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1.  Validation of a Molecular and Pathological Model for Five-Year Mortality Risk in Patients with Early Stage Lung Adenocarcinoma 
Journal of Thoracic Oncology  2014;10(1):67-73.
Introduction:
The aim of this study was to validate a molecular expression signature [cell cycle progression (CCP) score] that identifies patients with a higher risk of cancer-related death after surgical resection of early stage (I-II) lung adenocarcinoma in a large patient cohort and evaluate the effectiveness of combining CCP score and pathological stage for predicting lung cancer mortality.
Methods:
Formalin-fixed paraffin-embedded surgical tumor samples from 650 patients diagnosed with stage I and II adenocarcinoma who underwent definitive surgical treatment without adjuvant chemotherapy were analyzed for 31 proliferation genes by quantitative real-time polymerase chain reaction. The prognostic discrimination of the expression score was assessed by Cox proportional hazards analysis using 5-year lung cancer-specific death as primary outcome.
Results:
The CCP score was a significant predictor of lung cancer-specific mortality above clinical covariates [hazard ratio (HR) = 1.46 per interquartile range (95% confidence interval = 1.12–1.90; p = 0.0050)]. The prognostic score, a combination of CCP score and pathological stage, was a more significant indicator of lung cancer mortality risk than pathological stage in the full cohort (HR = 2.01; p = 2.8 × 10−11) and in stage I patients (HR = 1.67; p = 0.00027). Using the 85th percentile of the prognostic score as a threshold, there was a significant difference in lung cancer survival between low-risk and high-risk patient groups (p = 3.8 × 10−7).
Conclusions:
This study validates the CCP score and the prognostic score as independent predictors of lung cancer death in patients with early stage lung adenocarcinoma treated with surgery alone. Patients with resected stage I lung adenocarcinoma and a high prognostic score may be candidates for adjuvant therapy to reduce cancer-related mortality.
doi:10.1097/JTO.0000000000000365
PMCID: PMC4272230  PMID: 25396679
Carcinoma; Nonsmall cell lung cancer; Real-time polymerase chain reaction; Risk stratification
2.  Tsc1-Tp53 loss induces mesothelioma in mice, and evidence for this mechanism in human mesothelioma 
Oncogene  2013;33(24):3151-3160.
Mesothelioma is diagnosed in approximately 2,500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die from their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intra-peritoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, based on tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established which showed loss of both Tsc1 and Tp53, with mTORC1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival, and a 95–99% reduction in mesothelioma tumor volume, in comparison to vehicle-treated mice. To see if TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined 9 human mesothelioma cell lines, and found that 4 of 9 showed persistent activation of mTORC1 though none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma and suggesting that it is a potential therapeutic target.
doi:10.1038/onc.2013.280
PMCID: PMC3931745  PMID: 23851502
TSC1; TSC2; mesothelioma; rapamycin; mTOR
3.  MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma 
PLoS ONE  2014;9(9):e105919.
Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.
doi:10.1371/journal.pone.0105919
PMCID: PMC4164360  PMID: 25221930
4.  Detection of human telomerase reverse transcriptase mRNA in cells obtained by lavage of the pleura is not associated with worse outcome in patients with stage I/II non-small cell lung cancer; Results from CALGB 159902 
Introduction
Previous studies suggest cytologic analysis of cells obtained by lavage of the pleural surfaces at the time of resection of non-small cell lung cancer (NSCLC) can identify patients at risk for recurrence. Because telomerase gene expression has been associated with worse outcome in NSCLC, we hypothesized that identification of cells obtained from pleural lavage that express telomerase would identify patients at risk for recurrent disease.
Methods
Patients with presumed NSCLC underwent thoracotomy with curative intent. Cells obtained by lavage of the pleural surfaces were analyzed for telomerase catalytic subunit hTERT mRNA expression using RT-PCR.
Results
194 patients with stage I/II NSCLC had adequate samples and median follow-up was 60 months (17–91 mo). Using Cox models, no statistical differences were found between hTERT negative and positive patients in disease-free survival (HR=1.28, 95%CI 0.85–1.94; log-rank test, p-value=0.2349) or overall survival (HR=1.13, 95%CI 0.72–1.79; log-rank test, p-value=0.5912
Conclusion
Detection of hTERT in cells obtained from pleural lavage of patients with stage I/II NSCLC does not identify patients at risk for recurrent disease.
doi:10.1016/j.jtcvs.2012.08.059
PMCID: PMC3537860  PMID: 23026566
5.  Sequential Binary Gene-Ratio Tests Define a Novel Molecular Diagnostic Strategy for Malignant Pleural Mesothelioma 
Purpose
To develop a standardized approach for molecular diagnostics, we used the gene-expression ratio bioinformatic technique to design a molecular signature to diagnose MPM from among other potentially confounding diagnoses and differentiate the epithelioid from the sarcomatoid histological subtype of MPM. In addition, we searched for pathways relevant in MPM in comparison to other related cancers to identify unique molecular features in MPM.
