To report the clinical efficacy of sorafenib and to evaluate biomarkers associated with sorafenib clinical benefit in the BATTLE program.
Patients and Methods
Patients with previously treated non-small–cell lung cancer (NSCLC) received sorafenib until progression or unacceptable toxicity. Eight-week disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Prespecified biomarkers included K-RAS, EGFR, and B-RAF mutations, and EGFR gene copy number. Gene expression profiles from NSCLC cell lines and patient tumor biopsies with wild-type EGFR were used to develop a sorafenib sensitivity signature (SSS).
105 patients were eligible and randomized to receive sorafenib. Among 98 patients evaluable for 8-week DCR, the observed DCR was 58.2%. The median PFS and OS were 2.83 (95% confidence interval [CI], 2.04-3.58) and 8.48 months (95% CI, 5.78-10.97), respectively. Eight-week DCR was higher in patients with wt-EGFR than patients with EGFR mutation (P=0.012), and in patients with EGFR gene copy number gain (FISH positive) versus patients FISH negative (P=0.048). In wt-EGFR tumors, the SSS was associated with improved PFS (median PFS 3.61 months in high SSS versus 1.84 months in low SSS, P=0.026) but not with 8-week DCR. Increased expression of fibroblast growth factor-1, NF-kB and hypoxia pathways were identified potential drivers of sorafenib resistance.
Sorafenib demonstrates clinical activity in NSCLC, especially with wt-EGFR. SSS was associated with improved PFS. These data identify subgroups that may derive clinical benefit from sorafenib and merit investigation in future trials. ClinicalTrials.gov: NCT00411671.
multikinase inhibitor; non–small cell lung cancer; sorafenib; biomarkers; targeted treatment
Selenium has been reported to have chemopreventive benefits in lung cancer. We conducted a double-blind, placebo-controlled trial to evaluate the incidence of second primary tumors (SPTs) in patients with resected non–small-cell lung cancer (NSCLC) receiving selenium supplementation.
Patients and Methods
Patients with completely resected stage I NSCLC were randomly assigned to take selenized yeast 200 μg versus placebo daily for 48 months. Participation was 6 to 36 months postoperatively and required a negative mediastinal node biopsy, no excessive vitamin intake, normal liver function, negative chest x-ray, and no other evidence of recurrence.
The first interim analysis in October 2009, with 46% of the projected end points accumulated, showed a trend in favor of the placebo group with a low likelihood that the trial would become positive; thus, the study was stopped. One thousand seven hundred seventy-two participants were enrolled, with 1,561 patients randomly assigned. Analysis was updated in June 2011 with the maturation of 54% of the planned end points. Two hundred fifty-two SPTs (from 224 patients) developed, of which 98 (from 97 patients) were lung cancer (38.9%). Lung and overall SPT incidence were 1.62 and 3.54 per 100 person-years, respectively, for selenium versus 1.30 and 3.39 per 100 person-years, respectively, for placebo (P = .294). Five-year disease-free survival was 74.4% for selenium recipients versus 79.6% for placebo recipients. Grade 1 to 2 toxicity occurred in 31% of selenium recipients and 26% of placebo recipients, and grade ≥ 3 toxicity occurred in less than 2% of selenium recipients versus 3% of placebo recipients. Compliance was excellent. No increase in diabetes mellitus or skin cancer was detected.
Selenium was safe but conferred no benefit over placebo in the prevention of SPT in patients with resected NSCLC.
Lung cancer is the leading cancer cause of mortality worldwide; large-scale trials have failed to improve clinical outcomes of patients with chemorefractory non-small-cell lung cancer (NSCLC).
Following an initial equal randomization period, BATTLE adaptively randomized patients with chemorefractory NSCLC to erlotinib, vandetanib, erlotinib plus bexarotene, or sorafenib based on molecular biomarkers of NSCLC pathogenesis in fresh core needle biopsy specimens. The primary end point was disease control rate (DCR) at 8 weeks.
Of 255 patients randomly assigned to erlotinib (59 patients), vandetanib (54), erlotinib plus bexarotene (37), and sorafenib (105), 244 were eligible for the DCR analysis. Pneumothorax after lung biopsy occurred in 11.5% and treatment-related toxicities grade 3–4 in 6.5% of patients. Overall results were a 46% 8-week DCR, 1.9-month median progression-free survival, 9-month median overall survival, and 35% 1-year survival. Individual markers predicting a significantly superior DCR for a treatment included: epidermal growth factor receptor (EGFR) mutation (P=0.04) for erlotinib; cyclin D1 positivity (P=0.01) or EGFR amplification (P=0.006) for erlotinib plus bexarotene; vascular endothelial growth factor receptor 2 positivity (P=0.05) for vandetanib; and absence of EGFR mutation (P=0.01) or of EGFR high polysomy (P=0.05) for sorafenib. A better 8-week DCR occurred with sorafenib versus all other regimens (64% versus 33%; P<0.001) among EGFR wild-type patients and versus all other regimens (61% versus 32%; P=0.11) among mutant-KRAS patients. The prespecified biomarker groups were less predictive than the individual biomarkers analyzed in this study.
