We propose a randomized phase II clinical trial design based on Bayesian adaptive randomization and predictive probability monitoring. Adaptive randomization assigns more patients to a more efficacious treatment arm by comparing the posterior probabilities of efficacy between different arms. We continuously monitor the trial by using the predictive probability. The trial is terminated early when it is shown that one treatment is overwhelmingly superior to others or that all the treatments are equivalent. We develop two methods to compute the predictive probability by considering the uncertainty of the sample size of the future data. We illustrate the proposed Bayesian adaptive randomization and predictive probability design by using a phase II lung cancer clinical trial, and we conduct extensive simulation studies to examine the operating characteristics of the design. By coupling adaptive randomization and predictive probability approaches, the trial can treat more patients with a more efficacious treatment and allow for early stopping whenever sufficient information is obtained to conclude treatment superiority or equivalence. The design proposed also controls both the type I and the type II errors and offers an alternative Bayesian approach to the frequentist group sequential design.
Adaptive randomization; Bayesian inference; Clinical trial ethics; Group sequential method; Posterior predictive distribution; Randomized trial; Type I error; Type II error
Although the frequentist paradigm has been the predominant approach to clinical trial design since the 1940s, it has several notable limitations. The alternative Bayesian paradigm has been greatly enhanced by advancements in computational algorithms and computer hardware. Compared to its frequentist counterpart, the Bayesian framework has several unique advantages, and its incorporation into clinical trial design is occurring more frequently. Using an extensive literature review to assess how Bayesian methods are used in clinical trials, we find them most commonly used for dose finding, efficacy monitoring, toxicity monitoring, diagnosis/decision making, and for studying pharmacokinetics/pharmacodynamics. The additional infrastructure required for implementing Bayesian methods in clinical trials may include specialized software programs to run the study design, simulation, and analysis, and Web-based applications, which are particularly useful for timely data entry and analysis. Trial success requires not only the development of proper tools but also timely and accurate execution of data entry, quality control, adaptive randomization, and Bayesian computation. The relative merit of the Bayesian and frequentist approaches continues to be the subject of debate in statistics. However, more evidence can be found showing the convergence of the two camps, at least at the practical level. Ultimately, better clinical trial methods lead to more efficient designs, lower sample sizes, more accurate conclusions, and better outcomes for patients enrolled in the trials. Bayesian methods offer attractive alternatives for better trials. More such trials should be designed and conducted to refine the approach and demonstrate its real benefit in action.
adaptive trial design; Bayesian paradigm; clinical trial conduct; frequentist paradigm; trial efficiency; trial ethics
Outcome-adaptive randomization (AR) allocates more patients to the better treatments as the information accumulates in the trial. Is it worth to apply outcome-AR in clinical trials? Different views permeate the medical and statistical communities. We provide additional insights to the question by conducting extensive simulation studies. Trials are designed to maintain the type I error rate, achieve a specified power, and provide better treatment to patients. Generally speaking, equal randomization (ER) requires a smaller sample size and yields a smaller number of non-responders than AR by controlling type I and type II errors. Conversely, AR produces a higher overall response rate than ER with or without expanding the trial to the same maximum sample size. When there exist substantial treatment differences, AR can yield a higher overall response rate as well as a lower average sample size and a smaller number of non-responders. Similar results are found for the survival endpoint. The differences between AR and ER quickly diminish with early stopping of a trial due to efficacy or futility. In summary, ER maintains balanced allocation throughout the trial and reaches the specified statistical power with a smaller number of patients in the trial. If the trial’s result is positive, ER may lead to early approval of the treatment. AR focuses on treating patients best in the trial. AR may be preferred when the difference in efficacy between treatments is large or when limited patients are available.
Adaptive and fixed randomization; Bayesian clinical trial design; Efficacy and futility early stopping; Type I error and statistical power; Patient population; Sample size
This study’s objectives were to determine whether tumor response measured by CT and evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) correlated with overall survival (OS) in patients with non-small cell lung cancer (NSCLC) after neoadjuvant chemotherapy and surgical resection.
