated high levels of IGF-IR expression. CONCLUSIONS: IGF-IR expression may be useful as a predictive marker for gefitinib treatment of NSCLC. Suppression of IGF-IR signaling pathways may prevent or delay development of gefitinib resistance in patients with NSCLC.

Identification and validation of differences in protein levels in normal, premalignant, and malignant lung cells and tissues using high-throughput Western Array and immunohistochemistry.

The identification of proteins, which exhibit different levels in normal, premalignant, and malignant lung cells, could improve early diagnosis and intervention. We compared the levels of proteins in normal human bronchial epithelial (NHBE) and tumorigenic HBE cells (1170-I) by high-throughput immunoblotting (PowerBlot Western Array) using 800 monoclonal antibodies. This analysis revealed that 87 proteins increased by >2-fold, and 45 proteins decreased by >2-fold, in 1170-I compared with NHBE cells. These proteins are involved in DNA synthesis and repair, cell cycle regulation, RNA transcription and degradation, translation, differentiation, angiogenesis, apoptosis, cell adhesion, cytoskeleton and cell motility, and the phosphatidylinositol 3-kinase signaling pathway. Conventional Western blotting using lysates of normal, immortalized, transformed, and tumorigenic HBEs and non-small cell lung cancer cell lines confirmed some of these changes. The expression of several of these proteins has been then analyzed by immunohistochemistry in tissue microarrays containing 323 samples, including normal bronchial epithelium, hyperplasia, squamous metaplasia, dysplasias, squamous cell carcinomas, atypical adenomatous hyperplasia, and adenocarcinomas from 144 patients. The results of the immunohistochemical studies correlated with the Western blotting findings and showed gradual increases (caspase-8, signal transducers and activators of transcription 5, and p70s6K) or decrease (E-cadherin) in levels with tumor progression. These results indicate that the changes in proteins detected in this study may occur early in lung carcinogenesis and persist in lung cancer. In addition, some of the proteins detected by this approach may be novel biomarkers for early detection of lung cancer and novel targets for chemoprevention or therapy.

Heterodimerization of insulin-like growth factor receptor/epidermal growth factor receptor and induction of survivin expression counteract the antitumor action of erlotinib.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been used to treat non-small cell lung cancer (NSCLC). However, the overall response rate to EGFR TKIs is limited, and the mechanisms mediating resistance to the drugs are poorly understood. Here, we report that insulin-like growth factor-I receptor (IGF-IR) activation interferes with the antitumor activity of erlotinib, an EGFR TKI. Treatment with erlotinib increased the levels of EGFR/IGF-IR heterodimer localized on cell membrane, activated IGF-IR and its downstream signaling mediators, and stimulated mammalian target of rapamycin (mTOR)-mediated de novo protein synthesis of EGFR and survivin in NSCLC cells. Inhibition of IGF-IR activation, suppression of mTOR-mediated protein synthesis, or knockdown of survivin expression abolished resistance to erlotinib and induced apoptosis in NSCLC cells in vitro and in vivo. Our data suggest that enhanced synthesis of survivin protein mediated by the IGFR/EGFR heterodimer counteracts the antitumor action of erlotinib, indicating the needs of integration of IGF-IR-targeted agents to the treatment regimens with EGFR TKI for patients with lung cancer.

Inhibition of c-Src expression and activation in malignant pleural mesothelioma tissues leads to apoptosis, cell cycle arrest, and decreased migration and invasion.

Malignant pleural mesothelioma (MPM) is a deadly disease with few systemic treatment options. One potential therapeutic target, the non-receptor tyrosine kinase c-Src, causes changes in proliferation, motility, invasion, survival, and angiogenesis in cancer cells and may be a valid therapeutic target in MPM. To test this hypothesis, we determined the effects of c-Src inhibition in MPM cell lines and examined c-Src expression and activation in tissue samples. We analyzed four MPM cell lines and found that all expressed total and activated c-Src. Three of the four cell lines were sensitive by in vitro cytotoxicity assays to the c-Src inhibitor dasatinib, which led to cell cycle arrest and increased apoptosis. Dasatinib also inhibited migration and invasion independent of the cytotoxic effects, and led to the rapid and durable inhibition of c-Src and its downstream pathways. We used immunohistochemical analysis to determine the levels of c-Src expression and activation in 46 archived MPM tumor specimens. The Src protein was highly expressed in tumor cells, but expression did not correlate with survival. However, expression of activated Src (p-Src Y419) on the tumor cell membrane was higher in patients with advanced-stage disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Y419. Lower levels of membrane expression of inactive c-Src (p-Src Y530) correlated with advanced N stage (P = 0.02). Activated c-Src may play a role in survival, metastasis, and invasion of MPM, and targeting c-Src may be an important therapeutic strategy.

Biomarker-Integrated Neoadjuvant Dasatinib Trial in Resectable Malignant Pleural Mesothelioma.

