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1.  PILOT STUDY OF GEFITINIB AND FULVESTRANT IN THE TREATMENT OF POST-MENOPAUSAL WOMEN WITH ADVANCED NON-SMALL CELL LUNG CANCER 
SUMMARY
Introduction
Estrogen receptor beta (ERβ) has been detected in NSCLC cell lines and tumor specimens. The ER down-regulator, fulvestrant, blocked estradiol-stimulation of tumor growth and gene transcription in NSCLC preclinical models and showed additive effects with the EGFR inhibitor gefitinib. The safety and tolerability of combination therapy with the EGFR inhibitor, gefitinib, and fulvestrant was explored.
Methods
Post-menopausal women with advanced NSCLC received gefitinib 250 mg po daily and fulvestrant 250 mg IM monthly.
Results
Twenty-two patients were enrolled. Eight patients had adenocarcinoma, 6 NSCLC-NOS, 4 squamous cell, and 4 BAC. Seven patients were never smokers. Eight patients received ≥ 2 lines of prior chemotherapy, 6 received one prior chemotherapy, and 8 were treatment naive. One patient experienced grade 4 dyspnea possibly related to treatment; all other grade 3/4 toxicities were unrelated to treatment. Twenty patients were evaluable for response: 3 PRs were confirmed (response rate of 15%, 95% CI: 5% – 36%). The median PFS, OS, and estimated 1 yr OS were 12 wks (3–112 wks), 38.5 weeks (7–135 wks), and 41% (95% CI 20–62%), respectively. Survival outcomes did not differ by prior lines of therapy. A subset analysis revealed that OS in the 8 patients whose tumors exhibited at least 60% ERβ nuclear IHC staining measured 65.5 weeks, while that of the 5 patients with ERβ staining of less than 60% was 21 weeks. One patient with BAC and a PR had an EGFR L858R mutation in exon 21. There was no correlation between ERβ IHC expression and histology or smoking history.
Conclusions
Combination therapy with gefitinib and fulvestrant in this population was well-tolerated and demonstrated disease activity.
doi:10.1016/j.lungcan.2008.07.002
PMCID: PMC3164240  PMID: 18701186
epidermal growth factor receptor; hormonal treatment; non-small cell lung cancer; sex; age; estrogen receptor
2.  Smoking Out Reproductive Hormone Actions in Lung Cancer 
Experimental and population-based evidence has been steadily accumulating that steroid hormones are fundamentally involved in the biology of the lung. Both estrogen and progesterone receptors are present in normal and malignant lung tissue, and the reproductive hormones that bind these receptors have a role in lung development, lung inflammation, and lung cancer. The estrogen receptor β (ERβ) was discovered in the 1990's as a novel form of ER that is transcribed from a gene distinct from ERα, the receptor previously isolated from breast tissue. Interestingly, ERβ is the predominate ER expressed in normal and malignant lung tissue, while inflammatory cells that infiltrate the lung are known to express both ERα and ERβ. Although there is evidence from animal models for the preferential effects of ERβ in the lungs of females, human lung tumors from males often contain comparable numbers of ERβ-positive cells and male-derived lung cancer cell lines respond to estrogens. Lung tumors from both males and females also express CYP19 (aromatase), the rate-limiting enzyme in estrogen synthesis that converts testosterone to estrone and β-estradiol. Thus, testosterone acts as a precursor for local estrogen production within lung tumors, independent of reproductive organs. This review discusses the recent literature findings concerning the biology of the ERs, aromatase, and the progesterone receptor (PR) in lung cancer and highlights the ongoing clinical trials and future therapeutic implications of these findings.
doi:10.1158/1541-7786.MCR-13-0580
PMCID: PMC3972805  PMID: 24398390
3.  Diagnostic Accuracy of MALDI Mass Spectrometric Analysis of Unfractionated Serum in Lung Cancer 
Purpose
There is a critical need for improvements in the noninvasive diagnosis of lung cancer. We hypothesized that matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) analysis of the most abundant peptides in the serum may distinguish lung cancer cases from matched controls.
Patients and Methods
We used MALDI MS to analyze unfractionated serum from a total of 288 cases and matched controls split into training (n = 182) and test sets (n = 106). We used a training–testing paradigm with application of the model profile defined in a training set to a blinded test cohort.
Results
Reproducibility and lack of analytical bias was confirmed in quality-control studies. A serum proteomic signature of seven features in the training set reached an overall accuracy of 78%, a sensitivity of 67.4%, and a specificity of 88.9%. In the blinded test set, this signature reached an overall accuracy of 72.6 %, a sensitivity of 58%, and a specificity of 85.7%. The serum signature was associated with the diagnosis of lung cancer independently of gender, smoking status, smoking pack-years, and C-reactive protein levels. From this signature, we identified three discriminatory features as members of a cluster of truncated forms of serum amyloid A.
