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1.  Interfraction Displacement of Primary Tumor and Involved Lymph Nodes Relative to Anatomical Landmarks in Image–guided Radiotherapy of Locally Advanced Lung Cancer 
Image-guided radiotherapy for patients with locally advanced lung cancer relies on bony landmarks and carina or - if visible - the primary tumor (PT) for daily patient alignment, neglecting potential variations in the relative position of PT and involved lymph nodes (LN). This study analyzes PT and LN position changes relative to each other and relative to anatomical landmarks during conventionally fractionated radiotherapy.
Methods and Materials
In 12 patients with locally advanced non-small cell lung cancer PT, LN, carina and one thoracic vertebra were manually contoured on weekly 4D fan beam CTs. Systematic and random interfraction displacements of all contoured structures were identified in the three cardinal directions, resulting setup margins were calculated. Time trends and the effect of volume changes on displacements were analyzed.
Three-dimensional displacement vectors and systematic/random interfraction displacements were smaller for carina than vertebra both for PT and LN. For PT, mean 3D displacement vectors with carina-based alignment were 7 mm/SD 4 mm versus 9 mm/SD 5 mm with bony anatomy (p<0.0001). For LN, smaller displacements were found with carina- (5 mm/SD 3 mm, p<0.0001) and vertebra-based (6 mm/SD 3 mm, p=0.002) alignment compared to using PT for setup (8 mm/SD 5 mm). Primary tumor and LN displacements relative to bone and carina were independent (p>0.05). Displacements between PT and bone (p=0.04), and between PT and LN (p=0.01) were significantly correlated with PT volume regression. Displacements between LN and carina were correlated with LN volume change (p=0.03).
Carina-based setup results in a more reproducible PT and LN alignment than bony anatomy setup. Considering the independence of PT and LN displacement and the impact of volume regression on displacements over time, repeated CT imaging even with primary tumorbased alignment is recommended in locally advanced disease.
PMCID: PMC3895914  PMID: 24239387
Lung cancer; Mediastinal lymph nodes; Interfraction displacement; Conventional radiotherapy; Image guidance
2.  Novel Role of MDA-9/Syntenin in Regulating Urothelial Cell Proliferation by Modulating EGFR Signaling 
Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression.
Experimental Design
Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knock down was examined in multiple cells lines and key findings were validated in primary tumors.
Significantly higher (p= 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28/44) of primary tumors and an association was evident with stage (p=0.01), grade (p=0.03) and invasion status (p=0.02). MDA-9/Syntenin overexpression in non-tumorigenic HUC-1 cells increased proliferation (p=0.0012), invasion (p=0.0001) and EGFR, AKT, PI3K and c-Src expression. Alteration of Beta-catenin, E-Cadherin, Vimentin, Claudin-1, ZO-1 and TCF4 expression were also observed. MDA-9/Syntenin knock down in 3 UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and co-localization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin: AKT expressions with stage (p=0.04, EGFR), (p=0.01, AKT). A correlation between MDA-9/Syntenin: β-catenin co-expression with stage (p=0.03) and invasion (p=0.04) was also evident.
Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring and therapeutic strategies for managing UCC.
PMCID: PMC3872137  PMID: 23873690
Urothelial cancer; MDA-9/Syntenin; invasion; EGFR signaling
3.  ATM kinase inhibition preferentially sensitizes p53 mutant glioma to ionizing radiation 
Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12–15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of GBM patients is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. ATM, ataxia telangiectasia (A-T) mutated, is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, growth, and potently radiosensitized human glioma cells in vitro.
Experimental Design
Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intra-cranially, and KU-60019 was administered intra-tumorally by convection-enhanced delivery or osmotic pump.
Our results demonstrate that the combined effect of KU-60019 and radiation significantly increased survival of mice 2–3 fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma.
Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers.
PMCID: PMC3687028  PMID: 23620409
4.  Staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion via angiotensin II type 1 receptor (AT1R) and TGFβ signaling 
FEBS Open Bio  2014;4:353-361.