INTRODUCTION: Window of opportunity trials in malignant pleural mesothelioma (MPM) are challenging but can yield important translational information about a novel agent. METHODS: We treated patients with MPM (N = 24) with 4 weeks of oral dasatinib followed by surgery with or without radiotherapy and then an optional 2 years of maintenance dasatinib. The primary end point was biomarker modulation of phosphorylated (p) SrcTyr419. RESULTS: For all patients, the median progression-free survival (PFS) was 7.5 months and the median overall survival was 19.1 months. No significant responses were seen after 4 weeks of dasatinib therapy; however, modulation of median p-SrcTyr419 immunohistochemistry (IHC) scores was seen: the median pretreatment score was 70 (interquartile range 37.5-110), and the median posttreatment score was 41.9 (interquartile range 4.2-60) (p = 0.004). A decrease in p-SrcTyr419 levels after dasatinib correlated with improved median PFS (6.9 months versus 0.94 months [p = 0.03]), suggesting that p-SrcTyr419 is a viable pharmacodynamic biomarker for dasatinib in MPM. Platelet-derived growth factor receptor (PDGFR) pathway analysis correlated high PDGFR beta [PDGFRB) level (in the cytoplasm [hazard ratio] (HR) = 2.54, p = 0.05], stroma [HR = 2.79, p = 0.03], and nucleus [HR = 6.79, p = 0.023]) with a shorter PFS. Low (less than the median) cytoplasmic p-PDGFR alpha IHC levels were predictive of a decrease in positron emission tomography/computed tomography standard uptake values levels after dasatinib therapy (p = 0.04), whereas higher-than-median IHC scores of PDGFRB (cytoplasmic [HR = 2.8, p = 0.03] and nuclear [HR = 6.795, p = 0.02]) were correlated with rising standard uptake values levels. CONCLUSIONS: In conclusion, there was no significant efficacy signal, and dasatinib monotherapy will not continue to be studied in MPM. However, our study demonstrated that PDGFR subtypes (platelet-derived growth factor receptor alpha and PDGFRB) may have differential roles in prognosis and resistance to antiangiogenic tyrosine kinase inhibitors and are important potential therapeutic targets that require further investigation.

Met Receptor Tyrosine Kinase and Chemoprevention of Oral Cancer.

Background: We have previously shown that gene expression profiles of oral leukoplakia (OL) may improve the prediction of oral cancer (OC) risk. To identify new targets for prevention, we performed a systematic survey of transcripts associated with an increased risk of oral cancer and overexpressed in OC vs normal mucosa (NM). Methods: We used gene expression profiles of 86 patients with OL and available outcomes from a chemoprevention trial of OC and NM. MET expression was evaluated using immunohistochemistry in 120 OL patients, and its association with OC development was tested in multivariable analysis. Sensitivity to pharmacological Met inhibition was tested invitro in premalignant and OC cell lines (n = 33) and invivo using the 4-NQO model of oral chemoprevention (n = 20 mice per group). All statistical tests were two-sided. Results: The overlap of 693 transcripts associated with an increased risk of OC with 163 transcripts overexpressed in OC compared with NM led to the identification of 23 overlapping transcripts, including MET. MET overexpression in OL was associated with a hazard ratio of 3.84 (95% confidence interval = 1.59 to 9.27, P = .003) of developing OC. Met activation was found in OC and preneoplastic cell lines. Crizotinib activity in preneoplastic and OC cell lines was comparable. ARQ 197 was more active in preneoplastic compared with OC cell lines. In the 4-NQO model, squamous cell carcinoma, dysplasia, and hyperkeratosis were observed in 75.0%, 15.0%, and 10.0% in the control group, and in 25.0%, 70.0%, and 5.0% in the crizotinib group (P < .001). Conclusion: Together, these data suggest that MET activation may represent an early driver in oral premalignancy and a target for chemoprevention of OC.

Integrative proteomic and transcriptomic analysis provides evidence for TrkB (NTRK2) as a therapeutic target in combination with tyrosine kinase inhibitors for non-small cell lung cancer.

While several molecular targets have been identified for adenocarcinoma (ACA) of the lung, similar drivers with squamous cell carcinoma (SCC) are sparse. We compared signaling pathways and potential therapeutic targets in lung SCC and ACA tumors using reverse phase proteomic arrays (RPPA) from two independent cohorts of resected early stage NSCLC patients: a testing set using an MDACC cohort (N=140) and a validation set using the Cancer Genome Atlas (TCGA) cohorts. We identified multiple potentially targetable proteins upregulated in SCC, including NRF2, Keap1, PARP, TrkB, and Chk2. Of these potential targets, we found that TrkB also had significant increases in gene expression in SCC as compared to adenocarcinoma. Thus, we next validated the upregulation of TrkB both in vitro and in vivo and found that it was constitutively expressed at high levels in a subset of SCC cell lines. Furthermore, we found that TrkB inhibition suppressed tumor growth, invasiveness and sensitized SCC cells to tyrosine kinase EGFR inhibition in a cell-specific manner.

Concomitant targeting of the mTOR/MAPK pathways: novel therapeutic strategy in subsets of RICTOR/KRAS-altered non-small cell lung cancer.

Despite a therapeutic paradigm shift into targeted-driven medicinal approaches, resistance to therapy remains a hallmark of lung cancer, driven by biological and molecular diversity. Using genomic and expression data from advanced non-small cell lung cancer (NSCLC) patients enrolled in the BATTLE-2 clinical trial, we identified RICTOR alterations in a subset of lung adenocarcinomas and found RICTOR expression to carry worse overall survival. RICTOR-altered cohort was significantly enriched in KRAS/MAPK axis mutations, suggesting a co-oncogenic driver role in these molecular settings. Using NSCLC cell lines, we showed that, distinctly in KRAS mutant backgrounds, RICTOR blockade impairs malignant properties and generates a compensatory enhanced activation of the MAPK pathway, exposing a unique therapeutic vulnerability. In vitro and in vivo concomitant pharmacologic inhibition of mTORC1/2 and MEK1/2 resulted in synergistic responses of anti-tumor effects. Our study provides evidence of a distinctive therapeutic opportunity in a subset of NSCLC carrying concomitant RICTOR/KRAS alterations.