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1.  In Vivo DPP-4 Inhibition to Enhance Engraftment of Single-Unit Cord Blood Transplants in Adults with Hematological Malignancies 
Stem Cells and Development  2012;22(7):1007-1015.
Delayed engraftment is a significant limitation of umbilical cord blood (UCB) transplantation due to low stem cell numbers. Inhibition of dipeptidyl peptidase (DPP)-4 enhanced engraftment in murine transplants. We evaluated the feasibility of systemic DPP-4 inhibition using sitagliptin to enhance engraftment of single-unit UCB grafts in adults with hematological malignancies. Twenty-four patients (21–58 years) received myeloablative conditioning, followed by sitagliptin 600 mg orally days −1 to +2, and single UCB grafts day 0. Seventeen receiving red cell-depleted (RCD) grafts, matched at 4 (n=10) or 5 (n=7) of 6 human leucocyte antigen (HLA) loci with median nucleated cell dose 3.6 (2.5–5.2)×107/kg, engrafted at median of 21 (range, 13–50) days with cumulative incidence of 94% (95% confidence interval, 84%–100%) at 50 days. Plasma DDP-4 activity was reduced to 23%±7% within 2 h. Area under DPP-4 activity-time curve (AUCA) correlated with engraftment; 9 of 11 with AUCA <6,000 activity·h engrafted within ≤21 days, while all 6 with higher AUCA engrafted later (P=0.002). Seven patients receiving red cell replete grafts had 10-fold lower colony forming units after thawing compared with RCD grafts, with poor engraftment. Systemic DPP-4 inhibition was well tolerated and may enhance engraftment. Optimizing sitagliptin dosing to achieve more sustained DPP-4 inhibition may further improve outcome.
doi:10.1089/scd.2012.0636
PMCID: PMC3607909  PMID: 23270493
2.  Small-Molecule Inhibition of the uPAR·uPA Interaction: Synthesis, Biochemical, Cellular, in vivo Pharmacokinetics and Efficacy Studies in Breast Cancer Metastasis 
Bioorganic & medicinal chemistry  2013;21(7):2145-2155.
The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5 hours and peak concentration of 5 μM. Similar levels of the inhibitor were detected in tumor tissue up to 10 hours. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.
doi:10.1016/j.bmc.2012.12.047
PMCID: PMC3625246  PMID: 23411397
3.  RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia 
Nature genetics  2014;46(2):116-125.
The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL), is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near the breakpoints; incorporation of non-templated sequence at the junction; ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B-cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single cell tracking shows that this mechanism is active throughout leukemic evolution with evidence of localized clustering and re-iterated deletions. Integration of point mutation and rearrangement data identifies ATF7IP and MGA as two new tumor suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1 lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B-cell differentiation.
doi:10.1038/ng.2874
PMCID: PMC3960636  PMID: 24413735
4.  BRMS1 suppresses lung cancer metastases through an E3 ligase function on histone acetyltransferase p300 
Cancer research  2012;73(4):1308-1317.
The mechanisms through which the metastasis suppressor gene BRMS1 functions are poorly understood. Herein, we report the identification of a previously undescribed E3 ligase function of BRMS1 on the histone acetyltransferase p300. BRMS1 induces polyubiquitination of p300 resulting in its proteasome-mediated degradation. We identify BRMS1 as the first eukaryote structural mimic of the bacterial IpaH E3 ligase family, and establish that the evolutionarily conserved CXD motif located in in BRMS1 is responsible for its E3 ligase function. Mutation of this E3 ligase motif not only abolishes BRMS1-induced p300 polyubiquitination and degradation, but importantly, dramatically reduces the metastasis suppressor function of BRMS1 in both in vitro and in vivo models of lung cancer metastasis.
doi:10.1158/0008-5472.CAN-12-2489
PMCID: PMC3578176  PMID: 23269275
BRMS1; metastasis suppressor; p300; E3 ligase; polyubiquitination
5.  Teaching video-assisted thoracic surgery (VATS) lobectomy 
Journal of Thoracic Disease  2013;5(Suppl 3):S207-S211.
Video-assisted thoracic surgery (VATS) lobectomy has become the standard of care for early stage lung cancer throughout the world. Teaching this complex procedure requires adequate case volume, adequate instrumentation, a committed operating room team and baseline experience with open lobectomy. We outline what key maneuvers and steps are required to teach and learn VATS lobectomy. This is most easily performed as part of a thoracic surgery training program, but with adequate commitment and proctoring, there is no reason experienced open surgeons cannot become proficient VATS surgeons. We provide videos showing the key portions of a subcarinal lymph node dissection, posterior hilar dissection of the right upper lobe, fissureless right middle lobectomy, and fissureless left lower lobectomy. These videos highlight what we feel are important principals in VATS lobectomy, i.e., N2 and N1 lymph node dissection, fissureless techniques, and progressive responsibility of the learner. Current literature in simulation of VATS lobectomy is also outlined as this will be the future of teaching in VATS lobectomy.