•SND1 augments AT1R receptor level by posttranscriptional regulation.•SND1 activates TGFβ signaling which promotes the epithelial–mesenchymal transition.•Migration and invasion by human hepatocellular carcinoma (HCC) cells are augmented by SND1.•A correlation is observed between SND1 and AT1R expression in HCC patients.
Staphylococcal nuclease domain containing-1 (SND1) is overexpressed in human hepatocellular carcinoma (HCC) patients and promotes tumorigenesis by human HCC cells. We now document that SND1 increases angiotensin II type 1 receptor (AT1R) levels by increasing AT1R mRNA stability. This results in activation of ERK, Smad2 and subsequently the TGFβ signaling pathway, promoting epithelial–mesenchymal transition (EMT) and migration and invasion by human HCC cells. A positive correlation was observed between SND1 and AT1R expression levels in human HCC patients. Small molecule inhibitors of SND1, alone or in combination with AT1R blockers, might be an effective therapeutic strategy for late-stage aggressive HCC.
PMCID: PMC4050181  PMID: 24918049
ACE, angiotensin-I converting enzyme; ACE-I, ACE inhibitors; AT1R, angiotensin II type 1 receptor; EMT, epithelial–mesenchymal transition; FDR, false discovery rate; HCC, human hepatocellular carcinoma; LP, losartan potassium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NASH, non-alcoholic steatohepatitis; PAI-1, plasminogen activator inhibitor-1; RISC, RNA-induced silencing complex; SND1, Staphylococcal nuclease domain containing-1; SND1; AT1R; TGFβ; PAI-1; Invasion
5.  Astrocyte elevated gene-1 (AEG-1) promotes hepatocarcinogenesis: novel insights from a mouse model 
Hepatology (Baltimore, Md.)  2012;56(5):1782-1791.
Astrocyte elevated gene-1 (AEG-1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG-1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte-specific expression of AEG-1 (Alb/AEG1) was developed. Treating Alb/AEG-1, but not Wild type (WT) mice, with N-nitrosodiethylamine (DEN), resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis and fatty acid synthesis. Hepatocytes isolated from Alb/AEG-1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of pro-survival signaling pathways. Alb/AEG-1 hepatocytes also exhibited marked resistance towards senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media (CM) from Alb/AEG-1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG-1 facilitated association of Factor XII (FXII) mRNA with polysomes resulting in increased translation. siRNA-mediated knockdown of FXII resulted in profound inhibition of AEG-1-induced angiogenesis.
We uncover novel aspects of AEG-1 functions, including induction of steatosis, inhibition of senescence and activation of coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG-1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC.
PMCID: PMC3449036  PMID: 22689379
Astrocyte elevated gene-1 (AEG-1); transgenic; hepatocellular carcinoma (HCC); senescence; angiogenesis
6.  The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing 
Nucleic Acids Research  2013;42(2):926-940.
Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53−/−:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53−/− mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.
PMCID: PMC3902934  PMID: 24163099
7.  SH3GL2 is frequently deleted in non-small cell lung cancer and downregulates tumor growth by modulating EGFR signaling 
The purpose of this study was to identify key genetic pathways involved in non-small cell lung cancer (NSCLC) and understand their role in tumor progression. We performed a genome wide scanning using paired tumors and corresponding 16 mucosal biopsies from four follow-up lung cancer patients on Affymetrix 250K-NSpI array platform. We found that a single gene SH3GL2 located on human chromosome 9p22 was most frequently deleted in all the tumors and corresponding mucosal biopsies. We further validated the alteration pattern of SH3GL2 in a substantial number of primary NSCLC tumors at DNA and protein level. We also overexpressed wild-type SH3GL2 in three NSCLC cell lines to understand its role in NSCLC progression. Validation in 116 primary NSCLC tumors confirmed frequent loss of heterozygosity of SH3GL2 in overall 51 % (49/97) of the informative cases. We found significantly low (p=0.0015) SH3GL2 protein expression in 71 % (43/60) primary tumors. Forced over-expression of wild-type (wt) SH3GL2 in three NSCLC cell lines resulted in a marked reduction of active epidermal growth factor receptor (EGFR) expression and an increase in EGFR internalization and degradation. Significantly decreased in vitro (p=0.0015–0.030) and in vivo (p=0.016) cellular growth, invasion (p=0.029–0.049), and colony formation (p=0.023–0.039) were also evident in the wt-SH3GL2-transfected cells accompanied by markedly low expression of activated AKT(Ser473), STAT3 (Tyr705), and PI3K. Downregulation of SH3GL2 interactor USP9X and activated β-catenin was also evident in the SH3GL2-transfected cells. Our results indicate that SH3GL2 is frequently deleted in NSCLC and regulates cellular growth and invasion by modulating EGFR function.
