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author:("salgiy, Ravi")
1.  Whole-Animal Mounts of Caenorhabditis elegans for 3D Imaging Using Atomic Force Microscopy 
The 3D surface of Caenorhabditis elegans was imaged at nanometer resolution using atomic force microscopy (AFM). Oscillation of a medium stiffness silicon AFM cantilever at the upper second amplitude peak, typically 6 times above the fundamental frequency, vastly improved image quality on the moist, sticky, and soft worms. Whole-animal mounts of normal and double-headed mutants of the nematode worm were prepared and scanned. Well-preserved anatomical features including annuli, furrows, alae, and rows of never before seen nanometer-sized pores dotting the molted worm's outermost surface coat were resolved. This AFM method represents a simple and rapid new approach for nanometer-resolved 3D imaging and analysis of whole-animal specimens of C. elegans.
Graphical Abstract
C. elegans is a useful model to study genetics as well as genomic and environmental interactions as we have shown. Even though a large amount of data exists on the various ‘normal’ and variant C. elegans, the structure of these organisms still remains a mystery. We have refined the understanding of C. elegans morphology by studying images generated by atomic force microscopy. We show that the skin has considerable cuticles and also the variant vab-1 (EPH mutation) has the notched head phenotype. (Graphical Abstract: Figure 2)
Allen, et. al., Scanning Whole-Animal Mounts of Caenorhabditis Elegans Using Atomic Force Microscopy
doi:10.1016/j.nano.2015.07.014
PMCID: PMC5051269  PMID: 26282382
C. elegans; atomic force microscopy; AFM
2.  Phase II Study of the AKT inhibitor MK-2206 plus Erlotinib in Patients with Advanced Non-Small Cell Lung Cancer who Previously Progressed on Erlotinib 
Purpose
Preclinical modeling in non-small cell lung cancer (NSCLC) showed that stimulation with hepatocyte growth factor (HGF), the ligand for MET, could reverse the cytostatic and cytotoxic effects of the epidermal-growth factor receptor (EGFR) inhibitor erlotinib in erlotinib-sensitive cell lines. Inhibitors of AKT signaling mitigated this HGF-mediated resistance, partially restoring erlotinib activity. We conducted a phase II trial of erlotinib plus MK2206, a highly selective inhibitor of AKT, in NSCLC patients.
Experimental Design
Eligible patients must have progressed following prior benefit from erlotinib, defined as response or stable disease > 12 weeks. Treatment consisted of erlotinib 150 mg po QD + MK-2206 45 mg po QOD on a 28 day cycle. Primary endpoints were RECIST response rate > 30% (stratum 1: EGFR mutant) and disease control rate (DCR) > 20% at 12 weeks (stratum 2: EGFR wild type).
Results
Eighty patients were enrolled, 45 and 35 in stratum 1 and 2, respectively. Most common attributable adverse events (all grade 3) were rash, diarrhea, fatigue, and mucositis. Response and DCR were respectively 9% and 40% in stratum 1; 3% and 47% in stratum 2. Median progression free survival was 4.4 months in stratum 1 and 4.6 months in stratum 2.
Conclusions
Combination MK2206 and erlotinib met its primary endpoint in erlotinib-pretreated patients with EGFR wild type NSCLC. While activity was seen in EGFR mutated NSCLC, this did not exceed a priori estimates. AKT pathway inhibition merits further clinical evaluation in EGFR wild type NSCLC.
doi:10.1158/1078-0432.CCR-14-3281
PMCID: PMC4592395  PMID: 26106072
3.  AXL is a logical molecular target in head and neck squamous cell carcinoma 
Purpose
Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard of care treatments for HNSCC patients include surgery, radiation and chemotherapy. Additionally, the anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult to treat malignancy. Thus, identification of new molecular targets is critical.
Experimental Design
In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient derived xenografts (PDXs), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard of care treatment regimes used in HNSCC.
Results
AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases and shorter relapse free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. Additionally, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance.
Conclusion
Collectively, this study provides evidence for the role of AXL in HNSCC pathogenesis and supports further pre-clinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC.
doi:10.1158/1078-0432.CCR-14-2648
PMCID: PMC5032632  PMID: 25767293
AXL; Molecular Targeting; Head and Neck Squamous Cell Carcinoma; HNSCC
4.  PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma 
Scientific Reports  2016;6:32992.
Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent.
doi:10.1038/srep32992
PMCID: PMC5021085  PMID: 27623107
5.  Lung cancer—a fractal viewpoint 
Nature reviews. Clinical oncology  2015;12(11):664-675.