We measured primary tumor size on CT before and after neoadjuvant chemotherapy in 160 NSCLC patients who underwent surgical resection. The relationship between CT-measured response (RECIST) and histopathologic response (≤10% viable tumor) and OS were assessed by Kaplan Meier survival, univariable and multivariable Cox proportional hazards regression.
There was a statistically significant association between CT-measured response (RECIST) and OS (p=0.03). However, histopathologic response was a stronger predictor of OS (p=0.002), with a more pronounced separation of the survival curves when compared to CT-measured response. In multivariable Cox regression analysis, only pathologic stage and histopathologic response were significant predictors of OS. A 41% overall discordance rate was noted between CT RECIST response and histopathologic response. CT RECIST classified as non-responders a subset of patients with histopathologic response (8/30 pts, 27%) who demonstrated prolonged survival after neoadjuvant chemotherapy.
We were unable to show that CT RECIST is a reliable predictor of OS in patients with NSCLC undergoing surgical resection after neoadjuvant chemotherapy. The failure of CT RECIST to predict long-term outcome may be due to the inability of CT imaging to consistently identify patients with histopathologic response. CT RECIST may have only a limited role as an efficacy endpoint after neoadjuvant chemotherapy in patients with resectable NSCLC.
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
PIK3CA mutations may predict response to PI3K/AKT/mTOR inhibitors in patients with advanced cancers, but the relevance of mutation subtype has not been investigated. Patients with diverse cancers referred to the Clinical Center for Targeted Therapy were analyzed for PIK3CA and, if possible, KRAS mutations. Patients with PIK3CA mutations were treated, whenever possible, with agents targeting the PI3K/AKT/mTOR pathway. Overall, 105 (10%) of 1,012 patients tested harbored PIK3CA mutations. Sixty-six (median 3 prior therapies) of the 105 PIK3CA-mutant patients (including 16 individuals (of 55 PIK3CA-mutant patients tested) with simultaneous KRAS mutations) were treated on a protocol that included a PI3K/AKT/mTOR pathway inhibitor; 17% (11/66) achieved a partial response (PR). Patients with a PIK3CA H1047R mutation compared to patients with other PIK3CA mutations or patients with wild-type PIK3CA treated on the same protocols had a higher PR rate (6/16, 38% vs. 5/50, 10% vs. 23/174, 13%, respectively; all p ≤ 0.02). None of the 16 patients with co-existing PIK3CA and KRAS mutations in codon 12 or 13 attained a PR (0/16, 0%). Patients treated with combination therapy vs. single-agent therapies had a higher PR rate (11/38, 29% vs. 0/28, 0%; p=0.002). Multivariate analysis showed that H1047R was the only independent factor predicting response (odds ratio (OR) 6.6, 95% CI 1.02–43.0, p = 0.047). Our data suggest that interaction between PIK3CA mutation H1047R vs. other aberrations and response to PI3K/AKT/mTOR axis inhibitors warrants further exploration.
Gene expression alterations in response to cigarette smoke have been characterized in normal-appearing bronchial epithelium of healthy smokers and it has been suggested that adjacent histologically normal tissue display tumor-associated molecular abnormalities. We sought to delineate the spatial and temporal molecular lung field of injury in smoker early stage non-small cell lung cancer (NSCLC) patients (n=19) who were accrued into a surveillance clinical trial for annual follow-up and bronchoscopies within one year after definitive surgery. Bronchial brushings and biopsies were obtained from six different sites in the lung at the time of inclusion in the study and at 12, 24 and 36 months after the first time point. Affymetrix Human Gene 1.0 ST arrays were used for whole-transcript expression profiling of airways (n=391). Microarray analysis identified gene features (n=1165) that were non-uniform by site and differentially expressed between airways adjacent to tumors relative to more distant samples as well as those (n=1395) that were significantly altered with time up to three years. In addition, gene-interaction networks mediated by PI3K and ERK1/2 were modulated in adjacent compared to contralateral airways and the latter network with time. Furthermore, phosphorylated AKT and ERK1/2 immunohistochemical expression were significantly increased with time (nuclear pAKT, p=0.03; cytoplasmic pAKT, p<0.0001; pERK1/2, p=0.02) and elevated in adjacent compared to more distant airways (nuclear pAKT, p=0.04; pERK1/2, p=0.03). This study highlights spatial and temporal cancer-associated expression alterations in the molecular field of injury of early stage NSCLC patients after definitive surgery that warrant further validation in independent studies.