Experimental Design
We performed microarray analysis on 113 specimens including MPMs and a spectrum of tumors and benign tissues comprising the differential diagnosis of MPM. We generated a sequential combination of binary gene-expression ratio tests able to discriminate MPM from other thoracic malignancies. We compared this method to other bioinformatic tools and validated this signature in an independent set of 170 samples. Functional enrichment analysis was performed to identify differentially expressed probes.
Results
A sequential combination of gene-expression ratio tests was the best molecular approach to distinguish MPM from all the other samples. Bioinformatic and molecular validations showed that the sequential gene ratio tests were able to identify the MPM samples with high sensitivity and specificity. In addition, the gene-ratio technique was able to differentiate the epithelioid from the sarcomatoid type of MPM. Novel genes and pathways specifically activated in MPM were identified.
Conclusions
New clinically relevant molecular tests have been generated using a small number of genes to accurately distinguish MPMs from other thoracic samples supporting our hypothesis that the gene-expression ratio approach could be a useful tool in the differential diagnosis of cancers.
doi:10.1158/1078-0432.CCR-12-2117
PMCID: PMC3644001  PMID: 23493352
Mesothelioma; gene expression ratio test; microarray; diagnosis; gene enrichment analysis
6.  Preoperative Cetuximab, Irinotecan, Cisplatin, and Radiation Therapy for Patients With Locally Advanced Esophageal Cancer 
The Oncologist  2013;18(3):281-287.
The efficacy and toxicity of weekly neoadjuvant cetuximab combined with irinotecan, cisplatin, and radiation therapy in patients with locally advanced esophageal or gastroesophageal junction cancer were evaluated. This schedule was toxic and did not achieve a sufficient pathologic complete response rate in patients with localized esophageal adenocarcinoma to undergo further evaluation.
Purpose.
To determine the efficacy and toxicity of weekly neoadjuvant cetuximab combined with irinotecan, cisplatin, and radiation therapy in patients with locally advanced esophageal or gastroesophageal junction cancer.
Methods and Materials.
Patients with stage IIA–IVA esophageal or gastroesophageal junction cancer were enrolled in a Simon's two-stage phase II study. Patients received weekly cetuximab on weeks 0–8 and irinotecan and cisplatin on weeks 1, 2, 4, and 5, with concurrent radiotherapy (50.4 Gy on weeks 1–6), followed by surgical resection.
Results.
In the first stage, 17 patients were enrolled, 16 of whom had adenocarcinoma. Because of a low pathologic complete response (pCR) rate in this cohort, the trial was discontinued for patients with adenocarcinoma but squamous cell carcinoma patients continued to be enrolled; two additional patients were enrolled before the study was closed as a result of poor accrual. Of the 19 patients enrolled, 18 patients proceeded to surgery, and 16 patients underwent an R0 resection. Three patients (16%) had a pCR. The median progression-free survival interval was 10 months, and the median overall survival duration was 31 months. Severe neutropenia occurred in 47% of patients, and severe diarrhea occurred in 47% of patients. One patient died preoperatively from sepsis, and one patient died prior to hospital discharge following surgical resection.
Conclusions.
This schedule of cetuximab in combination with irinotecan, cisplatin, and radiation therapy was toxic and did not achieve a sufficient pCR rate in patients with localized esophageal adenocarcinoma to undergo further evaluation.
doi:10.1634/theoncologist.2012-0208
PMCID: PMC3607524  PMID: 23429739
Cetuximab; Esophageal cancer; EGFR; Chemoradiation
7.  Molecular pathways and therapeutic targets in lung cancer 
Oncotarget  2014;5(6):1392-1433.
Lung cancer is still the leading cause of cancer death worldwide. Both histologically and molecularly lung cancer is heterogeneous. This review summarizes the current knowledge of the pathways involved in the various types of lung cancer with an emphasis on the clinical implications of the increasing number of actionable molecular targets. It describes the major pathways and molecular alterations implicated in the development and progression of non-small cell lung cancer (adenocarcinoma and squamous cancer), and of small cell carcinoma, emphasizing the molecular alterations comprising the specific blueprints in each group. The approved and investigational targeted therapies as well as the immune therapies, and clinical trials exploring the variety of targeted approaches to treatment of lung cancer are the main focus of this review.
PMCID: PMC4039220  PMID: 24722523
lung cancer; targeted therapy; immune therapy
8.  Circulating Mitochondrial DNA in Patients in the ICU as a Marker of Mortality: Derivation and Validation 
PLoS Medicine  2013;10(12):e1001577.
In this paper, Choi and colleagues analyzed levels of mitochondrial DNA in two prospective observational cohort studies and found that increased mtDNA levels are associated with ICU mortality, and improve risk prediction in medical ICU patients. The data suggests that mtDNA could serve as a viable plasma biomarker in MICU patients.
Background
Mitochondrial DNA (mtDNA) is a critical activator of inflammation and the innate immune system. However, mtDNA level has not been tested for its role as a biomarker in the intensive care unit (ICU). We hypothesized that circulating cell-free mtDNA levels would be associated with mortality and improve risk prediction in ICU patients.