Conclusions
We found a serum proteomic profile that discriminates lung cancer from matched controls. Proteomic analysis of unfractionated serum may have a role in the noninvasive diagnosis of lung cancer and will require methodological refinements and prospective validation to achieve clinical utility.
doi:10.1097/JTO.0b013e31814b8be7
PMCID: PMC4220686  PMID: 17909350
Mass spectrometry; Biomarker; Blood; Diagnosis
4.  Kinin B2 Receptor Mediates Induction of Cyclooxygenase-2 and Is Over-Expressed In Head and Neck Squamous Cell Carcinomas 
Molecular cancer research : MCR  2008;6(12):1946-1956.
Bradykinin (BK) has been shown to promote growth and migration of head and neck squamous cell carcinoma (HNSCC) cells via epidermal growth factor receptor (EGFR) transactivation. It has also been reported that BK can cause the induction of cyclooxygenase-2 (COX-2), a pro-tumorigenic enzyme, via the MAPK (Mitogen-Activated Protein Kinase) pathway in human airway cells. To determine whether COX-2 is up-regulated by BK in HNSCC, the current study investigated BK- induced EGFR transactivation, MAPK activation, and cyclooxygenase-2 (COX-2) expression in human HNSCC cells. BK induced a concentration- and time-dependent induction of COX-2 protein in HNSCC, which was preceded by phosphorylation of EGFR and MAPK. These effects were abolished by the B2 receptor (B2R) antagonist Hoe 140 but not the B1 receptor (B1R) antagonist, Lys-[Leu8]des-Arg9-BK. COX-2 induction was accompanied by increased release of PGE2. No effect of a B1R agonist (des-Arg9-BK) on p-MAPK or COX-2 expression was observed. B2R protein was found to be expressed in all four head and neck cell lines tested. Immunohistochemical analysis and immunoblot analysis revealed that B2R, but not B1R, was significantly over-expressed in HNSCC tumors compared to levels in normal mucosa from the same patient. In HNSCC cells, the BK-induced expression of COX-2 was inhibited by the EGFR kinase inhibitor gefitinib or mitogen activated protein kinase kinases (MEK) inhibitors (PD98059 or U0126). These results suggest that EGFR and MAPK are required for COX-2 induction by BK. Up-regulation of the B2R in head and neck cancers suggests this pathway is involved in HNSCC tumorigenesis.
doi:10.1158/1541-7786.MCR-07-2197
PMCID: PMC3575100  PMID: 19074839
Bradykinin; B2 receptor; EGFR; MAPK; cyclooxygenase-2; head and neck cancer
5.  Phase I Trial of Carboplatin and Etoposide in Combination with Panobinostat in Patients with Lung Cancer 
Anticancer research  2013;33(10):4475-4481.
A phase I trial consisting of panobinostat (a HDAC inhibitor), carboplatin and etoposide was condacted in patients with lung cancer. Patients and Methods: Patients received carboplatin AUC5 on day 1 and etoposide 100 mg/m2 on days 1, 2 and 3, every 21 days. Concurrent oral panobinostat was given 3 times weekly on a 2-weeks-on and 1-week-off schedule during the 4–6 cycles of chemotherapy and then continued as maintenance therapy. Results: Six evaluable patients were treated at the first dose level of panobinostat (10 mg). Dose-limiting toxicity occurred in two patients (33%) during the first cycle. One patient developed grade 4 thrombocytopenia and another grade 4 febrile neutropenia. Therefore, the study was suspended based on the pre-specified study design. No recommended phase II starting dose was established. Conclusion: The addition of panobinostat to carboplatin and etoposide was not tolerable at the lowest dose level tested in this trial. Further research and development into this combination is not recommended.
PMCID: PMC4157617  PMID: 24123018
Lung cancer; panobinostat; carboplatin; etoposide
6.  Dual blockade of EGFR and c-Met abrogates redundant signaling and proliferation in head and neck carcinoma cells 
Purpose
Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade.
Experimental Design
Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with anti-tumor responses to inhibitors of these pathways.
Results
Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, transforming growth factor-alpha (TGF-α), were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also demonstrated that TGF-α can induce phosphorylation of c-Met over 6-fold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared to single agent treatment in vivo.
Conclusions
Together these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC.
doi:10.1158/1078-0432.CCR-10-3339
PMCID: PMC3138116  PMID: 21622718
7.  HGF-independent Potentiation of EGFR Action by c-Met 
Oncogene  2011;30(33):3625-3635.