The first completed biopsy-mandated study in pretreated NSCLC, BATTLE confirmed our pre-specified hypotheses regarding biomarker and targeted treatment interactions, establishing a new paradigm for personalizing therapy for patients with NSCLC. (ClinicalTrials.gov numbers, NCT00409968, NCT00411671, NCT00411632, NCT00410059, NCT00410189.)
Oral premalignant lesions (OPLs) are precursors of oral squamous cell carcinoma (OSCC). Short telomeres in peripheral blood leukocytes are associated with increased risks of several cancers. However, whether short leukocyte telomere length (LTL) predisposes to OPL and OSCC is unclear.
LTLs were measured in PBLs of 266 patients with OPL (N=174) or OSCC (N=92) at diagnosis and 394 age- and gender-matched control subjects. The association between LTL and OPL or OSCC risk, as well as the interaction of telomere length, cigarette smoking and alcohol drinking on OPL or OSCC risk were analyzed.
The age-adjusted relative LTL was the shortest in OSCC (1.64±0.29), intermediate in OPL (1.75±0.43), and longest in controls (1.82±0.36) (P for trend < 0.001). When dichotomized at the median value in controls, adjusting for age, gender, smoking and alcohol drinking status, the odds ratio (OR) for OPL and OSCC risks associated with short LTL was 2.03 (95% CI = 1.29–3.21) and 3.47 (95% CI = 1.84–6.53), respectively, with significant dose-response effects for both associations. Among 174 OPL patients, 23 progressed to OSCC and the mean LTL was shorter than in progressors than non-progressors (1.66±0.35 vs. 1.77±0.44), although the difference did not reach statistical significance (P=0.258) likely due to the small number of progressors. Interaction analysis shows that short LTL, smoking, and alcohol drinking are independent risk factors for OPL and OSCC.
Short LTL is associated with increased risks of developing OPL and OSCC and likely predisposes to the malignant progression of OPL patients.
Telomere length; peripheral blood leukocyte; oral premalignant lesion; oral squamous cell carcinoma; smoking; alcohol drinking
In a previous trial, we found that combined 13-cis retinoic acid (13-cRA), interferon-α and α-tocopherol more effectively reversed advanced premalignant lesions of the larynx than of the oral cavity and that cyclin D1 (CD1)G/A870 single nucleotide polymorphism correlated with cancer risk. We conducted the present trial primarily to confirm the clinical activity of the combination in advanced laryngeal premalignancy and to confirm and extend our findings on CD1, both genotype and protein expression, in association with cancer risk in this setting. Twenty-seven moderate-to-severe laryngeal dysplasia patients underwent induction with combined 13-cRA daily, α-interferon twice weekly, and α-tocopherol daily for one year; 14 non-progressing patients then were randomized to maintenance fenretinide or placebo for two years. During induction, 2 patients had pathological complete responses, 6 had partial responses (30% overall response rate), and 5 developed laryngeal cancer. There were no significant differences between maintenance fenretinide and placebo in response or cancer rates. Ten patients developed cancer overall. Twenty-four patients were evaluated for the CD1 G/A870 genotype, and 23 for pre- and post-treatment CD1 protein expression. Consistent with our earlier report, shorter cancer-free survival was associated with the CD1 AA/AG genotype (p = 0.05). Extending our earlier work, high CD1 expression was associated with worse cancer-free survival overall (p= 0.04) and within each CD1 genotype group. These findings support CD1 genotype and protein expression as important risk markers for laryngeal cancer and suggest future trials targeting upstream regulators of CD1 transcription.
Premalignant lesions; larynx; biochemoprevention; cyclin D1 genotype; cyclin D1 protein expression
Several phase II/III trials of anti–insulin-like growth factor 1 receptor (IGF-1R) monoclonal antibodies (mAbs) have shown limited efficacy. The mechanisms of resistance to IGF-1R mAb-based therapies and clinically applicable strategies for overcoming drug resistance are still undefined.