doi:10.3978/j.issn.2072-1439.2013.07.31
PMCID: PMC3771628  PMID: 24040525
video-assisted thoracic surgery (VATS) lobectomy; teaching; simulation
6.  Hemodialysis does not alter in vitro hepatic CYP3A4 and CYP2D6 metabolic activity in uremic serum 
There is a paucity of studies evaluating the change in liver metabolism in subjects receiving hemodialysis. The purpose of this study was to compare the effect of uremic toxins on hepatic cytochrome P450 (CYP)3A4 and CYP2D6 metabolism before and after a 4-hour hemodialysis session. Midazolam and dextromethorphan were incubated with uremic serum collected from subjects before and after the 4-hour hemodialysis session. Analysis and quantification of the 1′-OH-midazolam and 4-OH-midazolam and dextrorphan metabolites were performed by high-pressure liquid chromatography/mass spectrometry. Statistical analysis using the Student’s t-test (paired) was used to compare the amount of metabolite formed. The mean amount of 1′-OH-midazolam, 4-OH-midazolam, and dextrorphan metabolites formed before and after hemodialysis did not significantly differ. There was no significant difference in CYP3A4 and CYP2D6 metabolic activity in uremic serum before and after hemodialysis.
doi:10.2147/CPAA.S54381
PMCID: PMC3862653  PMID: 24353447
hemodialysis; uremia; CYP3A4; CYP2D6; metabolism
7.  Phase I Trial of Induction Histone Deacetylase and Proteasome Inhibition Followed by Surgery in Non-small Cell Lung Cancer 
Introduction
Despite complete surgical resection survival in early stage non-small cell lung cancer (NSCLC) remains poor. Based on prior pre-clinical evaluations, we hypothesized that combined induction proteasome and histone deacetylase inhibitor therapy, followed by tumor resection, is feasible.
Methods
A phase I clinical trial using a two-staged multiple agent design of bortezomib and vorinostat as induction therapy followed by consolidative surgery in patients with NSCLC was performed. Standard toxicity and MTD were examined. Pre- and post-treatment tumor gene expression arrays were performed and analyzed. Pre- and post-treatment FDG-PET imaging was used to assess tumor metabolism. Finally, serum 20S proteasome levels were analyzed with ELISA, and selected intratumoral proteins were assessed via immunohistochemistry.
Results
Thirty-four patients were consented with 21 patients enrolling in the trial. One patient withdrew early secondary to disease progression. The MTD was bortezomib 1.3 mg/m2 and vorinostat 300 mg BID given. There were (2) grade III dose-limiting toxicities of fatigue and hypophosphatemia that were self-limited. There was no mortality. Thirty percent (6/20) of patients had greater than 60% histologic necrosis of their tumor following treatment, with two having ≥90% tumor necrosis. Tumor metabolism, 20S proteasome activity, and specific protein expression did not demonstrate consistent results. Gene expression arrays comparing pre- and post-therapy NSCLC specimens revealed robust intratumoral changes in specific genes.
Conclusions
Induction bortezomib and vorinostat therapy followed by surgery in patients with operable NSCLC is feasible. Correlative gene expression studies suggest new targets and cell signaling pathways that may be important in modulating this combined therapy.
doi:10.1097/JTO.0b013e318267928d
PMCID: PMC3473142  PMID: 23059775
Histone deacetylase; proteasome inhibitor; lung cancer
8.  A Highly Selective and Potent PTP-MEG2 Inhibitor with Therapeutic Potential for Type 2 Diabetes 
Journal of the American Chemical Society  2012;134(43):18116-18124.
Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that control the cellular levels of protein tyrosine phosphorylation. A detailed understanding of PTP functions in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific, cell-permeable small molecule agents. We present a stepwise focused library approach that transforms a weak and general nonhydrolyzable pTyr mimetic (F2Pmp, phosphonodifluoromethyl phenylalanine) into a highly potent and selective inhibitor of PTP-MEG2, an antagonist of hepatic insulin signaling. The crystal structures of the PTP-MEG2-inhibitor complexes provide direct evidence that potent and selective PTP inhibitors can be obtained by introducing molecular diversity into the F2Pmp scaffold to engage both the active site and unique nearby peripheral binding pockets. Importantly, the PTP-MEG2 inhibitor possesses highly efficacious cellular activity and is capable of augmenting insulin signaling and improving insulin sensitivity and glucose homeostasis in diet-induced obese mice. The results indicate that F2Pmp can be converted into highly potent and selective PTP inhibitory agents with excellent in vivo efficacy. Given the general nature of the approach, this strategy should be applicable to other members of the PTP superfamily.
doi:10.1021/ja308212y
PMCID: PMC3505080  PMID: 23075115
9.  Existing General Population Models Inaccurately Predict Lung Cancer Risk in Patients Referred for Surgical Evaluation 
The Annals of thoracic surgery  2011;91(1):227-233.