The c-Met receptor is a potential therapeutic target for non-small cell lung cancer (NSCLC). Signaling interactions between c-Met and the mutant Epidermal Growth Factor Receptor (EGFR) have been studied extensively, but signaling intermediates and biological consequences of lateral signaling to c-Met in EGFR wild-type tumors is minimally understood. Our observations indicate that delayed c-Met activation in NSCLC cell lines is initiated by wild-type EGFR, the receptor most often found in NSCLC tumors. EGFR ligands induce accumulation of activated c-Met which begins at 8 h continues for 48 h. This effect is accompanied by an increase in c-Met expression and phosphorylation of critical c-Met tyrosine residues without activation of MAPK or Akt. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of HGF or another secreted factor, supporting a ligand-independent mechanism. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activity of both receptors is required for this effect. Prolonged c-Src phosphorylation is observed, and c-Src pathway is essential for EGFR to c-Met communication. Pre-treatment with pan-SFK inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared to control, further, confirming a c-Src requirement. Inhibition of c-Met with PF2341066 and siRNA decreases EGF-induced phenotypes of invasion by ~86% and motility by ~81%, suggesting that a novel form of c-Met activation is utilized by EGFR to maximize these biological effects. Combined targeting of c-Met and EGFR leads to increased xenograft anti-tumor activity, demonstrating that inhibition of downstream and lateral signaling from the EGFR-c-Src-c-Met axis might be effective in treatment of NSCLC.
doi:10.1038/onc.2011.84
PMCID: PMC3126872  PMID: 21423210
c-Met; c-Src; EGFR; cross-talk
8.  Genetic variation in ESR2 and estrogen receptor-beta expression in lung tumors 
Cancer epidemiology  2013;37(4):518-522.
Objective
To investigate the association between inherited variation in the estrogen receptor beta (ERβ) gene (ESR2) and ERβ lung tumor expression, a phenotype that possibly affects survival differently in men and women.
Methods
We genotyped 135 lung cancer patients for 22 ESR2 single nucleotide polymorphisms (SNPs) and measured nuclear and cytoplasmic ERβ expression by immunohistochemistry (IHC) in their primary lung tumor. Distributing Allred ERβ IHC scores according to ESR2 genotype classified under a dominant genetic model, we used rank sum tests to identify ESR2 SNPs significantly associated (p<0.05) with ERβ expression.
Results
35%, 35%, and 29% of lung tumors showed no/low (Allred <6), intermediate (Allred 6 to 7), and maximal (Allred 8) cytoplasmic ERβ expression, whereas 13%, 27%, and 60% showed no/low, intermediate, and maximal nuclear ERβ expression. For SNPs rs8021944, rs1256061 and rs10146204, ERβ expression was higher according to the rank sum test in lung tumors from patients with at least one minor allele. For each of these three SNPs, the odds of maximal (Allred 8) relative to no/low (Allred <6) ERβ expression was 3-fold higher in tumors from patients with at least one minor allele than in tumors from patients homozygous for the common allele.
Conclusion
Inherited variability in ESR2 may determine ERβ lung tumor expression.
doi:10.1016/j.canep.2013.03.020
PMCID: PMC3684046  PMID: 23619141
lung cancer; genetic polymorphism; estrogen receptor
9.  Validation of a blood protein signature for non-small cell lung cancer 
Clinical Proteomics  2014;11(1):32.
Background
CT screening for lung cancer is effective in reducing mortality, but there are areas of concern, including a positive predictive value of 4% and development of interval cancers. A blood test that could manage these limitations would be useful, but development of such tests has been impaired by variations in blood collection that may lead to poor reproducibility across populations.
Results
Blood-based proteomic profiles were generated with SOMAscan technology, which measured 1033 proteins. First, preanalytic variability was evaluated with Sample Mapping Vectors (SMV), which are panels of proteins that detect confounders in protein levels related to sample collection. A subset of well collected serum samples not influenced by preanalytic variability was selected for discovery of lung cancer biomarkers. The impact of sample collection variation on these candidate markers was tested in the subset of samples with higher SMV scores so that the most robust markers could be used to create disease classifiers. The discovery sample set (n = 363) was from a multi-center study of 94 non-small cell lung cancer (NSCLC) cases and 269 long-term smokers and benign pulmonary nodule controls. The analysis resulted in a 7-marker panel with an AUC of 0.85 for all cases (68% adenocarcinoma, 32% squamous) and an AUC of 0.93 for squamous cell carcinoma in particular. This panel was validated by making blinded predictions in two independent cohorts (n = 138 in the first validation and n = 135 in the second). The model was recalibrated for a panel format prior to unblinding the second cohort. The AUCs overall were 0.81 and 0.77, and for squamous cell tumors alone were 0.89 and 0.87. The estimated negative predictive value for a 15% disease prevalence was 93% overall and 99% for squamous lung tumors. The proteins in the classifier function in destruction of the extracellular matrix, metabolic homeostasis and inflammation.
Conclusions
Selecting biomarkers resistant to sample processing variation led to robust lung cancer biomarkers that performed consistently in independent validations. They form a sensitive signature for detection of lung cancer, especially squamous cell histology. This non-invasive test could be used to improve the positive predictive value of CT screening, with the potential to avoid invasive evaluation of nonmalignant pulmonary nodules.
doi:10.1186/1559-0275-11-32
PMCID: PMC4123246  PMID: 25114662
Lung cancer; Biomarker; SOMAmer; Proteomic; Squamous cell carcinoma; Diagnosis; Preanalytic variability; Sample bias
10.  Combined analysis of estrogen receptor β–1 and progesterone receptor expression identifies lung cancer patients with poor outcome 
Purpose
Steroid hormones and growth factors affect lung cancer, and it is possible they act in concert to influence patient outcome.