IGF-1R mAb cixutumumab efficacy, alone or in combination with Src inhibitors, was evaluated in 10 human head and neck squamous cell carcinoma (HNSCC) and six non–small cell lung cancer (NSCLC) cell lines in vitro in two- or three-dimensional culture systems and in vivo in cell line– or patient-derived xenograft tumors in athymic nude mice (n = 6–9 per group). Cixutumumab-induced changes in cell signaling and IGF-1 binding to integrin β3 were determined by Western or ligand blotting, immunoprecipitation, immunofluorescence, and cell adhesion analyses and enzyme-linked immunosorbent assay. Data were analyzed by the two-sided Student t test or one-way analysis of variance.
Integrin β3–Src signaling cascade was activated by IGF-1 in HNSCC and NSCLC cells, when IGF-1 binding to IGF-1R was hampered by cixutumumab, resulting in Akt activation and cixutumumab resistance. Targeting integrin β3 or Src enhanced antitumor activity of cixutumumab in multiple cixutumumab-resistant cell lines and patient-derived tumors in vitro and in vivo. Mean tumor volume of mice cotreated with cixutumumab and integrin β3 siRNA was 133.7mm3 (95% confidence interval [CI] = 57.6 to 209.8mm3) compared with those treated with cixutumumab (1472.5mm3; 95% CI = 1150.7 to 1794.3mm3; P < .001) or integrin β3 siRNA (903.2mm3; 95% CI = 636.1 to 1170.3mm3; P < .001) alone.
Increased Src activation through integrin ανβ3 confers considerable resistance against anti–IGF-1R mAb-based therapies in HNSCC and NSCLC cells. Dual targeting of the IGF-1R pathway and collateral integrin β3–Src signaling module may override this resistance.
The initial report of the Selenium and Vitamin E Cancer Prevention Trial (SELECT) found no reduction in risk of prostate cancer with either selenium or vitamin E supplements but a non-statistically significant increase in prostate cancer risk with vitamin E. Longer follow-up and more prostate cancer events provide further insight into the relationship of vitamin E and prostate cancer.
To determine the long-term effect of vitamin E and selenium on risk of prostate cancer in relatively healthy men.
Design, Setting and Participants
SELECT randomized 35,533 men from 427 study sites in the United States, Canada and Puerto Rico in a double-blind manner between August 22, 2001 and June 24, 2004. Eligible men were 50 years or older (African Americans) or 55 years or older (all others) with a PSA ≤4.0 ng/mL and a digital rectal examination not suspicious for prostate cancer. Included in the analysis are 34,887 men randomly assigned to one of four treatment groups: selenium (n=8752), vitamin E (n=8737), both agents (n=8702), or placebo (n=8696). Data reflect the final data collected by the study sites on their participants through July 5, 2011.
Oral selenium (200 μg/day from L-selenomethionine) with matched vitamin E placebo, vitamin E (400 IU/d of all rac-α-tocopheryl acetate) with matched selenium placebo, both agents, or both matched placebos for a planned follow-up of a minimum of 7 and maximum of 12 years.
Main Outcome Measures
Prostate cancer incidence.
This report includes 54,464 additional person-years of follow-up since the primary report. Hazard ratios (99% confidence intervals [CI]) and numbers of prostate cancers were 1.17(99% CI 1.004-1.36, p=.008, n=620) for vitamin E, 1.09 (99% CI 0.93-1.27, p=.18, n=575) for selenium, 1.05 (99%CI 0.89-1.22, p=.46, n=555) for selenium + vitamin E vs. 1.00 (n=529) for placebo.The absolute increase in risk compared with placebo for vitamin E, selenium and the combination were 1.6, 0.9 and 0.4 cases of prostate cancer per 1,000 person-years.
Dietary supplementation with Vitamin E significantly increases the risk of prostate cancer among healthy men.
clinicaltrials.gov identifier: NCT00006392
In a previous phase II trial, we demonstrated that fenretinide 200 mg/day had limited activity in retinoid-refractory leukoplakia (34% response rate), possibly due to the lack of achievement of high serum levels which would be required to elicit retinoid-receptor independent apoptosis in pre-malignant cells. We therefore designed this single-arm, phase II trial to investigate whether fenretinide at a higher dose would improve the leukoplakia response rate from our previous study’s historical control.
Patients and Methods
Patients with high-risk leukoplakia were treated with 4 three-week cycles of fenretinide (900 mg/m2 orally twice daily, days 1–7). At week 12, objective clinical responses were determined and blood samples were collected for serum drug level assessment. The original sample size was 25 patients to detect a 55% response rate (90% power, one-sided 10% type I error rate). A futility analysis was planned after accrual of the first 16 patients to allow for early trial closure if ≤4 patients responded.