PMCID: PMC3691869  PMID: 22968441
Single nucleotide polymorphism array; Lung cancer; SH3GL2; Deletion
8.  Mitochondrial DNA Mutations in Respiratory Complex-I in Never-Smoker Lung Cancer Patients Contribute to Lung Cancer Progression and associated with EGFR gene mutation 
Journal of cellular physiology  2012;227(6):2451-2460.
Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never-smoker lung cancer patients including patients with EGFR and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never-smoker and 30 current-smoker lung cancer patients, and determined the mtDNA content. All the patients’ samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex-I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P=0.006) mtDNA mutation in the never-smokers compared to the current-smoker lung cancer patients. MtDNA mutation was significantly higher (P=0.026) in the never-smoker Asian compared to the current-smoker Caucasian patients’ population. MtDNA mutation was significantly (P=0.007) associated with EGFR gene mutation in the never-smoker patients. We also observed a significant increase (P=0.037) in mtDNA content among the never-smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex-I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never-smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never-smoker lung cancer patients.
PMCID: PMC3256258  PMID: 21830212
Lung cancer; never-smokers; MtDNA mutation; Respiratory Complex-I; EGFR mutation
9.  Tumor, Lymph node and Lymph Node-to-Tumor Displacements over a Radiotherapy Series: Analysis of Inter- and Intrafraction Variations using Active Breathing Control (ABC) in Lung Cancer 
To estimate errors in soft tissue-based image guidance due to relative changes between primary tumor (PT) and affected lymph node (LN) position and volume, and to compare the results to bony anatomy-based displacements of PTs and LNs during radiotherapy of lung cancer.
Materials and Methods
Weekly repeated breath hold CT scans were acquired in 17 lung cancer patients undergoing radiotherapy. PTs and affected LNs were manually contoured on all scans after rigid registration. Inter- and intrafraction displacements in the centers of mass of PTs and LNs relative to bone, and LNs relative to PTs (LN-PT) were calculated.
The mean volume after 5 weeks was 65% for PTs and 63% for LNs. Systematic/random interfraction displacements were 2.6 – 4.6 mm/2.7 – 2.9 mm for PTs, 2.4 – 3.8 mm/1.4 – 2.7 mm for LNs, and 2.3 – 3.9 mm/1.9 – 2.8 mm for LN-PT. Systematic/random intrafraction displacements were < 1 mm except in superior-inferior direction. Interfraction LN-PT displacements > 3 mm were observed in 67% of fractions and require a safety margin of 12 mm in lateral, 11 mm in anteroposterior and 9 mm in superior-inferior direction. LN-PT displacements displayed significant time trends (p<0.0001) and depended on the presence of pathoanatomical conditions of the ipsilateral lung, such as atelectasis.
Interfraction LN-PT displacements were mostly systematic and comparable to bony anatomy-based displacements of PTs or LNs alone. Time trends, large volume changes and the influence of pathoanatomical conditions underline the importance of soft tissue-based image guidance and the potential of plan adaptation.
PMCID: PMC3294098  PMID: 22197237
lung cancer; active breathing control; interfraction motion; intrafraction motion; image-guided radiotherapy
10.  An inferential framework for biological network hypothesis tests 
BMC Bioinformatics  2013;14:94.