Methods and Findings
Analyses of mtDNA levels were performed on blood samples obtained from two prospective observational cohort studies of ICU patients (the Brigham and Women's Hospital Registry of Critical Illness [BWH RoCI, n = 200] and Molecular Epidemiology of Acute Respiratory Distress Syndrome [ME ARDS, n = 243]). mtDNA levels in plasma were assessed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Medical ICU patients with an elevated mtDNA level (≥3,200 copies/µl plasma) had increased odds of dying within 28 d of ICU admission in both the BWH RoCI (odds ratio [OR] 7.5, 95% CI 3.6–15.8, p = 1×10−7) and ME ARDS (OR 8.4, 95% CI 2.9–24.2, p = 9×10−5) cohorts, while no evidence for association was noted in non-medical ICU patients. The addition of an elevated mtDNA level improved the net reclassification index (NRI) of 28-d mortality among medical ICU patients when added to clinical models in both the BWH RoCI (NRI 79%, standard error 14%, p<1×10−4) and ME ARDS (NRI 55%, standard error 20%, p = 0.007) cohorts. In the BWH RoCI cohort, those with an elevated mtDNA level had an increased risk of death, even in analyses limited to patients with sepsis or acute respiratory distress syndrome. Study limitations include the lack of data elucidating the concise pathological roles of mtDNA in the patients, and the limited numbers of measurements for some of biomarkers.
Conclusions
Increased mtDNA levels are associated with ICU mortality, and inclusion of mtDNA level improves risk prediction in medical ICU patients. Our data suggest that mtDNA could serve as a viable plasma biomarker in medical ICU patients.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Intensive care units (ICUs, also known as critical care units) are specialist hospital wards that provide care for people with life-threatening injuries and illnesses. In the US alone, more than 5 million people are admitted to ICUs every year. Different types of ICUs treat different types of problems. Medical ICUs treat patients who, for example, have been poisoned or who have a serious infection such as sepsis (blood poisoning) or severe pneumonia (inflammation of the lungs); trauma ICUs treat patients who have sustained a major injury; cardiac ICUs treat patients who have heart problems; and surgical ICUs treat complications arising from operations. Patients admitted to ICUs require constant medical attention and support from a team of specially trained nurses and physicians to prevent organ injury and to keep their bodies functioning. Monitors, intravenous tubes (to supply essential fluids, nutrients, and drugs), breathing machines, catheters (to drain urine), and other equipment also help to keep ICU patients alive.
Why Was This Study Done?
Although many patients admitted to ICUs recover, others do not. ICU specialists use scoring systems (algorithms) based on clinical signs and physiological measurements to predict their patients' likely outcomes. For example, the APACHE II scoring system uses information on heart and breathing rates, temperature, levels of salts in the blood, and other signs and physiological measurements collected during the first 24 hours in the ICU to predict the patient's risk of death. Existing scoring systems are not perfect, however, and “biomarkers” (molecules in bodily fluids that provide information about a disease state) are needed to improve risk prediction for ICU patients. Here, the researchers investigate whether levels of circulating cell-free mitochondrial DNA (mtDNA) are associated with ICU deaths and whether these levels can be used as a biomarker to improve risk prediction in ICU patients. Mitochondria are cellular structures that produce energy. Levels of mtDNA in the plasma (the liquid part of blood) increase in response to trauma and infection. Moreover, mtDNA activates molecular processes that lead to inflammation and organ injury.
What Did the Researchers Do and Find?
The researchers measured mtDNA levels in the plasma of patients enrolled in two prospective observational cohort studies that monitored the outcomes of ICU patients. In the Brigham and Women's Hospital Registry of Critical Illness study, blood was taken from 200 patients within 24 hours of admission into the hospital's medical ICU. In the Molecular Epidemiology of Acute Respiratory Distress Syndrome study (acute respiratory distress syndrome is a life-threatening inflammatory reaction to lung damage or infection), blood was taken from 243 patients within 48 hours of admission into medical and non-medical ICUs at two other US hospitals. Patients admitted to medical ICUs with a raised mtDNA level (3,200 or more copies of a specific mitochondrial gene per microliter of plasma) had a 7- to 8-fold increased risk of dying within 28 days of admission compared to patients with mtDNA levels of less than 3,200 copies/µl plasma. There was no evidence of an association between raised mtDNA levels and death among patients admitted to non-medical ICUs. The addition of an elevated mtDNA level to a clinical model for risk prediction that included the APACHE II score and biomarkers that are already used to predict ICU outcomes improved the net reclassification index (an indicator of the improvement in risk prediction algorithms offered by new biomarkers) of 28-day mortality among medical ICU patients in both studies.
What Do These Findings Mean?
These findings indicate that raised mtDNA plasma levels are associated with death in medical ICUs and show that, among patients in medical ICUs, measurement of mtDNA plasma levels can improve the prediction of the risk of death from the APACHE II scoring system, even when commonly measured biomarkers are taken into account. These findings do not indicate whether circulating cell-free mtDNA increased because of the underlying severity of illness or whether mtDNA actively contributes to the disease process in medical ICU patients. Moreover, they do not provide any evidence that raised mtDNA levels are associated with an increased risk of death among non-medical (mainly surgical) ICU patients. These findings need to be confirmed in additional patients, but given the relative ease and rapidity of mtDNA measurement, the determination of circulating cell-free mtDNA levels could be a valuable addition to the assessment of patients admitted to medical ICUs.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001577.