Early stage NSCLC; gene expression profiling; lung airway epithelium; chemoprevention
A major barrier to oral cancer prevention has been the lack of validated risk predictors for oral premalignant lesions (OPLs). In 2000, we proposed a loss of heterozygosity (LOH) risk model in a retrospective study. This paper validated the previously reported LOH profiles as risk predictors and developed refined models via the largest longitudinal study to date of low-grade OPLs from a population-based patient group. Analysis involved a prospective cohort of 296 patients with primary mild/moderate oral dysplasia enrolled in the Oral Cancer Prediction Longitudinal Study. LOH status was determined in these OPLs. Patients were classified into high-risk or low-risk profiles to validate the 2000 model. Risk models were refined using recursive partitioning and Cox regression analyses. The prospective cohort validated that the high-risk lesions (3p &/or 9p LOH) had a 22·6 - fold increase in risk (P = 0·002) compared to low-risk lesions (3p & 9p retention). Addition of another two markers (loci on 4q/17p) further improved the risk prediction, with five-year progression rates of 3·1%, 16·3%, and 63·1% for the low-, intermediate-, and high-risk lesions, respectively. Compared to the low-risk group, intermediate- and high-risk groups had 11·6-fold and 52·1-fold increase in risk (P < 0·001). LOH profiles as risk predictors in the refined model were validated in the retrospective cohort. Multi-covariate analysis with clinical features showed LOH models to be the most significant predictors of progression. LOH profiles can reliably differentiate progression risk for OPLs. Potential uses include increasing surveillance for patients with elevated risk, improving target intervention for high-risk patients while sparing a large number of low-risk patients from needless screening and treatment.
Platinum resistance is a major limitation in the treatment of advanced non–small-cell lung cancer (NSCLC). Reduced intracellular drug accumulation is one of the most consistently identified features of platinum-resistant cell lines, but clinical data are limited. We assessed the effects of tissue platinum concentrations on response and survival in NSCLC.
Patients and Methods
We measured total platinum concentrations by flameless atomic absorption spectrophotometry in 44 archived fresh-frozen NSCLC specimens from patients who underwent surgical resection after neoadjuvant platinum-based chemotherapy. Tissue platinum concentration was correlated with percent reduction in tumor size on post- versus prechemotherapy computed tomography scans. The relationship between tissue platinum concentration and survival was assessed by univariate and multicovariate Cox proportional hazards regression model analysis and Kaplan-Meier analysis.
Tissue platinum concentration correlated significantly with percent reduction in tumor size (P < .001). The same correlations were seen with cisplatin, carboplatin, and all histology subgroups. Furthermore, there was no significant impact of potential variables such as number of cycles and time lapse from last chemotherapy on platinum concentration. Patients with higher platinum concentration had longer time to recurrence (P = .034), progression-free survival (P = .018), and overall survival (P = .005) in the multicovariate Cox model analysis after adjusting for number of cycles.
This clinical study established a relationship between tissue platinum concentration and response in NSCLC. It suggests that reduced platinum accumulation might be an important mechanism of platinum resistance in the clinical setting. Further studies investigating factors that modulate intracellular platinum concentration are warranted.