Networks are ubiquitous in modern cell biology and physiology. A large literature exists for inferring/proposing biological pathways/networks using statistical or machine learning algorithms. Despite these advances a formal testing procedure for analyzing network-level observations is in need of further development. Comparing the behaviour of a pharmacologically altered pathway to its canonical form is an example of a salient one-sample comparison. Locating which pathways differentiate disease from no-disease phenotype may be recast as a two-sample network inference problem.
We outline an inferential method for performing one- and two-sample hypothesis tests where the sampling unit is a network and the hypotheses are stated via network model(s). We propose a dissimilarity measure that incorporates nearby neighbour information to contrast one or more networks in a statistical test. We demonstrate and explore the utility of our approach with both simulated and microarray data; random graphs and weighted (partial) correlation networks are used to form network models. Using both a well-known diabetes dataset and an ovarian cancer dataset, the methods outlined here could better elucidate co-regulation changes for one or more pathways between two clinically relevant phenotypes.
Formal hypothesis tests for gene- or protein-based networks are a logical progression from existing gene-based and gene-set tests for differential expression. Commensurate with the growing appreciation and development of systems biology, the dissimilarity-based testing methods presented here may allow us to improve our understanding of pathways and other complex regulatory systems. The benefit of our method was illustrated under select scenarios.
PMCID: PMC3621801  PMID: 23496778
11.  Estimating statistical uncertainty of Monte Carlo efficiency-gain in the context of a correlated sampling Monte Carlo code for brachytherapy treatment planning with non-normal dose distribution 
Correlated sampling Monte Carlo methods can shorten computing times in brachytherapy treatment planning. Monte Carlo efficiency is typically estimated via efficiency gain, defined as the reduction in computing time by correlated sampling relative to conventional Monte Carlo methods when equal statistical uncertainties have been achieved. The determination of the efficiency gain uncertainty arising from random effects, however, is not a straightforward task specially when the error distribution is non-normal. The purpose of this study is to evaluate the applicability of the F distribution and standardized uncertainty propagation methods (widely used in metrology to estimate uncertainty of physical measurements) for predicting confidence intervals about efficiency gain estimates derived from single Monte Carlo runs using fixed-collision correlated sampling in a simplified brachytherapy geometry. A bootstrap based algorithm was used to simulate the probability distribution of the efficiency gain estimates and the shortest 95% confidence interval was estimated from this distribution. It was found that the corresponding relative uncertainty was as large as 37% for this particular problem. The uncertainty propagation framework predicted confidence intervals reasonably well; however its main disadvantage was that uncertainties of input quantities had to be calculated in a separate run via a Monte Carlo method. The F distribution noticeably underestimated the confidence interval. These discrepancies were influenced by several photons with large statistical weights which made extremely large contributions to the scored absorbed dose difference. The mechanism of acquiring high statistical weights in the fixed-collision correlated sampling method was explained and a mitigation strategy was proposed.
PMCID: PMC3242326  PMID: 21992844
Monte Carlo; correlated sampling; efficiency; uncertainty; bootstrap
12.  Clinical evaluation of soft tissue organ boundary visualization on cone-beam computed tomographic (CBCT) imaging 
Cone-beam computed tomographic images (CBCTs) are increasingly used for set up correction, soft tissue targeting and image-guided adaptive radiotherapy (IGART). However, CBCT image quality is limited by low contrast and imaging artifacts. This analysis investigates the detectability of soft tissue boundaries in CBCT by performing a multiple-observer segmentation study.
Material and Methods
In 4 prostate cancer patients prostate, bladder and rectum were repeatedly delineated by 5 observers on CBCTs and fan beam CTs (FBCTs). A volumetric analysis of contouring variations was performed by calculating coefficients of variation (COV: standard deviation/average volume). The topographical distribution of contouring variations was analyzed using an average surface mesh-based method.