Fenretinide was well tolerated, with only one grade 3 toxicity (diarrhea) observed. However, only 3 of the initial 15 patients (20%) had a partial response, leading to early trial termination due to lack of efficacy. Serum levels of fenretinide rose from 0 (baseline) to 0.122 μM ± 0.093 (week 12), indicating that high serum levels of the drug were achieved during the initial days of the cycle.
Despite high serum levels, fenretinide for oral leukoplakia, at the dose and schedule studied herein, failed to improve historical response rates.
fenretinide; oral pre-malignant lesion; leukoplakia; chemoprevention
As therapy for non-small cell lung cancer (NSCLC) patients becomes more personalized, additional tissue in the form of core needle biopsies (CNBs) for biomarker analysis is increasingly required for determining appropriate treatment and for enrollment into clinical trials. We report our experience with small-caliber percutaneous transthoracic (PT) CNBs for the evaluation of multiple molecular biomarkers in BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination), a personalized, targeted therapy NSCLC clinical trial.
The medical records of patients who underwent PTCNB for consideration of enrollment in BATTLE, were reviewed for diagnostic yield of 11 predetermined molecular markers, and procedural complications. Univariate and multivariate analyses of factors related to patient and lesion characteristics were performed to determine possible influences on diagnostic yield.
One hundred and seventy PTCNBs were performed using 20-gauge biopsy needles in 151 NSCLC patients screened for the trial. 82.9% of the biopsy specimens were found to have adequate tumor tissue for analysis of the required biomarkers. On multivariate analysis, metastatic lesions were 5.4 times more likely to yield diagnostic tissue as compared to primary tumors (p = 0.0079). Pneumothorax and chest tube insertion rates were 15.3% and 9.4%, respectively.
Image-guided 20-gauge PTCNB is safe and provides adequate tissue for analysis of multiple biomarkers in the majority of patients being considered for enrollment into a personalized, targeted therapy NSCLC clinical trial. Metastatic lesions are more likely to yield diagnostic tissue as compared to primary tumors.
research biopsy; biomarker analysis; percutaneous transthoracic biopsy
This study was designed to identify TGF-β signaling pathway-related serum microRNAs (miRNAs) as predictors of survival in advanced non-small cell lung cancer (NSCLC). Serum samples from 391 patients with advanced NSCLC were collected prior to treatment. Global miRNA microarray expression profiling based on sera from four patients with good survival (>24 months) and four patients with poor survival (<6 months) was used to identify 140 highly expressed serum miRNAs, among which 35 miRNAs had binding sites within the 3’-untranslated regions of a panel of 11 genes in the TGF-β signaling pathway and were assayed by quantitative RT-PCR for their associations with survival in a training (n=192) and testing set (n=191). Out of the 35 miRNAs, survival analysis using Cox regression model identified 17 miRNAs significantly associated with 2-year patient survival. MiR-16 exhibited the most statistically significant association: high expression of miR-16 was associated with a significantly better survival (adjusted hazard ratio = 0.4, 95% confidence interval: 0.3–0.5). A combined 17-miRNA risk score was created that was able to identify patients at the highest risk of death. Those with a high risk score had a 2.5-fold increased risk of death compared to those with a low risk score (95% CI=1.8–3.4, P=1.1×10−7). This increase in risk of death was corresponding to an 7.8 month decrease in median survival time (P=9.5×10−14). Our results suggest that serum miRNAs could serve as predictors of survival for advanced NSCLC.
serum miRNA; TGF-β; survival; NSCLC
Secondary analyses of two randomized controlled trials (RCTs) and supportive epidemiologic and preclinical indicated the potential of selenium and vitamin E for preventing prostate cancer.
To determine whether selenium or vitamin E or both could prevent prostate cancer with little or no toxicity in relatively healthy men.
Design, Setting, and Participants
Randomization of a planned 32,400 men to selenium, vitamin E, selenium plus vitamin E, and placebo in a double-blinded fashion. Participants were recruited and followed in community practices, local hospitals and HMOs, and tertiary cancer centers in the United States, Canada and Puerto Rico. Baseline eligibility included 50 years or older (African American) or 55 years or older (all others), a serum prostate-specific antigen (PSA) ≤ 4 ng/mL, and a digital rectal examination (DRE) not suspicious for prostate cancer. Between 2001 and 2004, 35,533 men (10% more than planned because of a faster-than-expected accrual rate) were randomly assigned to the four study arms, which were well balanced with respect to all potentially important risk factors.