Background
atients undergoing resections for suspicious pulmonary lesions have a 9-55% benign rate. Validated prediction models exist to estimate the probability of malignancy in a general population and current practice guidelines recommend their use. We evaluated these models in a surgical population to determine the accuracy of existing models to predict benign or malignant disease.
Methods
We conducted a retrospective review of our thoracic surgery quality improvement database (2005-2008) to identify patients who underwent resection of a pulmonary lesion. Patients were stratified into subgroups based on age, smoking status and fluorodeoxyglucose positron emission tomography (PET) results. The probability of malignancy was calculated for each patient using the Mayo and SPN prediction models. Receiver operating characteristic (ROC) and calibration curves were used to measure model performance.
Results
89 patients met selection criteria; 73% were malignant. Patients with preoperative PET scans were divided into 4 subgroups based on age, smoking history and nodule PET avidity. Older smokers with PET-avid lesions had a 90% malignancy rate. Patients with PET- non-avid lesions, or PET-avid lesions with age<50 years or never smokers of any age had a 62% malignancy rate. The area under the ROC curve for the Mayo and SPN models was 0.79 and 0.80, respectively; however, the models were poorly calibrated (p<0.001).
Conclusions
Despite improvements in diagnostic and imaging techniques, current general population models do not accurately predict lung cancer among patients ref erred for surgical evaluation. Prediction models with greater accuracy are needed to identify patients with benign disease to reduce non-therapeutic resections.
doi:10.1016/j.athoracsur.2010.08.054
PMCID: PMC3748597  PMID: 21172518
Lung Cancer; Lung Cancer; diagnosis; cancer staging; Positron Emission Tomography (PET)
10.  Phosphorylation of RelA/p65 promotes DNMT-1 recruitment to chromatin and represses transcription of the tumor metastasis suppressor gene BRMS1 
Oncogene  2011;31(9):1143-1154.
The majority of patients with lung cancer present with metastatic disease. Chronic inflammation and subsequent activation of NF-κB have been associated the development of cancers. The RelA/p65 subunit of NF-κB is typically associated with transcriptional activation. In this report we show that RelA/p65 can function as an active transcriptional repressor through enhanced methylation of the BRMS1 metastasis suppressor gene promoter via direct recruitment of DNMT-1 to chromatin in response to TNF. TNF-mediated phosphorylation of S276 on RelA/p65 is required for RelA/p65-DNMT-1 interactions, chromatin loading of DNMT-1, and subsequent BRMS1 promoter methylation and transcriptional repression. The ability of RelA/65 to function as an active transcriptional repressor is promoter specific as the NF-κB-regulated gene cIAP2 is transcriptionally activated while BRMS1 is repressed under identical conditions. Small molecule inhibition of either of the minimal interacting domains between RelA/p65-DNMT-1 and RelA/p65-BRMS1 promoter abrogates BRMS1 methylation and its transcriptional repression. The ability of RelA/p65 to directly recruit DNMT-1 to chromatin resulting in promoter-specific methylation and transcriptional repression of tumor metastasis suppressor gene BRMS1 highlights a new mechanism through which NF-κB can regulate metastatic disease, and offers a potential target for newer generation epigenetic oncopharmaceuticals.
doi:10.1038/onc.2011.308
PMCID: PMC3219802  PMID: 21765477
DNMT-1; Phosphorylation; RelA-p65; TNF; Transcription
11.  Fluorodeoxyglucose positron emission tomography and tumor marker expression in non–small cell lung cancer 
Objective
The best current noninvasive surrogate for tumor biology is fluorodeoxyglucose positron emission tomography (FDG–PET). Both FDG–PET maximal standardized uptake values and selected tumor markers have been shown to correlate with stage, nodal disease, and survival in non–small cell lung cancer (NSCLC). However, there are limited data correlating FDG–PET with tumor markers. The purpose of this study was to determine the correlation of tumor marker expression with FDG–PET maximal standardized uptake values in NSCLC.
Methods
FDG–PET maximal standardized uptake values were calculated in patients with NSCLC (n = 149). No patient had induction chemoradiotherapy. Intraoperative NSCLC tissue was obtained and tissue microarrays were created. Immunohistochemical analysis was performed for 5 known NSCLC tumor markers (glucose transporter 1, p53, cyclin D1, epidermal growth factor receptor, and vascular endothelial growth factor). Each tumor marker was assessed independently by two pathologists using common grading criteria. Subgroup analysis based on histologic characteristics and regional nodal status was performed.
Results
FDG–PET correlated with T classification (P<.0001), N stage (P = .002), and greatest tumor dimension (P<.0001). In addition, increasing maximal standardized uptake values correlated with increased expression of glucose transporter 1 (P<.0001) and p53 (P =.04) in adenocarcinoma. Epidermal growth factor receptor expression correlated with maximal standardized uptake values without predilection for histologic subtype (P = .004).