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
RECIST is used to quantify tumor changes during exposure to anticancer agents. Responses are categorized as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Clinical trials dictate a patient's management options based on the category into which his or her response falls. However, the association between response and survival is not well studied in the early trial setting.
Patients and Methods
To study the correlation between response as quantified by RECIST and overall survival (OS, the gold-standard survival outcome), we analyzed 570 participants of 24 phase I trials conducted between October 2004 and May 2009, of whom 468 had quantifiable changes in tumor size. Analyses of Kaplan-Meier estimates of OS by response and null Martingale residuals of Cox models were the primary outcome measures. All analyses are landmark analyses.
Kaplan-Meier analyses revealed strong associations between change in tumor size by RECIST and survival (P = 4.5 × 10−6 to < 1 × 10−8). The relationship was found to be near-linear (R2 = 0.75 to 0.92) and confirmed by the residual analyses. No clear inflection points were found to exist in the relationship between tumor size changes and survival.
RECIST quantification of response correlates with survival, validating RECIST's use in phase I trials. However, the lack of apparent boundary values in the relationship between change in tumor size and OS demonstrates the arbitrary nature of the CR/PR/SD/PD categories and questions emphasis placed on this categorization scheme. Describing tumor responses as a continuous variable may be more informative than reporting categoric responses when evaluating novel anticancer therapies.
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.
If detected early, oral cancer is eminently curable. However, survival rates for oral cancer patients remain low, largely due to late stage diagnosis and subsequent difficulty of treatment. To improve clinicians’ ability to detect early disease and to treat advanced cancers, we developed a multi-modal optical imaging system (MMIS) to evaluate tissue in situ, at macroscopic and microscopic scales. The MMIS was used to measure anatomical 100 sites in 30 patients, correctly classifying 98% of pathologically confirmed normal tissue sites, and 95% of sites graded as moderate dysplasia, severe dysplasia, or cancer. When used alone, MMIS classification accuracy was 35% for sites determined by pathology as mild dysplasia. However, MMIS measurements correlated with expression of candidate molecular markers in 87% of sites with mild dysplasia. These findings support the ability of non-invasive multi-modal optical imaging to accurately identify neoplastic tissue and pre-malignant lesions. This in turn may have considerable impact on detection and treatment of patients with oral cancer and other epithelial malignancies.
Optical imaging; clinical diagnostics; fluorescence imaging; biomarker imaging
The purpose of this study was to characterize insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) expression in patients with non-small cell lung cancer (NSCLC).
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.
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 to 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.
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.
Carcinoma; Non-Small-Cell Lung; Receptor; Insulin; Receptor; IGF Type 1; Prognosis; Survival
Folate receptor alpha (FRα) and reduced folate carrier-1 (RFC1) regulate uptake of folate molecules inside the cell. FRα is a potential biomarker of tumors response to antifolate chemotherapy and a target for therapy using humanized monocloncal antibody. Information on the protein expression of these receptors in non–small cell lung carcinoma (NSCLC) is limited.
Material and Methods
Expressions of FRα and RFC1 were examined by IHC in 320 surgically resected NSCLC (202 adenocarcinomas and 118 squamous cell carcinomas) tissue specimens and correlated with patients’ clinicopathologic characteristics. FOLR1 mRNA expression was examined using publicly available microarray datasets. FRα expression was correlated with thymidylate synthase (TS) and p53 expression in NSCLCs, and with EGFR and KRAS mutations in adenocarcinomas.
NSCLC overexpressed FRα and RFC1. In a multivariate analysis, lung adenocarcinomas were more likely to express FRα in the cytoplasm (odds ratio [OR] = 4.39; P<0.0001) and membrane (OR = 5.34; P<0.0001) of malignant cells than squamous cell carcinomas. Tumors from never-smokers were more likely to express cytoplasmic (OR = 3.35; P<0.03) and membrane (OR = 3.60; P=0.0005) FRα than those from smokers. In adenocarcinoma, EGFR mutations correlated with higher expression of membrane FRα and FOLR1 gene expressions. High levels of FRα expression was detected in 42 NSCLC advanced metastatic tumor tissues.