Oral selenium (200 µg/day from L-selenomethionine) and matched vitamin E placebo, vitamin E (400 IU/day of all rac-α-tocopheryl acetate) and matched selenium placebo, or the two combined or placebo plus placebo for a planned minimum of 7 and maximum of 12 years.
Main Outcome Measures
Prostate cancer (as determined by routine community diagnostic standards) and prespecified secondary outcomes including lung, colorectal and overall cancer.
Study supplements were discontinued at the recommendation of the Data and Safety Monitoring Committee at a planned 7-year interim analysis because the evidence convincingly demonstrated no benefit from either study agent (p < 0.0001) and no possibility of a benefit to the planned degree with additional follow-up. As of October 23, 2008, median overall follow-up was 5.46 years (range, 4.17 and 7.33). Hazard ratios (number of prostate cancers, 99% confidence intervals [CIs]) for prostate cancer were 1.13 for vitamin E (n=473; CI, 0.91–1.41), 1.04 for selenium (n=432; CI, 0.83–1.30), and 1.05 for the combination (n=437; CI, 0.83–1.31) compared with placebo (n=416). There were no significant differences (all p-values > 0.15) in any prespecified cancer endpoints. There were nonsignificant increased risks of prostate cancer in the vitamin E arm (p=0.06; relative risk [RR]=1.13; 99% CI, 0l95–1.35) and of Type 2 diabetes mellitus in the selenium arm (p=0.16; RR=1.07; 99% CI, 0.94–1.22), but they were not observed in the combination arm.
Selenium or vitamin E, alone or in combination, did not prevent prostate cancer in this population at the doses and formulations used.
Tumor cells, with stem-like properties, are highly aggressive and often display drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung, and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumor xenografts or in clinical specimens from lung cancer patients that had progressed on erlotinib. Mechanistically, αvβ3, in the unligated state, recruits KRAS and RalB to the tumor cell plasma membrane, leading to the activation of TBK-1/NFκB. In fact, αvβ3 expression and the resulting KRAS/RalB/NFκB pathway were both necessary and sufficient for tumor initiation, anchorage-independence, self-renewal, and erlotinib resistance. Pharmacological targeting of this pathway with Bortezomib reversed both tumor stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but they reveal a therapeutic strategy to sensitize such tumors to RTK inhibition.
To identify the genetic factors that influence overall survival in never smokers who have non-small cell lung cancer (NSCLC), we performed a consistency meta-analysis study utilizing genome-wide association approaches for overall survival in 327 never smoker NSCLC patients from the MD Anderson Cancer Center and 293 cases from the Mayo Clinic. We then performed a two-pronged validation of the top 25 variants that included additional validation in 1,256 NSCLC patients from Taiwan and assessment of expression quantitative trait loci (eQTL) and differential expression of genes surrounding the top loci in 70 tumors and matched normal tissues. A total of 94 loci were significant for overall survival in both MD Anderson and Mayo studies in the consistency meta-analysis phase, with the top 25 variants reaching a p-value of 10−6. Two variants of these 25 were also significant in the Taiwanese population: rs6901416 (HR:1.44, 95%CI:1.01-2.06) and rs10766739 (HR:1.23, 95%CI:1.00-1.51). These loci resulted in a reduction in median survival time of at least 8 and 5 months in three populations, respectively. An additional six variants (rs4237904, rs7976914, rs4970833, rs954785, rs485411, and rs10906104) were validated through eQTL analysis that identified significant correlations with expression levels of six genes (LEMD3, TMBIM, ATXN7L2, SHE, ITIH2, and NUDT5, respectively) in normal lung tissue. These genes were also significantly differentially expressed between the tumor and normal lung. These findings identify several novel, candidate prognostic markers for NSCLC in never smokers, with eQTL analysis suggesting a potential biological mechanism for a subset of these observed associations.
Given the density of single nucleotide polymorphisms (SNPs) in the human genome and the sensitivity of single nucleotide changes in microRNA (miRNA) functionality and processing, we asked whether polymorphisms within miRNA processing pathways and binding sites may influence non-small cell lung cancer (NSCLC) patients’ prognosis. We genotyped 240 miRNA-related SNPs in 535 stage I and II NSCLC patients to determine associations with overall recurrence and survival, as well as effect in specific treatment subgroups. After correcting for multiple comparisons, the G allele of FZD4:rs713065 displayed a significant association with decreased risk of death in surgery-only patients (HR:0.46, 95%CI:0.32-0.65). DROSHA:rs6886834 variant A allele (HR:6.38, 95%CI:2.49-16.31) remained significant for increased risk of recurrence in the overall and surgery-only populations, respectively. FAS:rs2234978 G allele remained significantly associated with survival in all patients (HR:0.59, 95%CI:0.44-0.77), while borderline significant in subgroups (surgery only: HR:0.59, 95%CI:0.42-0.84; surgery plus chemo: HR:0.19, 95%CI:0.07-0.46). Luciferase assays demonstrated that the FAS SNP created a miR-651 functional binding site. Survival tree analysis was performed to classify patients into distinct risk subgroups based on their risk genotype combinations. These results indicate that miRNA-related polymorphisms may be associated with NSCLC patients’ clinical outcomes through altered miRNA regulation of target genes.