Conclusion
FDG–PET maximal standardized uptake values correlate with an increased expression of glucose transporter 1 and p53 in lung adenocarcinoma, but not squamous cell cancer. Future studies attempting to correlate FDG–PET with tumor biology will need to consider the effect of different tumor histologic types.
doi:10.1016/j.jtcvs.2008.10.014
PMCID: PMC3632077  PMID: 19154901
12.  Predicted Risk of Mortality Models: Surgeons Need to Understand Limitations of the University Health System Consortium Models 
BACKGROUND
The University HealthSystem Consortium (UHC) mortality risk adjustment models are increasingly being used as benchmarks for quality assessment. But these administrative database models may include postoperative complications in their adjustments for preoperative risk. The purpose of this study was to compare the performance of the UHC with the Society of Thoracic Surgeons (STS) risk-adjusted mortality models for adult cardiac surgery and evaluate the contribution of postoperative complications on model performance.
STUDY DESIGN
We identified adult cardiac surgery patients with mortality risk estimates in both the UHC and Society of Thoracic Surgeons databases. We compared the predictive performance and calibration of estimates from both models. We then reestimated both models using only patients without any postoperative complications to determine the relative contribution of adjustments for postoperative events on model performance.
RESULTS
In the study population of 2,171 patients, the UHC model explained more variability (27% versus 13%, p < 0.001) and achieved better discrimination (C statistic = 0.88 versus 0.81, p < 0.001). But when applied in the population of patients without complications, the UHC model performance declined severely. The C statistic decreased from 0.88 to 0.49, a level of discrimination equivalent to random chance. The discrimination of the Society of Thoracic Surgeons model was unchanged (C statistic of 0.79 versus 0.81).
CONCLUSIONS
Although the UHC model demonstrated better performance in the total study population, this difference in performance reflects adjustments for conditions that are postoperative complications. The current UHC models should not be used for quality benchmarks.
doi:10.1016/j.jamcollsurg.2009.08.008
PMCID: PMC3627222  PMID: 19854393
13.  Multi-gene expression-based predictors for sensitivity to Vorinostat and Velcade in non-small cell lung cancer 
Molecular cancer therapeutics  2010;9(10):2834-2843.
The ability to predict the efficacy of molecularly-targeted therapies for non-small cell lung cancer (NSCLC) for an individual patient remains problematic. The purpose of this study was to identify tumor biomarkers, using a refined “coexpression extrapolation (COXEN)” algorithm with a continuous spectrum of drug activity, that predict drug sensitivity and therapeutic efficacy in NSCLC to Vorinostat, a histone deacetylase inhibitor, and Velcade, a proteasome inhibitor. Using our refined COXEN algorithm, biomarker prediction models were discovered and trained for Vorinostat and Velcade based on in vitro drug activity profiles of 9 NSCLC cell lines (NCI-9). Independently, a panel of 40 NSCLC cell lines (UVA-40) was treated with Vorinostat or Velcade to obtain 50% growth inhibition values. Genome-wide expression profiles for both the NCI-9 and UVA-40 cell lines were determined using HG-U133A Affymetrix platform. Modeling generated multi-gene expression signatures for Vorinostat (45-gene, p=0.002) and Velcade (15-gene, p=0.0002), with one overlapping gene (CFLAR). Examination of Vorinostat gene ontogeny revealed a predilection for cellular replication and death, whereas those of Velcade suggested involvement in cellular development and carcinogenesis. Multivariate regression modeling of the refined COXEN scores significantly predicted the activity of combination therapy in NSCLC cells (p=0.007). Through the refinement of the COXEN algorithm, we provide an in silico method to generate biomarkers that predict tumor sensitivity to molecularly-targeted therapies. Use of this refined COXEN methodology has significant implications for the a priori examination of targeted therapies to more effectively streamline subsequent clinical trial design and cost.
doi:10.1158/1535-7163.MCT-10-0327
PMCID: PMC2953585  PMID: 20713531
Lung cancer; histone deacetylase inhibitor; proteasome inhibitor; tumor biomarker; molecularly-targeted agents; chemotherapy
14.  S-Nitrosoglutathione Reductase in Human Lung Cancer 
S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.
doi:10.1165/rcmb.2011-0147OC
PMCID: PMC3262657  PMID: 21816964
lung cancer; S-nitrosoglutathione reductase; Ras
15.  Hydrogen Peroxide in Adaptation 
doi:10.1155/2012/596019
PMCID: PMC3544357  PMID: 23326624
16.  Subcutaneous 5-Azacitidine Treatment of Naturally Occurring Canine Urothelial Carcinoma: A Novel Epigenetic Approach to Human Urothelial Carcinoma Drug Development 
The Journal of urology  2011;187(1):302-309.