The UK National Health Service Choices website provides information about intensive care
The Society of Critical Care Medicine provides information for professionals, families, and patients about all aspects of intensive care
MedlinePlus provides links to other resources about intensive care (in English and Spanish)
The UK charity ICUsteps supports patients and their families through recovery from critical illness; its booklet Intensive Care: A Guide for Patients and Families is available in English and ten other languages; its website includes patient experiences and relative experiences of treatment in ICUs
Wikipedia has a page on ICU scoring systems (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1001577
PMCID: PMC3876981  PMID: 24391478
9.  Smoking cessation is challenging even for patients recovering from lung cancer surgery with curative intent 
Background
Although it is recommended that smokers undergoing surgery for lung cancer quit smoking to reduce post-operative complications, few studies have examined patterns of smoking in the peri-operative period. The goals of this study were to determine: 1) patterns of smoking during post-operative recovery, 2) types of cessation strategies used to quit smoking, and 3) factors related to smoking after lung cancer surgery.
Methods
Data were collected from 94 patients through chart review, tobacco, health-status, and symptom questionnaires at 1, 2, and 4-months after surgery. Smoking status was assessed through self-report and urinary cotinine measurement.
Results
Eighty-four patients (89%) were ever-smokers and 35 (37%) reported smoking at diagnosis. Thirty-nine (46%) ever-smokers remained abstinent, 13 (16%) continued smoking at all time-points, and 32 (38%) relapsed. Ten (46%) of those who relapsed were former-smokers and had not smoked for at least 1-year. Sixteen (46%) of those who were smoking at diagnosis received cessation assistance with pharmacotherapy being the most common strategy. Factors associated with smoking during recovery were younger age and quitting smoking ≤ six-months before the diagnosis of lung cancer. Factors that were marginally significant were lower educational level, male gender, lower number of comorbidities, and the presence of pain
Conclusion
Only half of those who were smoking received assistance to quit prior to surgery. Some patients were unable to quit and relapse rates post-surgery were high even among those who quit more than 1-year prior. Innovative programs incorporating symptom management and relapse prevention may enhance smoking abstinence during post-operative care.
doi:10.1016/j.lungcan.2009.01.021
PMCID: PMC3805262  PMID: 19321223
lung cancer; thoracic surgery; smoking cessation; symptom management
10.  Factors associated with smoking abstinence among smokers and recent-quitters with lung and head and neck cancer 
Introduction
Smoking cessation among cancer patients is critical for improving outcomes. Understanding factors associated with smoking abstinence after the diagnosis of cancer can provide direction to develop and test interventions to enhance cessation rates. The purpose of this study was to identify determinants of smoking outcomes among cancer patients.
Methods
Standardized questionnaires were used to collect data from 163 smokers or recent-quitters (quit ≤ 6 mo) at study entry of which 132 and 121 had data collected at 3 and 6-months. Biochemical verification was conducted with urinary cotinine and carbon monoxide. Descriptive statistics, Cronbach alpha coefficients, Pearson correlations, Fisher’s exact test, and multivariable logistic regression were used for analyses.
Results
Seven-day-point-prevalence-abstinence (PPA) rates were 90/132 (68%) at 3-months; 46/71 (65%) among lung and 44/61 (72%) among head and neck cancer patients, whereas 7-day-PPA rates were 74/121 (61%) at 6-months; 31/58 (53%) among lung and 43/63 (68%) among head and neck cancer patients. Continuous abstinence rates were 63/89 (71%) at 3-months; 32/45 (71%) among lung and 31/44 (70%) among head and neck cancer patients, whereas continuous abstinence rates were 46/89 (52%) at 6-months; 18/45 (40%) among lung and 28/44 (64%) among head and neck cancer patients. Lower cancer-related, psychological and nicotine withdrawal symptoms were associated with increased 7-D-PPA abstinence rates at 3 and 6 months in univariate models. In multivariable models, however, decreased craving was significantly related with 7-day-PPA at 3-months and decreased craving and increased self-efficacy were associated with 7-D-PPA at 6-months. Decreased craving was the only factor associated with continuous abstinence at 6-months.
Conclusions
Smoking outcomes among lung and head and neck cancer patients appear to have remained the same over the last two decades despite the availability of an increased number of pharmacotherapy options to treat tobacco dependence. Decreased craving and increased self-efficacy were the most consistent factors associated with improved smoking outcomes but symptom control may also play a role in optimal management. Use of combined, and/or higher doses of pharmacotherapy along with behavioral interventions that increase self-efficacy and manage symptoms may promote enhanced cessation rates.
doi:10.1016/j.lungcan.2011.10.005
PMCID: PMC3322288  PMID: 22093155
smoking cessation; tobacco dependence; lung cancer; head and neck cancer; smoking cessation interventions; symptom management; craving
11.  Vorinostat Eliminates Multicellular Resistance of Mesothelioma 3D Spheroids via Restoration of Noxa Expression 
PLoS ONE  2012;7(12):e52753.