NSCLC; recurrence; overall survival; early stage; miRNA; binding site; single nucleotide polymorphism
CXCR2 in non-small cell lung cancer (NSCLC) has been studied mainly in stromal cells and is known to increase tumor inflammation and angiogenesis. Here, we examined the prognostic importance of CXCR2 in NSCLC and the role of CXCR2 and its ligands in lung cancer cells. The effect of CXCR2 expression on tumor cells was studied using stable knockdown clones derived from a murine KRAS/p53-mutant lung adenocarcinoma cell line with high metastatic potential and an orthotopic syngeneic mouse model and in vitro using a CXCR2 small molecule antagonist (SB225002). CXCR2 protein expression was analyzed in tumor cells from 262 NSCLC. Gene expression profiles for CXCR2 and its ligands (CXCR2 axis) were analyzed in 52 human NSCLC cell lines and 442 human lung adenocarcinomas. Methylation of CXCR2 axis promoters was determined in 70 human NSCLC cell lines. Invasion and metastasis were decreased in CXCR2 knockdown clones in vitro and in vivo. SB225002 decreased invasion in vitro. In lung adenocarcinomas, CXCR2 expression in tumor cells was associated with smoking and poor prognosis. CXCR2 axis gene expression profiles in human NSCLC cell lines and lung adenocarcinomas defined a cluster driven by CXCL5 and associated with smoking, poor prognosis and RAS pathway activation. Expression of CXCL5 was regulated by promoter methylation. The CXCR2 axis may be an important target in smoking-related lung adenocarcinoma.
lung cancer; prognosis; metastasis; CXCR2; chemokine
EMT has been associated with metastatic spread and EGFR inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using NSCLC cell lines and patients treated in the BATTLE study.
We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from NSCLC patients. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination) study, and potential therapeutic targets associated with EMT were identified.
Compared with epithelial cells, mesenchymal cells demonstrated significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend towards greater sensitivity to the Axl inhibitor SGI-7079, while the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In NSCLC patients, the EMT signature predicted 8-week disease control in patients receiving erlotinib, but not other therapies.
We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype
lung cancer; EMT; EGFR inhibition; PI3K inhibition; Axl
Epidemiologic data support an inverse association between green tea intake and breast cancer risk and numerous experimental studies have demonstrated the anti-tumor effects of its main component, epigallocatechin gallate (EGCG). We conducted a phase IB dose escalation trial in women with a history of stage I-III hormone receptor-negative breast cancer of an oral green tea extract, Polyphenon E (Poly E) 400mg, 600mg, 800mg bid or matching placebo for 6 months. The primary endpoint was to determine the maximum tolerated dose (MTD), defined as the dose that causes 25% dose limiting toxicity (DLT, grade≥2). Assignment to dose level was based upon an adaptive design, the continual reassessment method. A mammogram and random core biopsy of the contralateral breast were obtained at baseline and 6 months and serial blood/urine collections every 2 months for biomarker analyses. Forty women were randomized: 10 to placebo, 30 to Poly E (16 at 400mg, 11 at 600mg, 3 at 800mg). There was 1 DLT at 400mg (grade 3 rectal bleeding), 3 DLTs at 600mg (grade 2 weight gain, grade 3 indigestion and insomnia), and 1 DLT at 800mg (grade 3 liver function abnormality). The DLT rate at 600mg was 27% (3/11). Pharmacologic levels of total urinary tea polyphenols were achieved with all three dose levels of Poly E. Using a novel phase I trial design, we determined the MTD for Poly E to be 600mg bid. This study highlights the importance of assessing toxicity for any chemopreventive agent being developed for chronic use in healthy individuals.
green tea; chemoprevention; breast cancer; biomarkers
Finasteride, an inhibitor of 5 α-reductase (Type II), lowers intraprostatic dihydrotestosterone (DHT), which is reflected in serum as reduced 5α-androstane-3α,17β-diol glucuronide (3α-dG). It also modestly increases serum testosterone (T), estrone (E1) and estradiol (E2). In this altered hormonal milieu, it is unknown whether serum concentrations of these hormones are associated with prostate cancer risk.