Purpose
We determined the efficacy, biological activity, pharmacokinetics and safety of the hypomethylating agent 5-azacitidine (Celgene Corp., Summit, New Jersey) in dogs with naturally occurring invasive urothelial carcinoma.
Materials and Methods
We performed a preclinical phase I trial in dogs with naturally occurring invasive urothelial carcinoma to examine once daily subcutaneous administration of 5-azacitidine in 28-day cycles at doses of 0.10 to 0.30 mg/kg per day according to 2 dose schedules, including days 1 to 5 (28-day cohort) or days 1 to 5 and 15 to 19 (14-day cohort). Clinical efficacy was assessed by serial cystosonography, radiography and cystoscopy. Urinary 5-azacitidine pharmacokinetic analysis was also done. Pretreatment and posttreatment peripheral blood mononuclear cell and invasive urothelial carcinoma DNA, respectively, was analyzed for global and gene specific [CDKN2A (p14ARF)] methylation changes.
Results
Enrolled in the study were 19 dogs with naturally occurring invasive urothelial carcinoma. In the 28-day cohort the maximum tolerated dose was 0.20 mg/kg per day with higher doses resulting in grade 3 or 4 neutropenia in 4 of 6 dogs. In the 14-day cohort the maximum tolerated dose was 0.10 mg/kg per day with grade 3 or 4 neutropenia seen in 2 of 3 dogs treated at higher doses. No grade 3 or 4 nonhematological toxicity was observed during either dosing schedule. Of 18 dogs evaluable for tumor response partial remission, stable disease and progressive disease were observed in 4 (22.2%), 9 (50.0%) and 4 (22.2%), respectively. Consistent 5-azacitidine levels (205 to 857 ng/ml) were detected in urine. Pretreatment and posttreatment methylation analysis revealed no significant correlation with clinical response.
Conclusions
Subcutaneous 5-azacitidine showed promising clinical activity in a canine invasive urothelial carcinoma model, thus meriting further development in humans with urothelial carcinoma.
doi:10.1016/j.juro.2011.09.010
PMCID: PMC3508763  PMID: 22099988
urinary bladder; urothelium; carcinoma; azacitidine; dogs
17.  Occult Metastases in Lymph Nodes Predict Survival in Resectable Non–Small-Cell Lung Cancer: Report of the ACOSOG Z0040 Trial 
Journal of Clinical Oncology  2011;29(32):4313-4319.
Purpose
The survival of patients with non–small-cell lung cancer (NSCLC), even when resectable, remains poor. Several small studies suggest that occult metastases (OMs) in pleura, bone marrow (BM), or lymph nodes (LNs) are present in early-stage NSCLC and are associated with a poor outcome. We investigated the prevalence of OMs in resectable NSCLC and their relationship with survival.
Patients and Methods
Eligible patients had previously untreated, potentially resectable NSCLC. Saline lavage of the pleural space, performed before and after pulmonary resection, was examined cytologically. Rib BM and all histologically negative LNs (N0) were examined for OM, diagnosed by cytokeratin immunohistochemistry (IHC). Survival probabilities were estimated using the Kaplan-Meier method. The log-rank test and Cox proportional hazards regression model were used to compare survival of groups of patients. P < .05 was considered significant.
Results
From July 1999 to March 2004, 1,047 eligible patients (538 men and 509 women; median age, 67.2 years) were entered onto the study, of whom 50% had adenocarcinoma and 66% had stage I NSCLC. Pleural lavage was cytologically positive in only 29 patients. OMs were identified in 66 (8.0%) of 821 BM specimens and 130 (22.4%) of 580 LN specimens. In univariate and multivariable analyses OMs in LN but not BM were associated with significantly worse disease-free survival (hazard ratio [HR], 1.50; P = .031) and overall survival (HR, 1.58; P = .009).
Conclusion
In early-stage NSCLC, LN OMs detected by IHC identify patients with a worse prognosis. Future clinical trials should test the role of IHC in identifying patients for adjuvant therapy.
doi:10.1200/JCO.2011.35.2500
PMCID: PMC3221530  PMID: 21990404
18.  Thirty and Ninety Day Outcomes after Sublobar Resection with and without brachytherapy for Non-small cell lung cancer: Results from a Multicenter Phase III study 
Introduction
Sublobar resection (SR) is commonly used for patients considered high-risk for lobectomy. Non-operative therapies are increasingly being reported for similar risk patients because of perceived lower morbidity. We report 30 and 90 day adverse events (AEs) from ACOSOG Z4032; a multicenter phase III study for high-risk stage I non-small cell lung cancer (NSCLC) patients.