Fractals are mathematical constructs that show self-similarity over a range of scales and non-integer (fractal) dimensions. Owing to these properties, fractal geometry can be used to efficiently estimate the geometrical complexity, and the irregularity of shapes and patterns observed in lung tumour growth (over space or time), whereas the use of traditional Euclidean geometry in such calculations is more challenging. The application of fractal analysis in biomedical imaging and time series has shown considerable promise for measuring processes as varied as heart and respiratory rates, neuronal cell characterization, and vascular development. Despite the advantages of fractal mathematics and numerous studies demonstrating its applicability to lung cancer research, many researchers and clinicians remain unaware of its potential. Therefore, this Review aims to introduce the fundamental basis of fractals and to illustrate how analysis of fractal dimension (FD) and associated measurements, such as lacunarity (texture) can be performed. We describe the fractal nature of the lung and explain why this organ is particularly suited to fractal analysis. Studies that have used fractal analyses to quantify changes in nuclear and chromatin FD in primary and metastatic tumour cells, and clinical imaging studies that correlated changes in the FD of tumours on CT and/or PET images with tumour growth and treatment responses are reviewed. Moreover, the potential use of these techniques in the diagnosis and therapeutic management of lung cancer are discussed.
doi:10.1038/nrclinonc.2015.108
PMCID: PMC4989864  PMID: 26169924
6.  Post-crizotinib management of effective ceritinib therapy in a patient with ALK-positive non-small cell lung cancer 
BMC Cancer  2016;16:568.
Background
We report the re-biopsied diagnosis of a patient with anaplastic lymphoma receptor tyrosine kinase (ALK)-positive lung adenocarcinoma successfully treated with ceritinib 450 mg/day taken with food following disease progression and gastrointestinal intolerance to crizotinib.
Case presentation
A 74-year old female patient initially diagnosed with ALK-negative lung adenocarcinoma responded to initial standard chemotherapy. The patient was subsequently re-tested by next generation sequencing (NGS) and found to have ALK EIF2AK3-ALK fusion, and responded to crizotinib, but ultimately progressed and showed intolerance to this ALK inhibitor. She was then successfully treated with ceritinib 450 mg/day taken with food, has not suffered from any further gastrointestinal side-effects, and remains on ceritinib treatment after 12 months.
Conclusions
Second-line ceritinib treatment, when administered at 450 mg/day with food, is both well tolerated and efficacious in a patient with previously treated lung adenocarcinoma who had discontinued crizotinib due to disease progression and gastrointestinal adverse effects (AEs).
doi:10.1186/s12885-016-2636-z
PMCID: PMC4969677  PMID: 27480287
ALK-positive NSCLC; Ceritinib; Gastrointestinal tolerance; Dose; Case report
7.  Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin 
Journal of molecular biology  2015;427(15):2532-2547.
Focal adhesion protein paxillin links integrin and growth factor signaling to actin cytoskeleton. Most of paxillin signaling activity is regulated via leucine-rich LD motifs (LD1-LD5) located at the N-terminus. Here, we demonstrate a method to engineer highly selective synthetic antibodies (sABs) against LD2 and LD4 which are binding sites for focal adhesion kinase (FAK) and other proteins. Phage display selections against peptides were used to generate sABs recognizing each LD motif. In the obtained X-ray crystal structures of the LD-sAB complexes the LD motifs are helical and bind sABs through a hydrophobic side, similarly as in the structures with natural paxillin partners. The sABs are capable of pulling down endogenous paxillin in complex with FAK and can visualize paxillin in focal adhesions in cells. They were also used as selective inhibitors to effectively compete with focal adhesion targeting domain of FAK for the binding to LD2 and LD4. The sABs are tools for investigation of paxillin LD binding “platforms” and are capable of inhibiting paxillin interactions; and thereby useful as potential therapeutics in the future.
Graphical abstract
doi:10.1016/j.jmb.2015.06.004
PMCID: PMC4521624  PMID: 26087144
synthetic antigen binder; phage display; paxillin; focal adhesion; protein-protein interaction
8.  A New PET/CT Volumetric Prognostic Index for Non-small Cell Lung Cancer 
Objectives
Whole-body metabolic tumor volume (MTVWB) has been shown of prognostic value for non-small cell lung cancer (NSCLC) beyond that of TNM stage, age, gender, performance status, and treatment selection. The current TNM staging system does not incorporate tumor volumetric information. We propose a new PET/CT volumetric prognostic (PVP) index that combines the prognostic value of MTVWB and TNM stage.