Experimental Design
Primary lung tumors and normal lung tissue were analyzed for expression and localization of estrogen receptor α and β–1 ERα and ERβ), aromatase, progesterone receptor (PR), and epidermal growth factor receptor (EGFR).
Results
Tumors expressed higher levels of ERβ compared to matched normal lung, while the reverse was true of PR. High cytoplasmic ERβ expression was identified as an independent negative prognostic predictor of overall survival (OS) (HR=1.67), and low total PR was identified as an independent negative predictor of time to progression (TTP) (HR=1.59). After adjusting for stage, age, sex and smoking, combined high cytoplasmic ERβ and low total PR showed enhanced effects on OS (HR=2.64) and on TTP (HR=6.02). Further effects on OS were observed when EGFR expression was included (HR=5.32). Patients with low cytoplasmic ERβ, low aromatase, low EGFR and high total PR had shorter OS than patients with the opposite pattern (HR= 6.60). Contribution of these markers to survival showed no significant sex differences in a multivariable model. ERα was elevated in tumors but was not predictive of survival, and appears to represent a variant ERα protein that is only recognized by a C-terminal antibody.
Conclusions
Hormonal and EGFR pathways together may contribute to lung cancer prognosis. Lung tumors with high ERβ–1 /low PR may define patients with aggressive biology. A validation study is necessary to fully assess the predictive value of these markers.
doi:10.1158/1078-0432.CCR-10-0992
PMCID: PMC3064257  PMID: 21062926
estrogen receptor; progesterone receptor; aromatase; epidermal growth factor receptor; lung cancer
11.  HGF and c-Met Participate in Paracrine Tumorigenic Pathways in Head and Neck Squamous Cell Cancer 
Purpose
We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling.
Experimental Design
Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice.
Results
c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa.
Conclusions
These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.
doi:10.1158/1078-0432.CCR-08-3252
PMCID: PMC3159511  PMID: 19470725
12.  Early Changes in Pulmonary Gene Expression Following Tobacco Exposure Shed Light on the Role of Estrogen Metabolism in Lung Carcinogenesis 
This perspective on Meireles et al. (beginning on p. XXX in this issue of the journal) discusses the increasing evidence for the role of female steroid hormones in lung-cancer development and progression. The novel work of Meireles et al. is the first evidence for the rapid upregulation by tobacco smoke of a key cytochrome P450 gene that can metabolize estrogens such as β-estradiol to potentially carcinogenic catechol and quinine forms, as well as the first evidence for the colocalization of β-estradiol and estrogen receptors in murine airway epithelium. Actions of estrogens that contribute to lung carcinogenesis, especially in the presence of tobacco smoke, may involve both reactive intermediates that damage DNA and steroid hormone-receptor signaling that promotes growth.
doi:10.1158/1940-6207.CAPR-10-0093
PMCID: PMC2881174  PMID: 20515955
13.  The Prognostic and Predictive Value of KRAS Oncogene Substitutions in Lung Adenocarcinoma 
Cancer  2013;119(12):2268-2274.
Background
The prognostic and therapeutic implications of the spectrum of KRAS oncogene substitutions in lung cancer remain poorly understood. The objective of this study was to determine if KRAS oncogene substitutions differed with regard to prognosis or predictive value in lung adenocarcinoma.
Methods
KRAS oncogene substitutions and mutant-allele specific imbalance (MASI) were determined in patients with lung adenocarcinoma and associations with overall survival (OS) and recurrence free survival (RFS), and chemotherapy interactions were assessed.
Results
KRAS mutational analysis was performed on 988 lung adenocarcinomas, and 318 KRAS mutations were identified. In this predominantly early stage cohort (78.6% stage I–III), OS and RFS did not differ by the type of KRAS substitution (OS, p=0.612; RFS P=0.089). There was a trend toward better OS in the subset of patients with KRAS codon 13 mutations (p=0.052), which was not significant in multivariate analysis (p=0.076). RFS did not differ by codon type in univariate analysis (p=0.322). There was a marked difference in RFS based on the presence of MASI in univariate (p=0.004) and multivariate analysis (p=0.009). A test for interaction was performed in order to determine if the effect of chemotherapy on OS and RFS differed based on the type of KRAS substitution, codon type or the presence of MASI. There were no differences in the effects of chemotherapy for any of variables examined.
Conclusions
KRAS codon 13 mutations and MASI are candidate biomarkers for prognosis that may be useful to incorporate in prospective studies evaluating novel therapies in KRAS mutant lung adenocarcinoma.
doi:10.1002/cncr.28039
PMCID: PMC3674175  PMID: 23526491
KRAS; lung adenocarcinoma; mutant allele-specific imbalance; prognosis; prediction
14.  Estrogen Receptor Signaling in Lung Cancer 
Seminars in oncology  2009;36(6):524.