Methods
Data from 222 evaluable patients randomized to SR (n=114) or SR with brachytherapy (SRB) (n=108) are reported. AEs were recorded using the Common Terminology Criteria for Adverse Events, Version 3.0 at 30 and 90 days post surgery. Risk factors (age, baseline DLCO%, and FEV1%, upper lobe versus lower lobe resections, performance status, surgery approach; VATS versus open and extent ; wedge versus segmentectomy) were analyzed using a multivariable logistic model for their impact on the incidence of Grade 3 (G3+) and higher AEs. Respiratory AEs were also specifically analyzed
Results
Median age, FEV1% and DLCO% were similar for the two treatment groups. There was no difference in the location of resection (upper versus lower lobe) or in the use of segmental or wedge resections. There were no differences between the groups with respect to “respiratory” G3+ (30 days: 14.9% vs. 19.4%; p=0.35; 0–90 days: 19.3% vs. 25%; p=0.31) and “any” G3+AEs (30 days: 25.4% vs. 30.6%; p=0.37; 0–90 days: 29.8% vs. 37%; p=0.25). Further analysis combined the two groups. Mortality occurred in 3 (1.4%) patients by 30 days and in 6 (2.7%) patients by 90 days. Four of the six deaths were felt to be attributable to surgery. When considered as continuous variables, FEV1% was associated with “any” grade 3 + AE at days 0–30 (p=0.03; OR=0.98), and days 0–90 (p=0.05; OR=0.98) respectively; and DLCO% was associated with “respiratory” grade 3+AE at days 0–30 (p=0.03; OR=0.97), and days 0–90 (p=0.05; OR=0.98) respectively. Segmental resection was associated with a higher incidence of any G3+ AE compared to wedge at days 0–30(40.3% versus 22.7%; OR=2.56; p<0.01) and days 0–90 (41.5% versus 29.7%; OR=1.96; p=0.04). The median FEV1% was 50% and the median DLCO% was 46%. Using these median values as potential cutpoints, only a DLCO% of less than 46% was significantly associated with an increased risk of “respiratory” and “any” grade 3+ AE for days 0–30, and 0–90.
Conclusions
In a multicenter setting, SRB was not associated with increased morbidity compared to SR alone. SR/SRB can be performed safely in high-risk patients with NSCLC with low 30 and 90 day mortality and acceptable morbidity. Segmental resection was associated with increased “any” G3+ AE, and DLCO% less than 46% was associated with “any” G3+AE as well as “respiratory” G3+ AE at both 30 and 90 days.
doi:10.1016/j.jtcvs.2011.07.051
PMCID: PMC3199353  PMID: 21872277
19.  Geographic Distance Between Donor and Recipient Does Not Influence Outcomes After Lung Transplantation 
The Annals of thoracic surgery  2011;92(5):1847-1853.
Background
The shortage in organ donation is a major limiting factor for patients with end-stage lung disease. Expanding the donor pool would be beneficial. We investigated the importance of geographic distance between the donor and recipient and hypothesized that it would not be a critical determinant of outcomes after lung transplantation.
Methods
We retrospectively reviewed the United Network for Organ Sharing lung transplant database from 2000 to 2005 to allow sufficient time for bronchiolitis obliterans syndrome (BOS) development. Allograft recipients were stratified by geographic distance from their donors (local, regional, and national) and had yearly follow-up. The primary end points were the development of BOS and 1-year and 3-year mortality. Posttransplant outcomes were compared using a multivariable Cox proportional hazard model. Kaplan-Meier curves were compared by log-rank test.
Results
Of 6,055 allograft recipients, donors were local in 59%, regional in 19.3%, and national in 21.7%. BOS-free survival did not differ by geographic distance. Geographic distance did not independently predict BOS (hazard ratio, 1.03; 95% confidence interval, 0.96 to 1.10). Similarly, Kaplan-Meier survival curves were not significantly worse for recipients with national donors. Geographic distance did not independently predict 3-year mortality (hazard ratio, 0.95; 95% confidence interval, 0.89 to 1.01).
Conclusions
With appropriate donor selection, moderately long geographic distance (average ischemic time < 6 hours) between the donor and recipient is not associated with the development of BOS or increased death after lung transplantation. By placing less emphasis on distance, more donors could potentially be used to expand the donor pool.
doi:10.1016/j.athoracsur.2011.06.086
PMCID: PMC3470835  PMID: 22051280
20.  Virtual Screening Targeting the Urokinase Receptor, Biochemical and Cell-Based Studies, Synthesis, Pharmacokinetic Characterization, and Effect on Breast Tumor Metastasis 
Journal of Medicinal Chemistry  2011;54(20):7193-7205.
Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.
doi:10.1021/jm200782y
PMCID: PMC3280887  PMID: 21851064
21.  BRMS1 transcriptional repression correlates with CpG island methylation and advanced pathological stage in non-small cell lung cancer 
The Journal of pathology  2010;221(2):229-237.