FRα and RFC1 proteins are overexpressed in NSCLC tumor tissues. The high levels of FRα in lung adenocarcinomas may be associated to these tumors’ better responses to antifolate chemotherapy and represents a potential novel target for this tumor type.
non–small cell lung carcinoma; EGFR; membrane transporter; FRα; FRC1
Mutations in the p53 gene are amongst the most frequent aberrations seen in human cancer. Our objective was to characterize the clinical characteristics associated with p53 mutation in patients with advanced cancer.
We retrospectively reviewed and analyzed the clinical features and response to standard systemic therapy of 145 patients with documented tumor p53 mutational status (mutant-type [mtp53] vs. wild-type [wtp53]) referred to the Clinical Center for Targeted Therapy.
Sixty-six (45.5%) patients had mtp53. Mutations in p53 occurred more frequently in older patients (p= 0.015) and in Caucasians (p=0.024). The incidence of liver metastases was 69.2% vs. 43%, p=0.002 in mtp53 and wtp53, respectively. PTEN loss by immunohistochemistry was found more frequently in mtp53-bearing tumors compared to wtp53 (33.3% vs. 10%, p=0.007). The best progression-free survival (PFS) on standard systemic therapy was significantly longer with bevacizumab-containing regimens as compared to non-bevacizumab containing regimen in patients with mtp53 (median 11.0 [95% CI 5.9-16.0], n=22 vs. 4.0 months [95% CI 3.6-5.7], n=35, p<0.0001) but not those with wtp53 (median 5.0 [95% CI 2.0-7.6] vs. 6.0 [95% CI 4.0-7.5] months, p=0.318. The median overall survival from diagnosis in patients with mtp53 and wtp53 was 7.4 [95% CI 6.3-9.8] vs. 11.8 [95% CI 2.9-21.5] years, respectively (p=0.365).
Patients with mtp53 tumors were older at diagnosis, had more incidence of liver metastasis, and more frequent PTEN loss. The best PFS on standard systemic therapy was significantly longer with bevacizumab-containing regimens in patients with mutant p53 tumors but not in those with wtp53.
P53 mutations; PTEN loss; bevacizumab; Li-Fraumeni syndrome
Applying the Emax model in a Lowe additivity model context, we analyze data from a combination study of trimetrexate (TMQ) and AG2034 (AG) in media of low and high concentrations of folic acid (FA). The Emax model provides a sufficient fit to the data. TMQ is more potent than AG in both low and high FA media. At low TMQ:AG ratios, when a smaller amount of the more potent drug (TMQ) is added to a larger amount of the less potent drug (AG), synergy results. When the TMQ:AG ratio reaches 0.4 or larger in low FA medium, or when the TMQ:AG ratio reaches 1 or larger in high FA medium, synergy is weakened and drug interaction becomes additive. In general, synergistic effect in a dilution series is stronger at higher doses that produce stronger effects (closer to 1−Emax) than at lower dose levels that produce weaker effects (closer to 1). The two drugs are more potent in the low compared to the high FA medium. Drug synergy, however, is stronger in the high FA medium.
additivity; antagonism; confidence interval estimation; Emax model; Loewe additivity model; nonlinear regression; synergy; trellis plot
Understanding oncogenes and tumor suppressor genes expression patterns is essential for characterizing lung cancer pathogenesis. We have previously demonstrated that mGprc5a/hGPRC5A is a lung-specific tumor suppressor evidenced by inflammation-mediated tumorigenesis in Gprc5a-knockout mice. The implication of GPRC5A in human lung cancer pathogenesis, including that associated with inflammatory chronic obstructive pulmonary disease (COPD), a risk factor for the malignancy, remains elusive.