Lung cancer has long been thought of as a cancer that mainly affects men, but over the past several decades, because of the high increase in tobacco use by women, there has been a corresponding dramatic increase in lung cancer among women. Since 1998, lung cancer deaths in women have surpassed those caused by breast cancer in the United States. Annual lung cancer deaths among women in the US also currently surpass those caused by breast, ovarian, and cervical cancers combined. Women are more likely than men to be diagnosed with adenocarcinoma and small-cell carcinoma of the lung compared to squamous cell carcinoma, and never smokers diagnosed with lung cancer are almost three times more likely to be female than male. These observations in the population, coupled to the findings that both estrogen receptors and aromatase, the enzyme that synthesizes 17β-estradiol, are expressed by lung tumors, suggest a role for female steroid hormones in control of lung cancer growth. Pre-clinical data and clinical data are increasingly emerging to support this concept, and to suggest that a local production of estrogen and expression of ERs occurs in lung tumors that rise in men as well as women. An additional protein that recognizes 17β-estradiol with high affinity, GPR30, is also expressed in lung tumors at high levels and may be responsible for some of the proliferation signals induced by estrogen.
doi:10.1053/j.seminoncol.2009.10.004
PMCID: PMC2818861  PMID: 19995644
15.  Optimal Threshold in CT Quantification of Emphysema 
European radiology  2012;23(4):975-984.
Objectives
To determine the optimal threshold by quantitatively assessing the extent of emphysema at the level of the entire lung and at the level of individual lobes using a large, diverse dataset of CT examinations.
Methods
This study comprises 573 chest CT examinations acquired from different subjects (222 none, 83 mild, 141 moderate, 63 severe, and 64 very severe obstruction). The extent of emphysema was quantified using the percentage of the low attenuation area (LAA%) divided by the total lung or lobe volume(s). The correlations between the extent of emphysema, and pulmonary functions and the five-category classification were assessed using Pearson and Spearman’s correlation coefficients, respectively. When quantifying emphysema using a density mask, a wide range of thresholds from −850 to −1000 HU were used.
Results
The highest correlations of LAA% with the five-category classification and PFT measures ranged from −925 to −965 HU for each individual lobe and the entire lung. However, the differences between the highest r and those obtained at −950 HU are relatively small.
Conclusion
Although there are variations in the optimal cut-off thresholds for individual lobes, the single threshold of −950 HU is still an acceptable threshold for density-based emphysema quantification.
doi:10.1007/s00330-012-2683-z
PMCID: PMC3573224  PMID: 23111815
Chronic obstructive pulmonary disease; computed tomography; pulmonary emphysema; density mask; lobe segmentation
16.  Computerized Identification of Airway Wall in CT Examinations Using a 3D Active Surface Evolution Approach 
Medical image analysis  2012;17(3):283-296.
Airway diseases (e.g., asthma, emphysema, and chronic bronchitis) are extremely common worldwide. Any morphological variations (abnormalities) of airways may physically change airflow and ultimately affect the ability of the lungs in gas exchange. In this study, we describe a novel algorithm aimed to automatically identify airway walls depicted on CT images. The underlying idea is to place a three-dimensional (3D) surface model within airway regions and thereafter allow this model to evolve (deform) under predefined external and internal forces automatically to the location where these forces reach a state of balance. By taking advantage of the geometric and the density characteristics of airway walls, the evolution procedure is performed in a distance gradient field and ultimately stops at regions with the highest contrast. The performance of this scheme was quantitatively evaluated from several perspectives. First, we assessed the accuracy of the developed scheme using a dedicated lung phantom in airway wall estimation and compared it with the traditional full-width at half maximum (FWHM) method. The phantom study shows that the developed scheme has an error ranging from 0.04 mm to 0.36 mm, which is much smaller than the FWHM method with an error ranging from 0.16 mm to 0.84 mm. Second, we compared the results obtained by the developed scheme with those manually delineated by an experienced (>30 years) radiologist on clinical chest CT examinations, showing a mean difference of 0.084 mm. In particular, the sensitivity of the scheme to different reconstruction kernels was evaluated on real chest CT examinations. For the ‘lung’, ‘bone’ and ‘standard’ kernels, the average airway wall thicknesses computed by the developed scheme were 1.302 mm, 1.333 mm and 1.339 mm, respectively. Our preliminary experiments showed that the scheme had a reasonable accuracy in airway wall estimation. For a clinical chest CT examination, it took around 4 minutes for this scheme to identify the inner and outer airway walls on a modern PC.
doi:10.1016/j.media.2012.11.003
PMCID: PMC3606689  PMID: 23260997
17.  Therapeutic targeting of human hepatocyte growth factor with a single neutralizing monoclonal antibody reduces lung tumorigenesis 
Molecular cancer therapeutics  2008;7(7):1913-1922.