Breast cancer metastasis suppressor gene-1 (BRMS1) mRNA and protein expression are significantly decreased in non-small cell lung cancer (NSCLC) and this is a poor prognostic indicator. Given that the BRMS1 promoter region contains a promoter-associated CpG island (CGI) that encompasses the transcriptional start site, we hypothesized that decreased BRMS1 mRNA and protein levels in NSCLC was secondary to increased BRMS1 promoter methylation. Methylation-specific PCR (MSP) of the two known CGIs (−3477 to −2214 and −531 to +608) in the BRMS1 genome was performed in NSCLC cells. This demonstrated a robust increase in methylation of the promoter-associated CGI (−531 to +608) but not of the upstream CGI (−3477 to −2214). To experimentally verify that methylation contributes to BRMS1 transcriptional repression, we cloned the BRMS1 promoter region, including the promoter-associated CGI, into a luciferase reporter gene and found that BRMS1 promoter activity was dramatically inhibited under methylated conditions. We then assessed the BRMS1 methylation profile with MSP and bisulphite-sequencing PCR in human NSCLC adenocarcinoma (n = 20) and squamous cell carcinoma (n = 20) relative to adjacent non-cancerous bronchial epithelium. There was a significant increase in BRMS1 promoter methylation in all NSCLC specimens relative to non-cancerous tissues, with the most dramatic difference in squamous cell cancer histology. Subsequent immunostaining demonstrated that nuclear BRMS1 expression is reduced in lung cancer specimens compared to normal bronchial epithelium. The association between BRMS1 promoter methylation and specific clinical and histopathological variables was examined using a general linear model. Pathological tumour stage was associated with increased BRMS1 methylation in squamous cell cancers. These observations demonstrate that methylation of the promoter-associated CGI in BRMS1 results in its transcriptional repression, and highlight the potential clinical relevance of this methylation event with respect to NSCLC tumour histology and pathological stage.
doi:10.1002/path.2707
PMCID: PMC2933360  PMID: 20455258
lung cancer; BRMS1; methylation; methylation-specific PCR; immunohistochemistry; pathological stage; squamous cell cancer
22.  Resource Requirements of the Pacific Leatherback Turtle Population 
PLoS ONE  2012;7(10):e45447.
The Pacific population of leatherback sea turtles (Dermochelys coriacea) has drastically declined in the last 25 years. This decline has been linked to incidental capture by fisheries, egg and meat harvesting, and recently, to climate variability and resource limitation. Here we couple growth rates with feeding experiments and food intake functions to estimate daily energy requirements of leatherbacks throughout their development. We then estimate mortality rates from available data, enabling us to raise food intake (energy requirements) of the individual to the population level. We place energy requirements in context of available resources (i.e., gelatinous zooplankton abundance). Estimated consumption rates suggest that a single leatherback will eat upward of 1000 metric tonnes (t) of jellyfish in its lifetime (range 924–1112) with the Pacific population consuming 2.1×106 t of jellyfish annually (range 1.0–3.7×106) equivalent to 4.2×108 megajoules (MJ) (range 2.0–7.4×108). Model estimates suggest 2–7 yr-old juveniles comprise the majority of the Pacific leatherback population biomass and account for most of the jellyfish consumption (1.1×106 t of jellyfish or 2.2×108 MJ per year). Leatherbacks are large gelatinous zooplanktivores with consumption to biomass ratios of 96 (up to 192 if feeding strictly on low energy density Cnidarians); they, therefore, have a large capacity to impact gelatinous zooplankton landscapes. Understanding the leatherback's needs for gelatinous zooplankton, versus the availability of these resources, can help us better assess population trends and the influence of climate induced resource limitations to reproductive output.
doi:10.1371/journal.pone.0045447
PMCID: PMC3465295  PMID: 23071518
23.  The Impact of Adjuvant Brachytherapy with Sublobar Resection on Pulmonary Function and Dyspnea in High-risk Operable Patients; Preliminary results from the ACOSOG Z4032 Trial 
Introduction
Z4032 is a randomized clinical trial conducted by the American College of Surgeons Oncology Group that compared sublobar resection alone (SR) to sublobar resection with brachytherapy (SRB) for high-risk operable patients with non-small cell lung cancer (NSCLC). This current report evaluate the early impact that adjuvant brachytherapy has on pulmonary function tests (PFT), dyspnea and perioperative (30-day) respiratory complications on this impaired patient population.
Methods
Eligible stage I NSCLC patients with tumors 3cm or less were randomized to SR or SRB. The outcomes measured included the % predicted forced expiratory volume (FEV1%), % predicted carbon monoxide diffusion capacity (DLCO%), dyspnea score using the UC San Diego Shortness of Breath Questionnaire. Pulmonary morbidity was assessed using the Common Terminology Criteria for Adverse Events (AE) Version 3.0 (CTCAE). Outcomes were measured at baseline, and at 3-months. A 10% change in PFT or a 10-point change in dyspnea score was deemed clinically meaningful.