When grown in 3D cultures as spheroids, mesothelioma cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found that resistance to the proteasome inhibitor bortezomib in 3D can be explained by a lack of upregulation of Noxa, the pro-apoptotic BH3 sensitizer that acts via displacement of the Bak/Bax-activator BH3-only protein, Bim. We hypothesized that the histone deacetylase inhibitor vorinostat might reverse this block to Noxa upregulation in 3D. Indeed, we found that vorinostat effectively restored upregulation of Noxa protein and message and abolished multicellular resistance to bortezomib in the 3D spheroids. The ability of vorinostat to reverse resistance was ablated by knockdown of Noxa or Bim, confirming the essential role of the Noxa/Bim axis in the response to vorinostat. Addition of vorinostat similarly increased the apoptotic response to bortezomib in another 3D model, the tumor fragment spheroid, which is grown from human mesothelioma ex vivo. In addition to its benefit when used with bortezomib, vorinostat also enhanced the response to cisplatin plus pemetrexed, as shown in both 3D models. Our results using clinically relevant 3D models show that the manipulation of the core apoptotic repertoire may improve the chemosensitivity of mesothelioma. Whereas neither vorinostat nor bortezomib alone has been clinically effective in mesothelioma, vorinostat may undermine chemoresistance to bortezomib and to other therapies thereby providing a rationale for combinatorial strategies.
doi:10.1371/journal.pone.0052753
PMCID: PMC3530471  PMID: 23300762
12.  The relationship between tumor MSLN methylation and serum mesothelin (SMRP) in mesothelioma 
Epigenetics  2011;6(8):1029-1034.
Malignant pleural mesothelioma (MPM) remains a cancer of poor prognosis. It is hoped that implementation of effective screening biomarkers will lead to earlier diagnoses and improved outcomes. Serum-measured soluble mesothelin-related peptide (SMRP) has been demonstrated to have excellent specificity for MPM, but poor sensitivity precludes its use as a screening biomarker. Using a case series of MPM patients from the International Mesothelioma Program at the Brigham and Women's hospital, we sought to determine whether epigenetic change at the MSLN gene in patient tumors is responsible for the poor sensitivity of SMRP. We identified three potential target regions for CpG methylation silencing in the MSLN promoter, one of which was amenable to bisulfite pyrosequencing and located 214 bp upstream of the transcription start site. MSLN promoter methylation was significantly higher in normal pleura than tumor tissue (p < 6.0 × 10−9). Next, we compared cases according to serum SMRP status and observed that MSLN methylation was significantly higher among tumors from patients testing negative for SMRP (<1.5 nM) versus those that were SMRP positive (p < 0.03). These results demonstrate that MSLN is normally methylated in the pleura, and that methylation is lost in most tumors. However, in a subset of tumors methylation is retained, and this mechanism explains the poor sensitivity of the SMRP assay. These results may lead to additional biomarker targets that will resolve the poor sensitivity of the SMRP assay and allow implementation of screening among exposed populations.
doi:10.4161/epi.6.8.16074
PMCID: PMC3219084  PMID: 21775819
SMRP; MSLN; mesothelioma; methylation; screening
13.  Patient report of receipt and interest in smoking cessation interventions after a diagnosis of cancer 
Cancer  2011;117(13):2961-2969.
Background
Smoking cessation is essential after the diagnosis of cancer to enhance clinical outcomes. Although effective smoking cessation treatments are available, less than one-half of smokers with cancer report receiving treatment. Reasons for the low dissemination in treatment are unclear.
Methods
Data were collected from questionnaires and medical record reviews from 160 smokers or recent-quitters with lung or head and neck cancer. Descriptive statistics, Cronbach alpha coefficient and logistic regression were used in the analyses. The median age of participants was 57 years, 63% (n=101) were male, 93% (n=149) were white, and 57% (n=91) had lung cancer.
Results
Eight-six percent (n=44) of smokers and 75% (n=82) of recent-quitters reported that health care providers gave advice to quit smoking. Sixty-five percent (n=33) of smokers and 47% (n=51) of recent-quitters reported that they were offered assistance from their health care providers to quit smoking. Fifty-one percent (n=26) of smokers and 20% (n=22) of recent-quitters expressed an interest in a smoking cessation program. An individualized smoking cessation program was the preferred type of program. Among smokers, younger, early stage patients and those with partners who were smokers were more interested in programs.
Conclusions
While the majority of patients received advice and were offered assistance to quit smoking, one-half of smokers were interested in cessation programs. Innovative approaches to increase interest in cessation programs need to be developed and tested in this population
doi:10.1002/cncr.25828
PMCID: PMC3314027  PMID: 21692055
cancer; smoking cessation programs; evidence-based tobacco treatment; smoking-related malignancies; patient preference
14.  A Novel Classification of Lung Cancer into Molecular Subtypes 
PLoS ONE  2012;7(2):e31906.