The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in lung tumor growth and progression, and agents that target this pathway have clinical potential for lung cancer treatment. L2G7, a single potent anti-human HGF neutralizing monoclonal antibody (mAb), demonstrated profound inhibition of human HGF-induced P-MAPK induction, wound healing and invasion in lung tumor cells in vitro. Transgenic mice that overexpress human HGF in the airways were utilized to study the therapeutic efficacy of L2G7 for lung cancer prevention. Mice were treated with the tobacco carcinogen, nitrosamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), over 4 weeks. Beginning at week 3, intraperitoneal (i.p.) treatment with 100μg L2G7 or isotyped matched antibody control, 5G8, was initiated and continued through week 15. The mean number of tumors per mouse in the L2G7 treated group was significantly lower than in the control group (1.58 versus 3.19, P=0.0005). Proliferative index was decreased by 48% (P=0.013) in tumors from L2G7 treated mice versus 5G8 treated mice while extent of apoptosis was increased in these same tumors by 5–fold (P=0.0013). P-MAPK expression was also significantly decreased by 84% in tumors from L2G7 treated mice versus 5G8 treated mice (P=0.0003). Tumors that arose in HGF transgenic animals despite L2G7 treatment were more likely to contain mutant K-ras, suggesting that targeting the HGF/c-Met pathway may not be as effective if downstream signaling is activated by a K-ras mutation. These preclinical results demonstrate that blocking the HGF/c-Met interaction with a single mAb delivered systemically can have profound inhibitory effects on development of lung tumors.
doi:10.1158/1535-7163.MCT-07-2169
PMCID: PMC2556508  PMID: 18645002
Hepatocyte growth factor; non-small cell lung cancer
18.  Elevated gastrin-releasing peptide receptor mRNA expression in buccal mucosa: association with head and neck squamous cell carcinoma 
Head & neck  2012;35(2):270-279.
Background
Expression of gastrin-releasing peptide receptor (GRPR) is elevated in mucosa adjacent to head and neck squamous cell carcinoma (HNSCC) compared with mucosa from cancer-free controls, suggesting elevated GRPR expression may indicate presence of HNSCC.
Methods
We measured GRPR mRNA levels in histologically normal buccal mucosa from 65 surgical patients with HNSCC and 75 cancer-free control subjects using quantitative polymerase chain reaction (PCR). We tested for association between GRPR expression and HNSCC and evaluated differences in patient progression-free survival (PFS).
Results
Buccal GRPR expression was higher in cases but not controls who were active smokers (p = .04). High GRPR expression was associated with HNSCC (odds ratio [OR] = 3.55; 95% confidence interval [CI] = 1.15–10.93), even after adjustment for age, sex, tobacco use, and sample storage time. PFS did not differ between patients with HNSCC with high versus low GRPR expression (p = .22).
Conclusion
Elevated buccal GRPR expression was significantly associated with HNSCC independent of known risk factors but was not an indicator of disease prognosis.
doi:10.1002/hed.22963
PMCID: PMC3553276  PMID: 22431275
gastrin-releasing peptide receptor; head and neck cancer; case-control study; surrogate tissue biomarker; risk factor
19.  c-Src Activation Mediates Erlotinib Resistance in Head and Neck Cancer by Stimulating c-Met 
PURPOSE
Strategies to inhibit the epidermal growth factor receptor (EGFR) using the tyrosine kinase inhibitor (TKI) erlotinib have been associated with limited clinical efficacy in head and neck squamous cell carcinoma (HNSCC). Co-activation of alternative kinases may contribute to erlotinib resistance.
EXPERIMENTAL DESIGN
We generated HNSCC cells expressing dominant-active c-Src (DA-Src) to determine the contribution of c-Src activation to erlotinib response.
RESULTS
Expression of DA-Src conferred resistance to erlotinib in vitro and in vivo compared with vector-transfected control cells (VC). Phospho-Met was strongly upregulated by DA-Src, and DA-Src cells did not produce hepatocyte growth factor (HGF). Knockdown of c-Met enhanced sensitivity to erlotinib in DA-Src cells in vitro, as did combining a c-Met or c-Src inhibitor with erlotinib. Inhibiting EGFR resulted in minimal reduction of phospho-Met in DA-Src cells, whereas complete phospho-Met inhibition was achieved by inhibiting c-Src. A c-Met inhibitor significantly sensitized DA-Src tumors to erlotinib in vivo, resulting in reduced Ki67 labeling and increased apoptosis. In parental cells, knockdown of endogenous c-Src enhanced sensitivity to erlotinib, while treatment with HGF to directly induce phospho-Met resulted in erlotinib resistance. The level of endogenous phospho-c-Src in HNSCC cell lines was also significantly correlated with erlotinib resistance.
CONCLUSIONS
Ligand-independent activation of c-Met contributes specifically to erlotinib resistance, not cetuximab resistance, in HNSCC with activated c-Src, where c-Met activation is more dependent on c-Src than on EGFR, providing an alternate survival pathway. Addition of a c-Met or c-Src inhibitor to erlotinib may increase efficacy of EGFR inhibition in patients with activated c-Src.
doi:10.1158/1078-0432.CCR-12-1555
PMCID: PMC3549019  PMID: 23213056
20.  Gastrin-Releasing Peptide Receptor Mediates Activation of the Epidermal Growth Factor Receptor in Lung Cancer Cells1 
Neoplasia (New York, N.Y.)  2005;7(4):426-431.