In this nested case-control study of men in the finasteride arm of the Prostate Cancer Prevention Trial, sex steroid hormones and sex hormone binding globulin (SHBG) were measured at baseline and approximately 3-years post-treatment in 553 prostate cancer cases and 694 controls.
Median post-treatment changes in concentrations of 3α-dG, T, E1, and E2 were −73.8%, +10.1%, +11.2%, and +7.5% (all p<0.001), respectively. Neither the pre- nor post-treatment concentrations of 3α-dG, nor its change, were associated with risk. Pre-treatment, high concentrations of E1 and low concentrations of T were associated with increased cancer risk (Odds Ratio[95% CI] quartile 4 vs 1: 1.38[0.99–1.93] ptrend=0.03; 0.64 [0.43–0.93] ptrend=0.07, respectively). Post-treatment, high concentrations of both E1 and E2 and were associated with increased cancer risk (OR[95% CI] quartile 4 vs 1: 1.54[1.09–2.17] ptrend=0.03; 1.49[1.07–2.07] ptrend=0.02, respectively).
Among finasteride-treated men, concentrations of 3α-dG were not associated with total or Gleason grades 2–6, 7–10 or 8–10 cancer. High serum estrogens may increase cancer risk when intraprostatic DHT is pharmacologically lowered.
Low post-treatment serum estrogens may identify men more likely to benefit from use of finasteride to prevent prostate cancer.
PX-478 is a potent small-molecule inhibitor of HIF-1α. In preclinical studies, it had antitumor activity against various solid tumors in subcutaneous xenografts but had no measurable activity against a non-small cell lung cancer (NSCLC) xenograft. To determine the effectiveness of PX-478 against lung tumors, we investigated HIF-1α expression in several lung cancer cell lines, both in vitro and in vivo, and treated orthotopic mouse models of human lung cancer with PX-478.
Cells from two human lung adenocarcinoma cell models (PC14-PE6 and NCI-H441) or two human small cell lung cancer (SCLC) models (NCI-H187 and NCI-N417) were injected into the left lungs of nude mice and were randomized 16 to 18 days after injection with daily oral treatment with PX-478 or vehicle for 5 days.
In the PC14-PE6 NSCLC model, treatment with 20 mg/kg PX-478 significantly reduced the median primary lung tumor volume by 87% (p = 0.005) compared with the vehicle-treated group. PX-478 treatment also markedly reduced mediastinal metastasis and prolonged survival. Similar results were obtained in a second NSCLC model. In SCLC models, PX-478 was even more effective. In the NCI-H187 model, the median primary lung tumor volume was reduced by 99% (p = 0.0001). The median survival duration was increased by 132%. In the NCI-N417 model, the median primary lung tumor volume was reduced by 97% (p = 0.008).
We demonstrated that the PX-478, HIF-1α inhibitor, had significant antitumor activity against two orthotopic models of lung adenocarcinomas and two models of SCLC. These results suggest the inclusion of lung cancer patients in phase I clinical trials of PX-478.
Hypoxia; HIF-1α; PX-478; Orthotopic model; Lung cancer
Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLC and NSCLC. SCLC demonstrated lower levels of several receptor tyrosine kinases and decreased activation of PI3K and Ras/MEK pathways, but significantly increased levels of E2F1-regulated factors including EZH2, thymidylate synthase, apoptosis mediators, and DNA repair proteins. Additionally, poly (ADP-ribose) polymerase 1 (PARP1), a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLC. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than NSCLC, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy.
The Eph family of receptors is the largest family of receptor tyrosine kinases, but it remains poorly studied in lung cancer. Our aim was to systematically explore the human Eph receptors and their ligands, the ephrins, in lung adenocarcinoma. The prognostic impact of Eph receptor and ephrin gene expression was analyzed using 2 independent cohorts of lung adenocarcinoma. Gene expression profiles in lung adenocarcinoma versus normal adjacent lung were studied in 3 independent cohorts and in cell lines. Gene expression profiles were validated with quantitative polymerase chain reaction (qPCR) and Western blotting in cell lines. Functional studies to assess the role of Eph receptor A4 (EphA4) were performed in vitro. The biological effects of EphA4 in lung cancer cell lines were assayed following overexpression and knockdown. Of the 11 Eph receptors and 8 ephrins analyzed, only EphA4 and ephrin A1 gene expression were consistently associated with an improved outcome in patients with lung adenocarcinoma. Expression levels of EphA4 by microarray correlated well with expression levels measured by qPCR and Western blotting. EphA4 overexpression reduced cell migration and invasion but did not affect cell cycle, apoptosis, or drug sensitivity. Surprisingly, EphA4 was expressed at higher levels in cancer versus non-cancer tissues and cell lines. EphA4 gene expression is associated with an improved outcome in patients with resected lung adenocarcinoma, likely by affecting cancer cell migration and invasion.