We sought to examine GPRC5A immunohistochemical expression in histologically normal bronchial epithelia (NBE) from lung disease-free never- and ever-smokers (n = 13 and n = 18, respectively), from COPD patients with (n = 26) and without cancer (n = 24) and in non-small cell lung cancers (NSCLCs) (n = 474). Quantitative assessment of GPRC5A transcript expression in airways (n = 6), adjacent NBEs (n = 29) and corresponding tumors (n = 6) from 6 NSCLC patients was also performed.
GPRC5A immunohistochemical expression was significantly lower in tumors compared to uninvolved NBE (p < 0.0001) and was positively associated with adenocarcinoma histology (p < 0.001). GPRC5A airway expression was highest in lung disease-free NBE, decreased and intermediate in NBE of cancer-free COPD patients (p = 0.004) and further attenuated and lowest in epithelia of COPD patients with adenocarcinoma and SCC (p < 0.0001). Furthermore, GPRC5A mRNA was significantly decreased in NSCLCs and corresponding NBE compared to uninvolved normal lung (p = 0.03).
Our findings highlight decreased GPRC5A expression in the field cancerization of NSCLC, including that associated with lung inflammation. Assessment of the use of GPRC5A expression as a risk factor for NSCLC development in COPD patients is warranted.
Field cancerization; Chronic obstructive pulmonary disease; Non–small-cell lung cancer; g-protein coupled receptor family C; group 5; member A; gene expression
Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) play a critical role in cancer cell growth and resistance to therapy. Most mutations occur at codons 12 and 13. In colorectal cancer, the presence of any mutant KRas amino acid substitution is a negative predictor of patient response to targeted therapy. However, in non–small cell lung cancer (NSCLC), the evidence that KRAS mutation is a predictive factor is conflicting.
We used data from a molecularly targeted clinical trial for 215 patients with tissues available out of 268 evaluable patients with refractory NSCLC to examine associations between specific mutant KRas proteins and progression-free survival and tumor gene expression. Transcriptome microarray studies of patient tumor samples and reverse-phase protein array studies of a panel of 67 NSCLC cell lines with known substitutions in KRas and in immortalized human bronchial epithelial cells stably expressing different mutant KRas proteins were used to investigate signaling pathway activation. Molecular modeling was used to study the conformations of wild-type and mutant KRas proteins. Kaplan–Meier curves and Cox regression were used to analyze survival data. All statistical tests were two-sided.
Patients whose tumors had either mutant KRas-Gly12Cys or mutant KRas-Gly12Val had worse progression-free survival compared with patients whose tumors had other mutant KRas proteins or wild-type KRas (P = .046, median survival = 1.84 months) compared with all other mutant KRas (median survival = 3.35 months) or wild-type KRas (median survival = 1.95 months). NSCLC cell lines with mutant KRas-Gly12Asp had activated phosphatidylinositol 3-kinase (PI-3-K) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling, whereas those with mutant KRas-Gly12Cys or mutant KRas-Gly12Val had activated Ral signaling and decreased growth factor–dependent Akt activation. Molecular modeling studies showed that different conformations imposed by mutant KRas may lead to altered association with downstream signaling transducers.
Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.