Materials and Methods
Based on 328 consecutive NSCLC patients with a baseline PET/CT scan before treatment, from which MTVWB was measured semi-automatically, we estimated hazard ratios (HRs) for ln(MTVWB) and TNM stage from a Cox proportional hazard regression model that consisted of only ln(MTVWB) and TNM stage as prognostic variables of overall survival. We used the regression coefficients, which gave rise to the HRs, as weights to formulate the PET/CT volumetric prognostic (PVP) index. We also compared the prognostic value of the PVP index against that of TNM stage alone and ln(MTVWB) alone with univariate and multivariate survival analyses and C-statistics.
Results
Univariate analysis C-statistic for the PVP index (C = 0.71) was statistically significantly greater than those for TNM stage alone (C = 0.67, p < 0.001) and for ln(MTVWB) alone (C = 0.69, p = 0.033). Multivariate analyses showed that the PVP index yielded significantly greater discriminatory power (C = 0.74) than similar models based on either TNM stage (C = 0.72, p < 0.01) or ln(MTVWB) (C = 0.73, p < 0.01). Lower values of the PVP index were associated with significantly better overall survival (adjusted HR = 2.70, 95%CI [2.16, 3.37]).
Conclusion
The PVP index provides a practical means for clinicians to combine the prognostic value of MTVWB and TNM stage and offers significantly better prognostic accuracy for overall survival of NSCLC patients than the current TNM staging system or metabolic tumor burden alone.
doi:10.1016/j.lungcan.2015.03.023
PMCID: PMC4457565  PMID: 25936471
18F-FDG PET/CT; non-small cell lung cancer (NSCLC); TNM stage; tumor burden; metabolic tumor volume (MTV)
9.  Mutation testing for directing upfront targeted therapy and post-progression combination therapy strategies in lung adenocarcinoma 
ABSTRACT
Introduction: Advances in the biology of non-small-cell lung cancer, especially adenocarcinoma, reveal multiple molecular subtypes driving oncogenesis. Accordingly, individualized targeted therapeutics are based on mutational diagnostics.
Areas covered: Advances in strategies and techniques for individualized treatment, particularly of adenocarcinoma, are described through literature review. Approved therapies are established for some molecular subsets, with new driver mutations emerging that represent increasing proportions of patients. Actionable mutations are de novo oncogenic drivers or acquired resistance mediators, and mutational profiling is important for directing therapy. Patients should be monitored for emerging actionable resistance mutations. Liquid biopsy and associated multiplex diagnostics will be important means to monitor patients during treatment.
Expert commentary: Outcomes with targeted agents may be improved by integrating mutation screens during treatment to optimize subsequent therapy. In order for this to be translated into impactful patient benefit, appropriate platforms and strategies need to be optimized and then implemented universally.
doi:10.1080/14737159.2016.1181545
PMCID: PMC4926789  PMID: 27139190
NSCLC; oncogenic drivers; mutation; resistance; liquid biopsy; re-biopsy; diagnostics
10.  FAK and paxillin, two potential targets in pancreatic cancer 
Oncotarget  2016;7(21):31586-31601.
Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer in large part due to late diagnosis and a lack of effective screening tests. In spite of recent progress in imaging, surgery and new therapeutic options for pancreatic cancer, the overall five-year survival still remains unacceptably low. Numerous studies have shown that focal adhesion kinase (FAK) is activated in many cancers including PDAC and promotes cancer progression and metastasis. Paxillin, an intracellular adaptor protein that plays a key role in cytoskeletal organization, connects integrins to FAK and plays a key role in assembly and disassembly of focal adhesions. Here, we have reviewed evidence in support of FAK as a potential therapeutic target and summarized related combinatorial therapies.
doi:10.18632/oncotarget.8040
PMCID: PMC5058780  PMID: 26980710
FAK; paxillin; pancreatic cancer; integrins; P53
11.  Unique fractal evaluation and therapeutic implications of mitochondrial morphology in malignant mesothelioma 
Scientific Reports  2016;6:24578.
Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.
doi:10.1038/srep24578
PMCID: PMC4832330  PMID: 27080907
12.  Comprehensive genetic testing identifies targetable genomic alterations in most patients with non-small cell lung cancer, specifically adenocarcinoma, single institute investigation 
Oncotarget  2016;7(14):18876-18886.