Observer- and patient-averaged COVs for FBCT/CBCT were 0.09/0.19 for prostate, 0.05/0.08 for bladder and 0.09/0.08 for rectum. Contouring variations on FBCT were significantly smaller than on CBCT for prostate (p<0.03) and bladder (p<0.04), but not for rectum (p<0.37) (intermodality differences). Intraobserver variations from repeated contouring of the same image set were not significant for either FBCT or CBCT (p<0.05). Average standard deviations of individual observers’ contour differences from average surface meshes on FBCT versus CBCT were 1.5 versus 2.1 mm for prostate, 0.7 versus 1.4 mm for bladder, and 1.3 versus 1.5 mm for rectum. The topographical distribution of contouring variations was similar for FBCT and CBCT.
Contouring variations were larger on CBCT than FBCT, except for rectum. Given the well-documented uncertainty in soft tissue contouring in the pelvis, improvement of CBCT image quality and establishment of well-defined soft tissue identification rules are desirable for image-guided radiotherapy.
PMCID: PMC2946461  PMID: 20542644
Cone-beam CT; Image guided adaptive radiotherapy; Prostate cancer; Image quality; Segmentation
13.  Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression 
Hepatocellular carcinoma (HCC) is a highly aggressive vascular cancer characterized by diverse etiology, activation of multiple signal transduction pathways, and various gene mutations. Here, we have determined a specific role for astrocyte elevated gene-1 (AEG1) in HCC pathogenesis. Expression of AEG1 was extremely low in human hepatocytes, but its levels were significantly increased in human HCC. Stable overexpression of AEG1 converted nontumorigenic human HCC cells into highly aggressive vascular tumors, and inhibition of AEG1 abrogated tumorigenesis by aggressive HCC cells in a xenograft model of nude mice. In human HCC, AEG1 overexpression was associated with elevated copy numbers. Microarray analysis revealed that AEG1 modulated the expression of genes associated with invasion, metastasis, chemoresistance, angiogenesis, and senescence. AEG1 also was found to activate Wnt/β-catenin signaling via ERK42/44 activation and upregulated lymphoid-enhancing factor 1/T cell factor 1 (LEF1/TCF1), the ultimate executor of the Wnt pathway, important for HCC progression. Inhibition studies further demonstrated that activation of Wnt signaling played a key role in mediating AEG1 function. AEG1 also activated the NF-κB pathway, which may play a role in the chronic inflammatory changes preceding HCC development. These data indicate that AEG1 plays a central role in regulating diverse aspects of HCC pathogenesis. Targeted inhibition of AEG1 might lead to the shutdown of key elemental characteristics of HCC and could lead to an effective therapeutic strategy for HCC.
PMCID: PMC2648696  PMID: 19221438
14.  Identification of genes for complex disease using longitudinal phenotypes 
BMC Genetics  2003;4(Suppl 1):S58.
Using the simulated data set from Genetic Analysis Workshop 13, we explored the advantages of using longitudinal data in genetic analyses. The weighted average of the longitudinal data for each of seven quantitative phenotypes were computed and analyzed. Genome screen results were then compared for these longitudinal phenotypes and the results obtained using two cross-sectional designs: data collected near a single age (45 years) and data collected at a single time point. Significant linkage was obtained for nine regions (LOD scores ranging from 5.5 to 34.6) for six of the phenotypes. Using cross-sectional data, LOD scores were slightly lower for the same chromosomal regions, with two regions becoming nonsignificant and one additional region being identified. The magnitude of the LOD score was highly correlated with the heritability of each phenotype as well as the proportion of phenotypic variance due to that locus. There were no false-positive linkage results using the longitudinal data and three false-positive findings using the cross-sectional data. The three false positive results appear to be due to the kurtosis in the trait distribution, even after removing extreme outliers. Our analyses demonstrated that the use of simple longitudinal phenotypes was a powerful means to detect genes of major to moderate effect on trait variability. In only one instance was the power and heritability of the trait increased by using data from one examination. Power to detect linkage can be improved by identifying the most heritable phenotype, ensuring normality of the trait distribution and maximizing the information utilized through novel longitudinal designs for genetic analysis.
PMCID: PMC1866495  PMID: 14975126

Results 1-14 (14)