The remarkably heterogeneous nature of lung cancer has become more apparent over the last decade. In general, advanced lung cancer is an aggressive malignancy with a poor prognosis. The discovery of multiple molecular mechanisms underlying the development, progression, and prognosis of lung cancer, however, has created new opportunities for targeted therapy and improved outcome. In this paper, we define “molecular subtypes” of lung cancer based on specific actionable genetic aberrations. Each subtype is associated with molecular tests that define the subtype and drugs that may potentially treat it. We hope this paper will be a useful guide to clinicians and researchers alike by assisting in therapy decision making and acting as a platform for further study. In this new era of cancer treatment, the ‘one-size-fits-all’ paradigm is being forcibly pushed aside—allowing for more effective, personalized oncologic care to emerge.
doi:10.1371/journal.pone.0031906
PMCID: PMC3283716  PMID: 22363766
15.  Fine Needle Aspiration Biopsies for Gene Expression Ratio-based Diagnostic and Prognostic Tests in Malignant Pleural Mesothelioma 
Purpose
Malignant pleural mesothelioma (MPM) is an aggressive disease associated with median survival between 9 and 12 months. The correct diagnosis of MPM is sometimes challenging and usually requires solid tissue biopsies rather than fine needle aspiration biopsies (FNA). We postulated that the accuracy of FNA-based diagnosis might be improved by the addition of molecular tests using a gene expression ratio-based algorithm and that prognostic tests could be similarly performed.
Experimental Design
Two MPM and two lung cancer cell lines were used to establish the minimal RNA amount required for ratio tests. Based on these results, 276 ex-vivo FNA biopsies from 63 MPM patients, and 250 ex-vivo FNA samples from 92 lung cancer patients were analyzed using previously described diagnostic and prognostic tests based on gene expression ratios.
Results
We found that the sensitivity of the diagnostic test for MPM was 100% (95% CI: 95–100%), and the specificity in primary lung adenocarcinoma was 90% (95% CI: 81–95%). The FNA-based prognostic classification was concordant among 76% (95% CI: 65–87%) of patients with the risk assignment in a subset of the matched surgical specimens previously analyzed by the prognostic test.
Conclusions
Sufficient RNA can be extracted from most FNA biopsies to perform gene expression molecular tests. In particular, we show that the gene expression ratio algorithms performed well when applied to diagnosis and prognosis in MPM. This study provides support for the development of additional RNA molecular tests that may enhance the utility of FNA in the management of other solid cancers.
doi:10.1158/1078-0432.CCR-10-0806
PMCID: PMC3058309  PMID: 21088255
mesothelioma; FNA; diagnosis; prognosis; molecular tests
16.  Semi-supervised recursively partitioned mixture models for identifying cancer subtypes 
Bioinformatics  2010;26(20):2578-2585.
Motivation: Patients with identical cancer diagnoses often progress differently. The disparity we see in disease progression and treatment response can be attributed to the idea that two histologically similar cancers may be completely different diseases on the molecular level. Methods for identifying cancer subtypes associated with patient survival have the capacity to be powerful instruments for understanding the biochemical processes that underlie disease progression as well as providing an initial step toward more personalized therapy for cancer patients. We propose a method called semi-supervised recursively partitioned mixture models (SS-RPMM) that utilizes array-based genetic and patient-level clinical data for finding cancer subtypes that are associated with patient survival.
Results: In the proposed SS-RPMM, cancer subtypes are identified using a selected subset of genes that are associated with survival time. Since survival information is used in the gene selection step, this method is semi-supervised. Unlike other semi-supervised clustering classification methods, SS-RPMM does not require specification of the number of cancer subtypes, which is often unknown. In a simulation study, our proposed method compared favorably with other competing semi-supervised methods, including: semi-supervised clustering and supervised principal components analysis. Furthermore, an analysis of mesothelioma cancer data using SS-RPMM, revealed at least two distinct methylation profiles that are informative for survival.
Availability: The analyses implemented in this article were carried out using R (http://www.r.project.org/).
Contact: devin_koestler@brown.edu; e_andres_houseman@brown.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq470
PMCID: PMC2951086  PMID: 20834038
17.  Integrated profiling reveals a global correlation between epigenetic and genetic alterations in mesothelioma 
Cancer research  2010;70(14):5686-5694.
Development of mesothelioma is linked mainly to asbestos exposure but the combined contributions of genetic and epigenetic alterations are unclear. We investigated the potential relationships between gene copy number (CN) alterations and DNA methylation profiles in a case series of pleural mesotheliomas (n=23). There were no instances of significantly correlated CN alteration and methylation at probed loci, whereas averaging loci over their associated genes revealed only two genes with significantly correlated CN and methylation alterations. In contrast to the lack of discrete correlations, the overall extent of tumor CN alteration was significantly associated with DNA methylation profile when comparing CN alteration extent among methylation profile classes. Further, there was evidence that this association was partially attributable to prevalent allele loss at the DNA methyltransferase gene DNMT1. Our findings define a strong association between global genetic and global epigenetic dysregulation in mesothelioma, rather than a discrete, local coordination of gene inactivation.
doi:10.1158/0008-5472.CAN-10-0190
PMCID: PMC2905466  PMID: 20587528
Integrative genomics; DNA methylation; Copy number alteration; Mesothelioma
18.  Genes Associated With Prognosis After Surgery For Malignant Pleural Mesothelioma Promote Tumor Cell Survival In Vitro 
BMC Cancer  2011;11:169.