This study reviews extensive genetic analysis in advanced non-small cell lung cancer (NSCLC) patients in order to: describe how targetable mutation genes interrelate with the genes identified as variants of unknown significance; assess the percentage of patients with a potentially targetable genetic alterations; evaluate the percentage of patients who had concurrent alterations, previously considered to be mutually exclusive; and characterize the molecular subset of KRAS.
Thoracic Oncology Research Program Databases at the University of Chicago provided patient demographics, pathology, and results of genetic testing. 364 patients including 289 adenocarcinoma underwent genotype testing by various platforms such as FoundationOne, Caris Molecular Intelligence, and Response Genetics Inc. For the entire adenocarcinoma cohort, 25% of patients were African Americans; 90% of KRAS mutations were detected in smokers, including current and former smokers; 46% of EGFR and 61% of ALK alterations were detected in never smokers. 99.4% of patients, whose samples were analyzed by next-generation sequencing (NGS), had genetic alterations identified with an average of 10.8 alterations/tumor throughout different tumor subtypes. However, mutations were not mutually exclusive.
NGS in this study identified potentially targetable genetic alterations in the majority of patients tested, detected concurrent alterations and provided information on variants of unknown significance at this time but potentially targetable in the future.
doi:10.18632/oncotarget.7739
PMCID: PMC4951336  PMID: 26934441
non-small cell lung cancer; genetic testing; next-generation sequencing; genomic alteration
13.  Effective growth-suppressive activity of maternal embryonic leucine-zipper kinase (MELK) inhibitor against small cell lung cancer 
Oncotarget  2016;7(12):13621-13633.
Maternal embryonic leucine zipper kinase (MELK), that plays a critical role in maintenance of cancer stem cells (CSCs), is predominantly expressed in various types of human cancer including small cell lung cancer (SCLC). SCLC usually acquires resistance to anti-cancer drugs and portends dismal prognosis. We have delineated roles of MELK in development/progression of SCLC and examined anti-tumor efficacy of OTS167, a highly potent MELK inhibitor, against SCLC. MELK expression was highly upregulated in both SCLC cell lines and primary tumors. siRNA-mediated MELK knockdown induced significant growth inhibition in SCLC cell lines. Concordantly, treatment with OTS167 exhibited strong cytotoxicity against eleven SCLC cell lines with IC50 of < 10 nM. As similar to siRNA knockdown, OTS167 treatment induced cytokinetic defects with intercellular bridges, and in some cell lines we observed formation of neuronal protrusions accompanied with increase of a neuronal differentiation marker (CD56), indicating that the compound induced differentiation of cancer cells to neuron-like cells. Furthermore, the MELK inhibition decreased its downstream FOXM1 activity and Akt expression in SCLC cells, and led to apoptotic cell death. OTS167 appeared to be more effective to CSCs as measured by the sphere formation assay, thus MELK inhibition might become a promising treatment modality for SCLC.
doi:10.18632/oncotarget.7297
PMCID: PMC4924666  PMID: 26871945
small cell lung cancer; MELK; molecular target; kinase inhibitor; cancer stem cell
14.  The Chicago Thoracic Oncology Database Consortium: A Multisite Database Initiative 
Cureus  null;8(3):e533.
Objective: An increasing amount of clinical data is available to biomedical researchers, but specifically designed database and informatics infrastructures are needed to handle this data effectively. Multiple research groups should be able to pool and share this data in an efficient manner. The Chicago Thoracic Oncology Database Consortium (CTODC) was created to standardize data collection and facilitate the pooling and sharing of data at institutions throughout Chicago and across the world. We assessed the CTODC by conducting a proof of principle investigation on lung cancer patients who took erlotinib. This study does not look into epidermal growth factor receptor (EGFR) mutations and tyrosine kinase inhibitors, but rather it discusses the development and utilization of the database involved.
Methods:  We have implemented the Thoracic Oncology Program Database Project (TOPDP) Microsoft Access, the Thoracic Oncology Research Program (TORP) Velos, and the TORP REDCap databases for translational research efforts. Standard operating procedures (SOPs) were created to document the construction and proper utilization of these databases. These SOPs have been made available freely to other institutions that have implemented their own databases patterned on these SOPs.
Results: A cohort of 373 lung cancer patients who took erlotinib was identified. The EGFR mutation statuses of patients were analyzed. Out of the 70 patients that were tested, 55 had mutations while 15 did not. In terms of overall survival and duration of treatment, the cohort demonstrated that EGFR-mutated patients had a longer duration of erlotinib treatment and longer overall survival compared to their EGFR wild-type counterparts who received erlotinib.