Results
Z4032 permanently closed to patient accrual in January 2010 with a total of 224 patients. At 3-month follow-up, PFT data is currently available on 148 (74 SR/74 SRB) patients described in this report. There were no differences in baseline characteristics between the arms. In the SR arm, 9 (12%) patients reported grade-3 respiratory AE compared to 12 (16%) in the SRB arm (p=0.49). There was no significant change in the percent change in DLCO%, or dyspnea score from baseline to 3-month within either arm. In the case of FEV1%, the percent change from baseline to 3-month was significant within SR arm (p=0.03), with patients reporting an improvement in the FEV1% at month 3. Multivariable regression analysis (adjusted for baseline values) showed no significant impact of treatment arm, tumor location (upper versus other lobe), or surgical approach (VATS versus thoracotomy) on the 3-month values for FEV1%, DLCO% and dyspnea scores. There was no significant difference in the incidence of clinically meaningful (10% PFT change, or 10-point dyspnea score) change between the two arms. Twenty-two percent of patients with lower lobe tumors compared to 9% with upper lobe tumors demonstrated a 10% decline in FEV1% (odds ratio 2.79; 95 CI=1.07 – 7.25; p=0.04).
Conclusions
Adjuvant intraoperative brachytherapy performed in conjunction with sublobar resection does not significantly worsen pulmonary function, or dyspnea at 3-months in a high-risk population with NSCLC. SRB was not associated with increased perioperative pulmonary AE. Lower-lobe resection was the only factor that was significantly associated with a clinically meaningful decline in FEV1%.
doi:10.1016/j.jtcvs.2010.10.061
PMCID: PMC3156868  PMID: 21724195
24.  Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth 
Molecular cancer therapeutics  2011;10(9):1698-1708.
Pancreatic cancer is an especially deadly form of cancer with a survival rate <2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression and resistance to traditional therapy is therefore critical. Reduction-oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/ Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells as well as pancreatic cancer-associated endothelial cells (PCECs) in vitro. Modulation of APE1/Ref-1 using a specific inhibitor of APE1/Ref-1’s redox function, E3330 leads to a decrease in transcription factor activity for NFκB, AP-1, and HIF1 in vitro. This study aims to further establish the redox signaling protein APE1/Ref-1 as a molecular target in pancreatic cancer. Here, we show that inhibition of APE1/Ref-1 via E3330 results in tumor growth inhibition in cell lines as well as pancreatic cancer xenograft models in mice. Pharmacokinetic (PK) studies also demonstrate that E3330 attains >10 μM blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NFκB, AP-1, and HIF1α which are key transcriptional regulators involved in survival, invasion and metastasis is blocked. These data indicate that E3330, inhibitor of APE1/Ref-1, has potential in pancreatic cancer and clinical investigation of APE1/Ref-1 molecular target is warranted.
doi:10.1158/1535-7163.MCT-11-0107
PMCID: PMC3170439  PMID: 21700832
Pancreatic cancer; animal models of cancer; new targets; xenograft models; cellular responses to anticancer drugs; cellular pharmacology; pharmacokinetics and pharmacodynamics; agents with other mechanisms of action
25.  Gender, Race and Socioeconomic Status Affects Outcomes Following Lung Cancer Resections in the United States 
The Annals of Thoracic Surgery  2011;92(2):434-439.
Background
The effect of gender, race and socioeconomic status on contemporary outcomes following lung cancer resections has not been comprehensively evaluated nationwide. We hypothesized that risk-adjusted outcomes for lung cancer resections would not be influenced by these factors.
Methods
From 2003–2007, 129, 207 patients undergoing lung cancer resections were evaluated using the Nationwide Inpatient Sample (NIS). Multiple regression was utilized to estimate the effects of gender, race and socioeconomic status on risk-adjusted outcomes.
Results
Average patient age was 66.8±10.5 years. Females accounted for 5.0% of the total study population. Among racial groups, whites underwent the large majority of operations (86.2%) followed by Black (6.9%) and Hispanic (2.8%) races. Overall, the incidence of mortality was 2.9%, postoperative complications 30.4%, and pulmonary complications 22.0%. Female gender, race, and mean income were all multivariate correlates of adjusted mortality and morbidity. Black patients incurred decreased risk-adjusted morbidity and mortality compared to white patients. Hispanics and Asians demonstrated decreased risk-adjusted complication rates. Importantly, low-income status independently increased the adjusted odds of mortality.
Conclusions
Female gender is associated with decreased mortality and morbidity following lung cancer resections. Complication rates are lower for Black, Hispanic and Asian patients. Low socioeconomic status increases the risk of in-hospital death. These factors should be considered during patient risk stratification for lung cancer resection.
doi:10.1016/j.athoracsur.2011.04.048
PMCID: PMC3282148  PMID: 21704976
Gender; Race; Income; Lung Cancer; Surgery; Outcomes

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