Abstract
Gastrin-releasing peptide receptor (GRPR) and the epidermal growth factor receptor (EGFR) are expressed in several cancers including non-small cell lung cancer (NSCLC). Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP), in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-α or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP) inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18 µM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.
PMCID: PMC1501149  PMID: 15967120
EGFR; GRPR; MAPK; signal transduction; non-small cell lung cancer
21.  Prevention of tobacco carcinogen-induced lung cancer in female mice using antiestrogens 
Carcinogenesis  2012;33(11):2181-2189.
Increasing evidence shows that estrogens are involved in lung cancer proliferation and progression, and most human lung tumors express estrogen receptor β (ERβ) as well as aromatase. To determine if the aromatase inhibitor anastrozole prevents development of lung tumors induced by a tobacco carcinogen, alone or in combination with the ER antagonist fulvestrant, ovariectomized female mice received treatments with the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) along with daily supplements of androstenedione, the substrate for aromatase. Placebo, anastrozole and/or fulvestrant were administered in both an initiation and a promotion protocol of lung tumorigenesis. The combination of fulvestrant and anastrozole given during NNK exposure resulted in significantly fewer NNK-induced lung tumors (mean = 0.5) compared with placebo (mean = 4.6, P < 0.001), fulvestrant alone (mean = 3.4, P < 0.001) or anastrozole alone (mean = 2.8, P = 0.002). A significantly lower Ki67 cell proliferation index was also observed compared with single agent and control treatment groups. Beginning antiestrogen treatment after NNK exposure, when preneoplastic lesions had already formed, also yielded maximum antitumor effects with the combination. Aromatase expression was found mainly in macrophages infiltrating preneoplastic and tumorous areas of the lungs, whereas ERβ was found in both macrophages and tumor cells. Antiestrogens, especially in combination, effectively inhibited tobacco carcinogen-induced murine lung tumorigenesis and may have application for lung cancer prevention. An important source of estrogen synthesis may be inflammatory cells that infiltrate the lungs in response to carcinogens, beginning early in the carcinogenesis process. ERβ expressed by inflammatory and neoplastic epithelial cells in the lung may signal in response to local estrogen production.
doi:10.1093/carcin/bgs260
PMCID: PMC3584962  PMID: 22859269
22.  Genetic variability in DNA repair and cell cycle control pathway genes and risk of smoking-related lung cancer 
Molecular carcinogenesis  2011;51(Suppl 1):E11-E20.
DNA repair and cell cycle control play an important role in the repair of DNA damage caused by cigarette smoking. Given this role, functionally relevant single nucleotide polymorphisms (SNPs) in genes in these pathways may well affect the risk of smoking-related lung cancer. We examined the relationship between 240 SNPs in DNA repair and cell cycle control pathway genes and lung cancer risk in a case-control study of white current and ex-cigarette smokers (722 cases and 929 controls). Additive, dominant and recessive genetic models were evaluated for each SNP. A genetic risk summary score was also constructed. Odds ratios (OR) for lung cancer risk and 95% confidence intervals (95% CI) were estimated using logistic regression models. Thirty-eight SNPs were associated with lung cancer risk in our study population at P<0.05. The strongest associations were observed for rs2074508 in GTF2H4 (Padditive=0.003), rs10500298 in LIG1 (Precessive=2.7×10−4), rs747658 and rs3219073 in PARP1 (rs747658: Padditive=5.8×10−5; rs3219073: Padditive=4.6×10−5), and rs1799782 and rs3213255 in XRCC1 (rs1799782: Pdominant=0.006; rs3213255: Precessive=0.004). Compared to individuals with first quartile (lowest) risk summary scores, individuals with third and fourth quartile summary score results were at increased risk for lung cancer (OR: 2.21, 95% CI: 1.66–2.95 and OR: 3.44, 95% CI: 2.58–4.59, respectively; Ptrend<0.0001). Our data suggests that variation in DNA repair and cell cycle control pathway genes is associated with smoking-related lung cancer risk. Additionally, combining genotype information for SNPs in these pathways may assist in classifying current and ex-cigarette smokers according to lung cancer risk.
doi:10.1002/mc.20858
PMCID: PMC3289753  PMID: 21976407
SNP; case-control; lung cancer
23.  Sex-specific association of sequence variants in CBS and MTRR with risk for promoter hypermethylation in the lung epithelium of smokers 
Carcinogenesis  2012;33(8):1542-1547.