Discussion: The investigation successfully yielded data from all institutions of the CTODC. While the investigation identified challenges, such as the difficulty of data transfer and potential duplication of patient data, these issues can be resolved with greater cross-communication between institutions of the consortium.
Conclusion: The investigation described herein demonstrates the successful data collection from multiple institutions in the context of a collaborative effort. The data presented here can be utilized as the basis for further collaborative efforts and/or development of larger and more streamlined databases within the consortium.
doi:10.7759/cureus.533
PMCID: PMC4833499  PMID: 27092293
database; bioinformatics; lung cancer
15.  Concurrent EGFR Mutation and ALK Translocation in Non-Small Cell Lung Cancer 
Cureus  null;8(2):e513.
Epidermal growth factor receptor (EGFR) mutations and anaplastic large-cell lymphoma kinase (ALK) rearrangements are now routine biomarkers that have been incorporated into the practice of managing non-small cell lung cancer (NSCLC). Historically, the two molecular alterations have been viewed as mutually exclusive, but recent identified cases suggest otherwise. In this report, we describe cases of lung cancer with concurrent EGFR mutation and ALK rearrangement and identify their clinical characteristics.
Non-small cell lung cancer patients with multiple molecular alterations were retrospectively analyzed from an academic referral center from 2011–2013. An additional review was conducted of reported cases with dual alterations. Four cases of NSCLC with alterations in both EGFR and ALK were identified and evaluated with 16 published cases for a total of 20 cases. The age of patients ranged from 37 to 77 years. Nine patients were never smokers. The disease control rates in patients treated with EGFR inhibitors and ALK inhibitors were 46% (6/13) and 71% (5/7), respectively.
This series highlights the importance of comprehensive molecular profiling of newly diagnosed lung cancer, as NSCLC may be driven by concurrent molecular alterations. EGFR- and ALK-targeted therapies appear to have modest activity in patients with tumors possessing both alterations. Dual-altered NSCLC patients may have distinct clinical characteristics warranting further study. Combination targeted therapy or novel multi-targeted tyrosine kinase inhibitors may prove important in these patients, though necessary studies remain ongoing.
doi:10.7759/cureus.513
PMCID: PMC4807912  PMID: 27026837
personalized medicine; genomics; molecular subtypes; non-small-cell lung cancer; alk-positive adenocarcinoma; egfr mutations in lung adenocarcinoma
16.  MOLECULARLY TARGETED THERAPIES IN NON-SMALL CELL LUNG CANCER ANNUAL UPDATE 2014 
There have been significant advances in the understanding of the biology and treatment of non-small cell lung cancer (NSCLC) over the past few years. A number of molecularly targeted agents are in the clinic or in development for patients with advanced NSCLC (Table 1). We are beginning to understand the mechanisms of acquired resistance following exposure to tyrosine kinase inhibitors in patients with oncogene addicted NSCLC. The advent of next generation sequencing has enabled to study comprehensively genomic alterations in lung cancer. Finally, early results from immune checkpoint inhibitors are very encouraging. This review summarizes recent advances in the area of cancer genomics, targeted therapies and immunotherapy.
doi:10.1097/JTO.0000000000000405
PMCID: PMC4346098  PMID: 25535693
NSCLC; TARGETED THERAPIES; IMMUNOTHERAPY
17.  Role played by paxillin and paxillin tyrosine phosphorylation in hepatocyte growth factor/sphingosine-1-phosphate-mediated reactive oxygen species generation, lamellipodia formation, and endothelial barrier function 
Pulmonary Circulation  2015;5(4):619-630.
Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of lamellipodia formation, a prerequisite for endothelial cell migration. The role played by paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced lamellipodia formation and barrier function is unclear. HGF or S1P stimulated lamellipodia formation, tyrosine phosphorylation of paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of paxillin with small interfering RNA (siRNA) or transfection with paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced lamellipodia formation, translocation of p47phox to lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated lamellipodia formation, translocation of p47phox to lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates HGF- or S1P-mediated lamellipodia formation, ROS generation in lamellipodia, and endothelial permeability.
doi:10.1086/683693
PMCID: PMC4671736  PMID: 26697169
paxillin; tyrosine phosphorylation of paxillin; lamellipodia; c-Abl; endothelial barrier; reactive oxygen species
18.  Geographic Variation in Radiologist Capacity and Widespread Implementation of Lung Cancer CT Screening 
Journal of medical screening  2014;21(4):207-215.