PIK3CA mutations are frequently diagnosed in diverse cancers and may predict response to PI3K/AKT/mTOR inhibitors. It remains unclear whether they are associated with other characteristics. We analyzed characteristics and outcome of 90 consecutive patients with diverse advanced tumors and PIK3CA mutations and 180 wild-type PIK3CA controls matched by tumor type, gender, and age referred to the Clinical Center for Targeted Therapy. PIK3CA and MAPK mutations (KRAS, NRAS, and BRAF) were analyzed using polymerase chain reaction-based DNA sequencing. The most frequent PIK3CA mutations were E545K (31/90, 34%), E542K (16/90, 18%) in exon 9, and H1047R (20/90, 22%) in exon 20. PIK3CA mutations compared to wild-type PIK3CA were associated with simultaneous KRAS (p=0.047) and MAPK mutations (p=0.03), but only MAPK mutations were confirmed as having an independent association in multivariate analysis. Rates of lung, bone, liver and brain metastases were similar in PIK3CA-mutant and wild-type patients. Patients with PIK3CA mutations treated on trials with PI3K/AKT/mTOR inhibitors had a higher partial/complete response (PR/CR) rate than wild-type PIK3CA patients treated with their best phase I therapy (10/56, 18% vs. 12/152, 8%; p=0.045), but not a prolonged progression-free survival. Patients with H1047R PIK3CA mutations had a higher PR/CR rate with PI3K/AKT/mTOR inhibitors compared to wild-type PIK3CA patients treated with their best phase I therapy (6/16, 38% vs. 12/152, 8%; p=0.003). In conclusion, PIK3CA mutations in diverse cancers were not associated with clinical characteristics, but were correlated with MAPK mutations. PIK3CA mutations, especially, H1047R, were associated with attaining a PR/CR to PI3K/AKT/mTOR pathway inhibitors.
PIK3CA mutation; phenotypic taxonomy; clinical outcome
Lung cancer is the leading cause of cancer death, developing over prolonged periods through genetic and epigenetic changes induced and exacerbated by tobacco exposure. Many epigenetic changes including DNA methylation and histone methylation and acetylation are reversible, and agents that can modulate these aberrations are a potentially effective approach to cancer chemoprevention. Combined epigenetic-targeting agents have gained interest for their potential to increase efficacy and lower toxicity. The present study applied recently developed statistical methods to validate the combined effects of the demethylating agent 5-aza-2-deoxycytidine (5-AZA-CdR, or AZA, or decitabine) and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, or vorinostat). This validation compared AZA alone with SAHA alone and with their combinations (at later or earlier time points and in varying doses) for inhibiting the growth of cell lines of an in vitro lung carcinogenesis system. This system comprises isogenic premalignant and malignant cells that are immortalized (earlier premalignant), transformed (later premalignant), and tumorigenic human bronchial epithelial (HBE) cells (immortalized BEAS-2B and its derivatives 1799 [immortalized], 1198 [transformed], and 1170-I [tumorigenic]). AZA alone and SAHA alone produced a limited (< 50%) inhibition of cell growth, whereas combined AZA and SAHA inhibited cell growth more than did either agent alone, reaching 90% inhibition under some conditions. Results of drug-interaction analyses in the Emax model and semiparametric model supported the conclusion that the drug combinations exert synergistic effects, i.e., beyond additivity in the Loewe model. The present results demonstrate the applicability of our novel statistical methodology for quantitatively assessing drug synergy across a wide range of doses of agents with complex dose-response profiles, a methodology with great potential for advancing the development of chemopreventive combinations.
lung cancer; epigenetics; vorinostat; decitabine; premalignant; epithelial cells
We evaluated the ability of histopathologic response criteria to predict overall survival (OS) and disease-free survival (DFS) in patients with surgically resected non-small cell lung cancer (NSCLC) treated with or without neoadjuvant chemotherapy.
Tissue specimens from 358 patients with NSCLC were evaluated by pathologists blinded to the patient treatment and outcome. The surgical specimens were reviewed for various histopathologic features in the tumor including percentage of residual viable tumor cells, necrosis, and fibrosis. The relationship between the histopathologic findings and OS was assessed.
The percentage of residual viable tumor cells and surgical pathologic stage were associated with OS and DFS in 192 patients with NSCLC receiving neoadjuvant chemotherapy in multivariate analysis (p = 0.005 and p = 0.01, respectively). There was no association of OS or DFS with percentage of viable tumor cells in 166 patients with NSCLC who did not receive neoadjuvant chemotherapy (p = 0.31 and p = 0.45, respectively). Long-term OS and DFS were significantly prolonged in patients who had ≤10% viable tumor compared with patients with >10% viable tumor cells (5 years OS, 85% versus 40%, p < 0.0001 and 5 years DFS, 78% versus 35%, p < 0.001).