Background
Mesothelioma is an aggressive neoplasm with few effective treatments, one being cytoreductive surgery. We previously described a test, based on differential expression levels of four genes, to predict clinical outcome in prospectively consented mesothelioma patients after surgery. In this study, we determined whether any of these four genes could be linked to a cancer relevant phenotype.
Methods
We conducted a high-throughput RNA inhibition screen to knockdown gene expression levels of the four genes comprising the test (ARHGDIA, COBLL1, PKM2, TM4SF1) in both a human lung-derived normal and a tumor cell line using three different small inhibitory RNA molecules per gene. Successful knockdown was confirmed using quantitative RT-PCR. Detection of statistically significant changes in apoptosis and mitosis was performed using immunological assays and quantified using video-assisted microscopy at a single time-point. Changes in nuclear shape, size, and numbers were used to provide additional support of initial findings. Each experiment was conducted in triplicate. Specificity was assured by requiring that at least 2 different siRNAs produced the observed change in each cell line/time-point/gene/assay combination.
Results
Knockdown of ARHGDIA, COBLL1, and TM4SF1 resulted in 2- to 4-fold increased levels of apoptosis in normal cells (ARHGDIA only) and tumor cells (all three genes). No statistically significant changes were observed in apoptosis after knockdown of PKM2 or for mitosis after knockdown of any gene.
Conclusions
We provide evidence that ARHGDIA, COBLL1, and TM4SF1 are negative regulators of apoptosis in cultured tumor cells. These genes, and their related intracellular signaling pathways, may represent potential therapeutic targets in mesothelioma.
doi:10.1186/1471-2407-11-169
PMCID: PMC3112160  PMID: 21569526
mesothelioma; Cell biology/culture; Genes/polymorphisms; Genetics/genomics
19.  Peripheral blood gene expression profiles in COPD subjects 
To identify non-invasive gene expression markers for chronic obstructive pulmonary disease (COPD), we performed genome-wide expression profiling of peripheral blood samples from 12 subjects with significant airflow obstruction and an equal number of non-obstructed controls. RNA was isolated from Peripheral Blood Mononuclear Cells (PBMCs) and gene expression was assessed using Affymetrix U133 Plus 2.0 arrays.
Tests for gene expression changes that discriminate between COPD cases (FEV1< 70% predicted, FEV1/FVC < 0.7) and controls (FEV1> 80% predicted, FEV1/FVC > 0.7) were performed using Significance Analysis of Microarrays (SAM) and Bayesian Analysis of Differential Gene Expression (BADGE). Using either test at high stringency (SAM median FDR = 0 or BADGE p < 0.01) we identified differential expression for 45 known genes. Correlation of gene expression with lung function measurements (FEV1 & FEV1/FVC), using both Pearson and Spearman correlation coefficients (p < 0.05), identified a set of 86 genes. A total of 16 markers showed evidence of significant correlation (p < 0.05) with quantitative traits and differential expression between cases and controls. We further compared our peripheral gene expression markers with those we previously identified from lung tissue of the same cohort. Two genes, RP9and NAPE-PLD, were identified as decreased in COPD cases compared to controls in both lung tissue and blood. These results contribute to our understanding of gene expression changes in the peripheral blood of patients with COPD and may provide insight into potential mechanisms involved in the disease.
doi:10.1186/2043-9113-1-12
PMCID: PMC3164605  PMID: 21884629
Microarray; Biomarkers; PBMC
20.  Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma12 
Neoplasia (New York, N.Y.)  2011;13(1):12-22.
The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR) and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3) in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90). Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17-demethoxygeldanamycin (17-AAG) had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of downstream signaling intermediates (AKT, mitogen-activated protein kinase, and S6); upregulated the p53, p21, and p27 cell cycle checkpoints; induced G2 phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G1 apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.
PMCID: PMC3022424  PMID: 21245936
21.  Differentiation of lung adenocarcinoma, pleural mesothelioma, and non-malignant pulmonary tissues using DNA methylation profiles 
Cancer research  2009;69(15):6315-6321.
Pathologic differentiation of tissue of origin in tumors found in the lung can be challenging, with differentiation of mesothelioma and lung adenocarcinoma emblematic of this problem. Indeed, proper classification is essential for determination of treatment regimen for these diseases, making accurate and early diagnosis critical. Here we investigate the potential of epigenetic profiles of lung adenocarcinoma, mesothelioma, and non-malignant pulmonary tissues (n=285) as differentiation markers in an analysis of DNA methylation at 1413 autosomal CpG loci associated with 773 cancer-related genes. Using an unsupervised recursively-partitioned mixture modeling technique for all samples, the derived methylation profile classes were significantly associated with sample type (P < 0.0001). In a similar analysis restricted to tumors, methylation profile classes significantly predicted tumor type (P < 0.0001). Random forests classification of CpG methylation of tumors - which splits the data into training and test sets - accurately differentiated MPM from lung adenocarcinoma over 99% of the time (P < 0.0001). In a locus-by-locus comparison of CpG methylation between tumor types, 1266 CpG loci had significantly different methylation between tumors following correction for multiple comparisons (Q < 0.05); 61% had higher methylation in adenocarcinoma. Using the CpG loci with significant differential methylation in a pathways analysis revealed significant enrichment of methylated gene-loci in Cell Cycle Regulation, DNA Damage Response, PTEN Signaling, and Apoptosis Signaling pathways in lung adenocarcinoma when compared to mesothelioma. Methylation-profile-based differentiation of lung adenocarcinoma and mesothelioma is highly accurate, informs on the distinct etiologies of these diseases, and holds promise for clinical application.