Background
Newly released United States Preventive Services Task Force (USPSTF) recommendations for lung cancer screening are expected to increase demand for low-dose computed tomography scanning, but health system capacity constraints might threaten the scale-up of screening.
Objectives
To estimate the prevalence of capacity constraints in the radiologist workforce and resulting potential disparities in access to lung cancer screening.
Methods
We combined information from health interview surveys to estimate the numbers of smokers who meet the USPSTF eligibility criteria, and information from administrative datasets to estimate the numbers of radiologists and the numbers of scans they currently interpret in Health Service Areas (HSAs) nationwide. We estimated and mapped the prevalence of capacity constrained HSAs – those having a greater than 5% or greater than 25% projected increase in scans over current levels from scaling up screening – and used descriptive statistics and logistic regressions to identify HSA characteristics associated with capacity constraints.
Results
Scaling up lung cancer screening would increase imaging procedures by an average of 4% across HSAs. Of the 9.6 million eligible smokers, 1,023,943 lived in HSAs with increases of at least 5%. HSAs that were rural, with many eligible smokers, and disproportionately Hispanic or low-income, smokers had significantly higher odds of facing capacity constraints.
Conclusions
Disparities in access to lung cancer screening appear likely unless policy makers target HSAs with few radiologists for additional resources. Radiologists should be able to absorb the workload imposed by lung cancer screening in most areas of the country.
doi:10.1177/0969141314548055
PMCID: PMC4407799  PMID: 25118160
lung cancer; cancer screening; CT scan; health care capacity; radiologists
19.  Randomized Phase Ib/II Study of Gemcitabine Plus Placebo or Vismodegib, a Hedgehog Pathway Inhibitor, in Patients With Metastatic Pancreatic Cancer 
Journal of Clinical Oncology  2015;33(36):4284-4292.
Purpose
Sonic hedgehog (SHH), an activating ligand of smoothened (SMO), is overexpressed in > 70% of pancreatic cancers (PCs). We investigated the impact of vismodegib, an SHH antagonist, plus gemcitabine (GV) or gemcitabine plus placebo (GP) in a multicenter phase Ib/randomized phase II trial and preclinical PC models.
Patients and Methods
Patients with PC not amenable to curative therapy who had received no prior therapy for metastatic disease and had Karnofsky performance score ≥ 80 were enrolled. Patients were randomly assigned in a one-to-one ratio to GV or GP. The primary end point was progression-free-survival (PFS). Exploratory correlative studies included serial SHH serum levels and contrast perfusion computed tomography imaging. To further investigate putative biologic mechanisms of SMO inhibition, two autochthonous pancreatic cancer models (KrasG12D; p16/p19fl/fl; Pdx1-Cre and KrasG12D; p53R270H/wt; Pdx1-Cre) were studied.
Results
No safety issues were identified in the phase Ib portion (n = 7), and the phase II study enrolled 106 evaluable patients (n = 53 in each arm). Median PFS was 4.0 and 2.5 months for GV and GP arms, respectively (95% CI, 2.5 to 5.3 and 1.9 to 3.8, respectively; adjusted hazard ratio, 0.81; 95% CI, 0.54 to 1.21; P = .30). Median overall survival (OS) was 6.9 and 6.1 months for GV and GP arms, respectively (95% CI, 5.8 to 8.0 and 5.0 to 8.0, respectively; adjusted hazard ratio, 1.04; 95% CI, 0.69 to 1.58; P = .84). Response rates were not significantly different. There were no significant associations between correlative markers and overall response rate, PFS, or OS. Preclinical trials revealed no significant differences with vismodegib in drug delivery, tumor growth rate, or OS in either model.
Conclusion
The addition of vismodegib to gemcitabine in an unselected cohort did not improve overall response rate, PFS, or OS in patients with metastatic PC. Our preclinical and clinical results revealed no statistically significant differences with respect to drug delivery or treatment efficacy using vismodegib.
doi:10.1200/JCO.2015.62.8719
PMCID: PMC4678179  PMID: 26527777
20.  Crizotinib (PF02341066) as a ALK /MET inhibitor— Special Emphasis as a Therapeutic Drug Against Lung Cancer 
Drugs of the future  2011;36(2):91-99.