The percentages of residual viable tumor cells predict OS and DFS in patients with resected NSCLC after neoadjuvant chemotherapy even when controlled for pathologic stage. Histopathologic assessment of resected specimens after neoadjuvant chemotherapy could potentially have a role in addition to pathologic stage in assessing prognosis, chemotherapy response, and the need for additional adjuvant therapies.
Lung cancer; Neoadjuvant chemotherapy; Histopathology
Retinoids have shown antiproliferative and chemopreventive activity. We analyzed data from a randomized, placebo-controlled chemoprevention trial to determine whether a 3-month treatment with either 9-cis-retinoic acid (RA) or 13-cis-RA and α-tocopherol reduced Ki-67, a proliferation biomarker, in the bronchial epithelium.
Former smokers (n = 225) were randomly assigned to receive 3 months of daily oral 9-cis-RA (100 mg), 13-cis-RA (1 mg/kg) and α-tocopherol (1200 IU), or placebo. Bronchoscopic biopsy specimens obtained before and after treatment were immunohistochemically assessed for changes in the Ki-67 proliferative index (i.e., percentage of cells with Ki-67–positive nuclear staining) in the basal and parabasal layers of the bronchial epithelium. Per-subject and per–biopsy site analyses were conducted. Multicovariable analyses, including a mixed-effects model and a generalized estimating equations model, were used to investigate the treatment effect (Ki-67 labeling index and percentage of bronchial epithelial biopsy sites with a Ki-67 index ≥ 5%) with adjustment for multiple covariates, such as smoking history and metaplasia. Coefficient estimates and 95% confidence intervals (CIs) were obtained from the models. All statistical tests were two-sided.
In per-subject analyses, Ki-67 labeling in the basal layer was not changed by any treatment; the percentage of subjects with a high Ki-67 labeling in the parabasal layer dropped statistically significantly after treatment with 13-cis-RA and α-tocopherol treatment (P = .04) compared with placebo, but the drop was not statistically significant after 9-cis-RA treatment (P = .17). A similar effect was observed in the parabasal layer in a per-site analysis; the percentage of sites with high Ki-67 labeling dropped statistically significantly after 9-cis-RA treatment (coefficient estimate = −0.72, 95% CI = −1.24 to −0.20; P = .007) compared with placebo, and after 13-cis-RA and α-tocopherol treatment (coefficient estimate = −0.66, 95% CI = −1.15 to −0.17; P = .008).
In per-subject analyses, treatment with 13-cis-RA and α-tocopherol, compared with placebo, was statistically significantly associated with reduced bronchial epithelial cell proliferation; treatment with 9-cis-RA was not. In per-site analyses, statistically significant associations were obtained with both treatments.
Vascular endothelial growth factor-2 (VEGFR-2 or KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected NSCLC (n=248). KDR copy number gains (CNGs), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR=5.16; P=0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear HIF-1α in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11 and 21 by PCR-based sequencing; however, two variant SNP genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.
Agents can enter clinical development for cancer prevention either initially or after previous development for a different indication, such as arthritis, with both approaches consuming many years of development before an agent is fully evaluated for cancer prevention. We propose the following, third approach: Reverse migration, that is, importing agents, targets and study designs to personalize interventions, and concepts developed in advanced cancer to the setting of cancer prevention. Importing these “ready-made” features from therapy will allow reverse migration to streamline preventive-agent development. We recently reported the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial of personalized lung-cancer therapy and now propose the reverse-migration development of personalized lung-cancer prevention based on the BATTLE model.
chemoprevention; personalized; targeted; lung cancer