doi:10.1158/0008-5472.CAN-09-1073
PMCID: PMC2755616  PMID: 19638575
22.  MAKING THE CASE FOR MOLECULAR STAGING OF MPM 
doi:10.1053/j.semtcvs.2009.07.003
PMCID: PMC2904964  PMID: 19822292
23.  Second Generation Sequencing of the Mesothelioma Tumor Genome 
PLoS ONE  2010;5(5):e10612.
The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.
doi:10.1371/journal.pone.0010612
PMCID: PMC2869344  PMID: 20485525
24.  Four-Gene Expression Ratio Test for Survival in Patients Undergoing Surgery for Mesothelioma 
Background
Malignant pleural mesothelioma has few effective treatments, one being cytoreductive surgery. We previously developed a gene ratio test to predict outcome of malignant pleural mesothelioma patients undergoing surgery. In this study, we investigated the predictive value and technical assay performance of this test in patients with malignant pleural mesothelioma.
Methods
Clinical data were obtained prospectively from 120 consecutive patients with malignant pleural mesothelioma who were scheduled for debulking surgery at one institution. Specimens were obtained at surgery or by pleural biopsy examination. Expression data for four genes were collected from tumor specimens, and three ratios of gene expression (TM4SF1/PKM2, TM4SF1/ARHGDIA, and COBLL1/ARHGDIA) were determined by quantitative reverse transcriptase–polymerase chain reaction. Patients were assigned to good or poor outcome groups by the gene ratio test. Survival was estimated by the Kaplan–Meier method and the log-rank test in univariate analyses. A multivariable Cox proportional hazards model was used to control for prognostic factors. Technical robustness was determined by using up to 30 specimens per patient, two biopsy techniques, and two performance sites. All statistical tests were two-sided.
Results
The test predicted overall survival (P < .001) and cancer-specific survival (P = .007) in univariate analysis and overall survival in multivariable analysis (hazard ratio for death = 2.09, 95% confidence interval [CI] = 1.27 to 3.45, P = .004). The test was reproducible within patients and repeatable between two determinations for specimens with widely varying tumor cell contents. Repeatability between two determinations was 88.5% (95% CI = 84.0% to 92.2%) or, when technically unacceptable test values were excluded, 91.9% (95% CI = 87.4% to 95.1%). Reproducibility between two determinations was 96.1% (95% CI = 86.5% to 99.5%). Combining the gene ratio test and other prognostic factors allowed prospective discrimination between patients at high risk (median survival = 6.9 months, 95% CI = 2.6 to 8.9 months; 3-year survival = 0%) and low risk (median survival = 31.9 months, 95% CI = 21.9 to 41.7 months; 3-year survival = 42%).
Conclusion
The gene ratio test for survival of patients with malignant pleural mesothelioma has robust predictive value and technical assay performance.
doi:10.1093/jnci/djp061
PMCID: PMC2677573  PMID: 19401544
25.  Molecular Biomarkers for Quantitative and Discrete COPD Phenotypes 
Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disorder with complex pathological features and largely unknown etiology. The identification of biomarkers for this disease could aid the development of methods to facilitate earlier diagnosis, the classification of disease subtypes, and provide a means to define therapeutic response. To identify gene expression biomarkers, we completed expression profiling of RNA derived from the lung tissue of 56 subjects with varying degrees of airflow obstruction using the Affymetrix U133 Plus 2.0 array. We applied multiple, independent analytical methods to define biomarkers for either discrete or quantitative disease phenotypes. Analysis of differential expression between cases (n = 15) and controls (n = 18) identified a set of 65 discrete biomarkers. Correlation of gene expression with quantitative measures of airflow obstruction (FEV1%predicted or FEV1/FVC) identified a set of 220 biomarkers. Biomarker genes were enriched in functions related to DNA binding and regulation of transcription. We used this group of biomarkers to predict disease in an unrelated data set, generated from patients with severe emphysema, with 97% accuracy. Our data contribute to the understanding of gene expression changes occurring in the lung tissue of patients with obstructive lung disease and provide additional insight into potential mechanisms involved in the disease process. Furthermore, we present the first gene expression biomarker for COPD validated in an independent data set.
doi:10.1165/rcmb.2008-0114OC
PMCID: PMC2645534  PMID: 18849563
microarray; gene expression; emphysema; lung function

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