Gene promoter hypermethylation is now regarded as a promising biomarker for the risk and progression of lung cancer. The one-carbon metabolism pathway is postulated to affect deoxyribonucleic acid (DNA) methylation because it is responsible for the generation of S-adenosylmethionine (SAM), the methyl donor for cellular methylation reactions. This study investigated the association of single nucleotide polymorphisms (SNPs) in six one-carbon metabolism-related genes with promoter hypermethylation in sputum DNA from non-Hispanic white smokers in the Lovelace Smokers Cohort (LSC) (n = 907). Logistic regression was used to assess the association of SNPs with hypermethylation using a high/low methylation cutoff. SNPs in the cystathionine beta synthase (CBS) and 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) genes were significantly associated with high methylation in males [CBS rs2850146 (-8283G > C), 
OR = 4.9; 95% CI: 1.98, 12.2, P = 0.0006] and low methylation in females [MTRR rs3776467 (7068A > G), OR = 0.57, 95% CI: 0.42, 0.77, P = 0.0003]. The variant allele of rs2850146 was associated with reduced gene expression and increased plasma homocysteine (Hcy) concentrations. Three plasma metabolites, Hcy, methionine and dimethylglycine, were associated with increased risk for gene methylation. These studies suggest that SNPs in CBS and MTRR have sex-specific associations with aberrant methylation in the lung epithelium of smokers that could be mediated by the affected one-carbon metabolism and transsulfuration in the cells.
Abbreviations:CBScystathionine beta synthaseDNAdeoxyribonucleic acidHBEChuman bronchial epithelial cellHcyhomocysteineLD, linkage disequilibrium; LSClovelace Smokers CohortMAFminor allele frequencyMTHFRmethylenetetrahydrofolate reductaseMTRRmethyltransferase reductaseSNPsingle nucleotide polymorphismsSAHS-adenosylhomocysteineSAMS-adenosylmethionine
doi:10.1093/carcin/bgs194
PMCID: PMC3499054  PMID: 22665368
24.  Tumor epidermal growth factor receptor (EGFR) and EGFR PY1068 are independent prognostic indicators for head and neck squamous cell carcinoma 
Purpose
To assess the prognostic value of EGFR molecular characteristics of head and neck squamous cell carcinoma (HNSCC).
Patients and Methods
HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N=154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N=39) were evaluated for EGFR gene amplification by fluorescence in situ hybridization (FISH) and EGFR protein by immunohistochemical (IHC) staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (RPPA) (n=67). Tumor (n=50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR.
Results
EGFR expression by IHC was significantly higher in the EDRN tumors with EGFR gene amplification (P<0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (p=0.019 and p=0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort (HR=2.75; 95% CI=1.26–6.00 and HR=3.29; 95% CI=1.34–8.14, respectively).
Conclusions
In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR.
doi:10.1158/1078-0432.CCR-11-1593
PMCID: PMC3430124  PMID: 22351687
epidermal growth factor receptor; receptor tyrosine kinase; site-specific phosphorylation; prognosis; head and neck cancer
25.  A Multiplexed Serum Biomarker Immunoassay Panel Discriminates Clinical Lung Cancer Patients from High-Risk Individuals Found to be Cancer-Free by CT Screening 
Journal of Thoracic Oncology  2012;7(4):698-708.
Introduction
Clinical decision-making in the setting of CT screening could benefit from accessible biomarkers that help predict the level of lung cancer risk in high-risk individuals with indeterminate pulmonary nodules.
Methods
To identify candidate serum biomarkers, we measured 70 cancer-related proteins by Luminex xMAP® multiplexed immunoassays in a training set of sera from 56 patients with biopsy-proven primary non small cell lung cancer and 56 age-, sex- and smoking-matched CT-screened controls.
Results
We identified a panel of 10 serum biomarkers – prolactin, transthyretin, thrombospondin-1, E-selectin, C-C motif chemokine 5, macrophage migration inhibitory factor, plasminogen activator inhibitor, receptor tyrosine-protein kinase, Cyfra 21.1, and serum amyloid A – that distinguished lung cancer from controls with an estimated balanced accuracy (average of sensitivity and specificity) of 76.0%±3.8% from 20-fold internal cross-validation. We then iteratively evaluated this model in independent test and verification case/control studies confirming the initial classification performance of the panel. The classification performance of the 10-biomarker panel was also analytically validated using ELISAs in a second independent case/control population further validating the robustness of the panel.
Conclusions
The performance of this 10-biomarker panel based model was 77.1% sensitivity/76.2% specificity in cross-validation in the expanded training set, 73.3% sensitivity/93.3% specificity (balanced accuracy 83.3%) in the blinded verification set with the best discriminative performance in Stage I/II cases: 85% sensitivity (balanced accuracy 89.2%). Importantly, the rate of misclassification of CT-screened controls was not different in most control subgroups with or without airflow obstruction or emphysema or pulmonary nodules. These biomarkers have potential to aid in the early detection of lung cancer and more accurate interpretation of indeterminate pulmonary nodules detected by screening CT.
doi:10.1097/JTO.0b013e31824ab6b0
PMCID: PMC3308353  PMID: 22425918
Lung cancer; serum protein biomarkers; CT screening; Luminex xMAP® immunoassays; pulmonary nodules

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