There are a number of molecular abnormalities that can occur in normal cells to induce a malignant phenotype. Recently, the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) has been shown to have gain-of-function when partnered with different proteins. As an example, on chromosome 2p, with inversion, there is translocation with generation of EML4-ALK tyrosine kinase in lung cancer. In a phase I trial, EML4-ALK patients were selected to determine the response to a potent small molecule tyrosine kinase inhibitor crizotinib (previously identified as PF02341066). Dramatic durable responses were observed with crizotinib at 250 mg twice a day (orally). Interestingly, crizotinib also has activity against MET receptor tyrosine kinase. We have previously shown that MET can be overexpressed, sometimes mutated, or sometimes amplified in lung cancer. Thus, this review will emphasize the characteristics of crizotinib, and detail the clinical experience.
doi:10.1358/dof.2011.036.02.1584112
PMCID: PMC4582997  PMID: 26412935
21.  AXL mediates resistance to cetuximab therapy 
Cancer research  2014;74(18):5152-5164.
The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical problem. In this study we show that overexpression of the oncogenic receptor kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated and tightly associated with EGFR expression in cells resistant to cetuximab (CtxR cells). Using RNAi methods and novel AXL targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft assays, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL targeting drugs to treat cetuximab-resistant cancers.
doi:10.1158/0008-5472.CAN-14-0294
PMCID: PMC4167493  PMID: 25136066
AXL; EGFR; Cetuximab; Resistance
22.  Phase I Trial of Erlotinib-Based Multimodality Therapy for Inoperable Stage III Non-small Cell Lung Cancer 
Introduction
This Phase I trial aimed to determine the maximum-tolerated-dose of erlotinib administered with two standard chemoradiotherapy regimens for non-small cell lung cancer.
Methods
Unresectable stage III non-small cell lung cancer patients were enrolled in this 2-arm dose-escalation study. Erlotinib, given only during chemoradiotherapy, was escalated from 50 to 150 mg/d in 3 to 6 patient cohorts. Arm A: erlotinib with cisplatin (50 mg/m2 IV days 1, 8, 29, 36), etoposide (50 mg/m2 IV days 1–5, 29–33) and chest radiotherapy (66 Gy, 2 Gy/d) followed by docetaxel (75 mg/m2 IV Q21 d) for 3 cycles. Arm B: induction carboplatin (AUC 6) and paclitaxel (200 mg/m2) for two 21-d cycles then radiotherapy with erlotinib, carboplatin (AUC = 2/wk) and paclitaxel (50 mg/m2/wk).
Results
Seventeen patients were treated in each arm. Patient characteristics: performance status 0 to 24 patients, 1 to 10 patients, median age 63 years, adenocarcinoma 21% and female 14 patients. Dose-escalation of erlotinib to 150 mg/d was possible on both chemoradiotherapy regimens. Grade 3/4 leukopenia and neutropenia were predominant toxicities in both arms. Grade 3 chemoradiotherapy toxicities in arm A were esophagitis (3 patients), vomiting (1), ototoxicity (1), diarrhea (2), dehydration (3), pneumonitis (1); and arm B was esophagitis (6). Seven patients (21%) developed rash (all grade 1/2). Median survival times for patients on Arm A and B were 10.2 and 13.7 months, respectively. Three-year overall survival in patients with and without rash were 53% and 10%, respectively (log-rank P = 0.0807). Epidermal growth factor receptor IHC or FISH positive patients showed no significant overall survival difference.
Conclusion
Addition of standard-dose erlotinib to chemoradiotherapy is feasible without evident increase in toxicities. However, the survival data are disappointing in this unselected patient population and does not support further investigation of this approach.
doi:10.1097/JTO.0b013e31818396a4
PMCID: PMC4535721  PMID: 18758303
Non-small cell lung cancer; Chemoradiotherapy; Multimodality therapy; Erlotinib; Epidermal-growth factor inhibitor
23.  Dose-Finding and Pharmacokinetic Study to Optimize the Dosing of Irinotecan According to the UGT1A1 Genotype of Patients With Cancer 
Journal of Clinical Oncology  2014;32(22):2328-2334.
Purpose
The risk of severe neutropenia from treatment with irinotecan is related in part to UGT1A1*28, a variant that reduces the elimination of SN-38, the active metabolite of irinotecan. We aimed to identify the maximum-tolerated dose (MTD) and dose-limiting toxicity (DLT) of irinotecan in patients with advanced solid tumors stratified by the *1/*1, *1/*28, and *28/*28 genotypes.