non-small cell lung cancer; adenocarcinoma; Eph receptor; ephrin; prognosis
We adopted a two-stage study design to screen 927 single nucleotide polymorphisms (SNPs) located in 73 apoptotic-pathway genes in a case-control study and then performed a fast-track validation of the significant SNPs in a replication population to identify sequence variations in the apoptotic pathway modulating lung cancer risk. Fifty-five SNPs showed significant associations in the discovery population comprised of 661 lung cancer cases and 959 controls. Six of these SNPs located in three genes (Bcl-2, CASP9 and ANKS1B) were validated in a replication population with 1154 cases and 1373 controls. Additive model was the best-fitting model for five SNPs (rs1462129 and rs255102 of Bcl-2, rs6685648 of CASP9 and rs1549102, rs11110099 of ANKS1B) and recessive model was the best fit for one SNP (rs10745877 of ANKS1B). In the analysis of joint effects with subjects carrying no unfavorable genotypes as the reference group, those carrying one, two, and three or more unfavorable genotypes had an odds ratio (OR) of 2.22 [95% confidence interval (CI) = 1.08–4.57, P = 0.03], 2.70 (95% CI = 1.33–5.49; P = 0.006) and 4.13 (95% CI = 2.00–8.57; P = 0.0001), respectively (P for trend = 6.05E-06). The joint effect of unfavorable genotypes was also validated in the replication population. The SNPs identified are located in or near key genes known to play important roles in apoptosis regulation, supporting the strong biological relevance of our findings. Future studies are needed to identify the causal SNPs and elucidate the underlying molecular mechanisms.
Most patients with non–small cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). We investigated the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling in primary resistance to EGFR TKIs and the molecular determinants of resistance to IGF-1R TKIs.
Phosphorylated IGF-1R/insulin receptor (pIGF-1R/IR) was immunohistochemically evaluated in a NSCLC tissue microarray. We 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 MAPK/extracellular signal-regulated kinase kinase (MEK), in vitro and in vivo in NSCLC cells with variable histologic features and EGFR or K-Ras mutations.
pIGF-1R/IR expression in NSCLC specimens was associated with a history of tobacco smoking, squamous cell carcinoma histology, mutant (mut) 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 mut 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 mut K-Ras.
The mutation status of both EGFR and K-Ras could be predictive markers of response to IGF-1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF-1R TKIs.
EGFR; K-Ras; IGF-1R; lung cancer; TKI
The development of second primary tumors (SPT) or recurrence alters prognosis for curatively-treated head and neck squamous cell carcinoma (HNSCC) patients. 13-cis-retnoic acid (13-cRA) has been tested as a chemoprevention agent in clinical trials with mixed results. Therefore, we investigated if genetic variants in the PI3K/PTEN/AKT/MTOR pathway could serve as biomarkers to identify which patients are at high risk of an SPT/recurrence while also predicting response to 13-cRA chemoprevention.
A total of 137 pathway SNPs were genotyped in 440 patients from the Retinoid Head and Neck Second Primary Trial and assessed for SPT/recurrence risk and response to 13-cRA. Risk models were created based on epidemiology, clinical, and genetic data.
Twenty-two genetic loci were associated with increased SPT/recurrence risk with six also being associated with a significant benefit following chemoprevention. Combined analysis of these high-risk/high-benefit loci identified a significant (P = 1.54×10−4) dose-response relationship for SPT/recurrence risk, with patients carrying 4–5 high-risk genotypes having a 3.76-fold (95%CI:1.87–7.57) increase in risk in the placebo group (n=215). Patients carrying 4–5 high-risk loci showed the most benefit from 13-cRA chemoprevention with a 73% reduction in SPT/recurrence (95%CI:0.13–0.58) compared to those with the same number of high-risk genotypes who were randomized to receive placebo. Incorporation of these loci into a risk model significantly improved the discriminatory ability over models with epidemiology, clinical, and previously identified genetic variables.
These results demonstrate that loci within this important pathway could identify individuals with a high-risk/high-benefit profile and are a step towards personalized chemoprevention for HNSCC patients.