Patients and Methods
Sixty-eight patients received an intravenous flat dose of irinotecan every 3 weeks. Forty-six percent of the patients had the *1/*1 genotype, 41% had the *1/*28 genotype, and 13% had the *28/*28 genotype. The starting dose of irinotecan was 700 mg in patients with the *1/*1 and *1/*28 genotypes and 500 mg in patients with the *28/*28 genotype. Pharmacokinetic evaluation was performed at cycle 1.
Results
In patients with the *1/*1 genotype, the MTD was 850 mg (four DLTs per 16 patients), and 1,000 mg was not tolerated (two DLTs per six patients). In patients with the *1/*28 genotype, the MTD was 700 mg (five DLTs per 22 patients), and 850 mg was not tolerated (four DLTs per six patients). In patients with the *28/*28 genotype, the MTD was 400 mg (one DLT per six patients), and 500 mg was not tolerated (three DLTs per three patients). The DLTs were mainly myelosuppression and diarrhea. Irinotecan clearance followed linear kinetics. At the MTD for each genotype, dosing by genotype resulted in similar SN-38 areas under the curve (AUCs; r2 = 0.0003; P = .97), but the irinotecan AUC was correlated with the actual dose (r2 = 0.39; P < .001). Four of 48 patients with disease known to be responsive to irinotecan achieved partial response.
Conclusion
The UGT1A1*28 genotype can be used to individualize dosing of irinotecan. Additional studies should evaluate the effect of genotype-guided dosing on efficacy in patients receiving irinotecan.
doi:10.1200/JCO.2014.55.2307
PMCID: PMC4105486  PMID: 24958824
24.  Novel EPHB4 Receptor Tyrosine Kinase Mutations and Kinomic Pathway Analysis in Lung Cancer 
Scientific Reports  2015;5:10641.
Lung cancer outcomes remain poor despite the identification of several potential therapeutic targets. The EPHB4 receptor tyrosine kinase (RTK) has recently emerged as an oncogenic factor in many cancers, including lung cancer. Mutations of EPHB4 in lung cancers have previously been identified, though their significance remains unknown. Here, we report the identification of novel EPHB4 mutations that lead to putative structural alterations as well as increased cellular proliferation and motility. We also conducted a bioinformatic analysis of these mutations to demonstrate that they are mutually exclusive from other common RTK variants in lung cancer, that they correspond to analogous sites of other RTKs’ variations in cancers, and that they are predicted to be oncogenic based on biochemical, evolutionary, and domain-function constraints. Finally, we show that EPHB4 mutations can induce broad changes in the kinome signature of lung cancer cells. Taken together, these data illuminate the role of EPHB4 in lung cancer and further identify EPHB4 as a potentially important therapeutic target.
doi:10.1038/srep10641
PMCID: PMC4466581  PMID: 26073592
25.  Crizotinib in ROS1-Rearranged Non–Small-Cell Lung Cancer 
The New England journal of medicine  2014;371(21):1963-1971.
BACKGROUND
Chromosomal rearrangements of the gene encoding ROS1 proto-oncogene receptor tyrosine kinase (ROS1) define a distinct molecular subgroup of non–small-cell lung cancers (NSCLCs) that may be susceptible to therapeutic ROS1 kinase inhibition. Crizotinib is a small-molecule tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK), ROS1, and another proto-oncogene receptor tyrosine kinase, MET.
METHODS
We enrolled 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in an expansion cohort of the phase 1 study of crizotinib. Patients were treated with crizotinib at the standard oral dose of 250 mg twice daily and assessed for safety, pharmacokinetics, and response to therapy. ROS1 fusion partners were identified with the use of next-generation sequencing or reverse-transcriptase–polymerase-chain-reaction assays.
RESULTS
The objective response rate was 72% (95% confidence interval [CI], 58 to 84), with 3 complete responses and 33 partial responses. The median duration of response was 17.6 months (95% CI, 14.5 to not reached). Median progression-free survival was 19.2 months (95% CI, 14.4 to not reached), with 25 patients (50%) still in follow-up for progression. Among 30 tumors that were tested, we identified 7 ROS1 fusion partners: 5 known and 2 novel partner genes. No correlation was observed between the type of ROS1 rearrangement and the clinical response to crizotinib. The safety profile of crizotinib was similar to that seen in patients with ALK-rearranged NSCLC.
CONCLUSIONS
In this study, crizotinib showed marked antitumor activity in patients with advanced ROS1-rearranged NSCLC. ROS1 rearrangement defines a second molecular subgroup of NSCLC for which crizotinib is highly active.
doi:10.1056/NEJMoa1406766
PMCID: PMC4264527  PMID: 25264305

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