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1.  Polymorphisms Near TBX5 and GDF7 Are Associated With Increased Risk for Barrett’s Esophagus 
Palles, Claire | Chegwidden, Laura | Li, Xinzhong | Findlay, John M. | Farnham, Garry | Castro Giner, Francesc | Peppelenbosch, Maikel P. | Kovac, Michal | Adams, Claire L. | Prenen, Hans | Briggs, Sarah | Harrison, Rebecca | Sanders, Scott | MacDonald, David | Haigh, Chris | Tucker, Art | Love, Sharon | Nanji, Manoj | deCaestecker, John | Ferry, David | Rathbone, Barrie | Hapeshi, Julie | Barr, Hugh | Moayyedi, Paul | Watson, Peter | Zietek, Barbara | Maroo, Neera | Gay, Laura | Underwood, Tim | Boulter, Lisa | McMurtry, Hugh | Monk, David | Patel, Praful | Ragunath, Krish | Al Dulaimi, David | Murray, Iain | Koss, Konrad | Veitch, Andrew | Trudgill, Nigel | Nwokolo, Chuka | Rembacken, Bjorn | Atherfold, Paul | Green, Elaine | Ang, Yeng | Kuipers, Ernst J. | Chow, Wu | Paterson, Stuart | Kadri, Sudarshan | Beales, Ian | Grimley, Charles | Mullins, Paul | Beckett, Conrad | Farrant, Mark | Dixon, Andrew | Kelly, Sean | Johnson, Matthew | Wajed, Shahjehan | Dhar, Anjan | Sawyer, Elinor | Roylance, Rebecca | Onstad, Lynn | Gammon, Marilie D. | Corley, Douglas A. | Shaheen, Nicholas J. | Bird, Nigel C. | Hardie, Laura J. | Reid, Brian J. | Ye, Weimin | Liu, Geoffrey | Romero, Yvonne | Bernstein, Leslie | Wu, Anna H. | Casson, Alan G. | Fitzgerald, Rebecca | Whiteman, David C. | Risch, Harvey A. | Levine, David M. | Vaughan, Tom L. | Verhaar, Auke P. | van den Brande, Jan | Toxopeus, Eelke L. | Spaander, Manon C. | Wijnhoven, Bas P.L. | van der Laan, Luc J.W. | Krishnadath, Kausilia | Wijmenga, Cisca | Trynka, Gosia | McManus, Ross | Reynolds, John V. | O’Sullivan, Jacintha | MacMathuna, Padraic | McGarrigle, Sarah A. | Kelleher, Dermot | Vermeire, Severine | Cleynen, Isabelle | Bisschops, Raf | Tomlinson, Ian | Jankowski, Janusz
Gastroenterology  2015;148(2):367-378.
Background & Aims
Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations.
We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls.
We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09–1.18; P = 1.8 × 10−11) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86–0.93; P = 7.5 × 10−9). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87–0.93; P = 3.72 × 10−9).
We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.
PMCID: PMC4315134  PMID: 25447851
EAC; Intestinal Metaplasia; Susceptibility; Cancer; ASE, allele-specific expression; BE, Barrett’s esophagus; BEACON, Barrett's and Esophageal Adenocarcinoma Consortium; CI, confidence interval; EAC, esophageal adenocarcinoma; eQTL, expression quantitative trait locus; GWAS, genome-wide association study; LD, linkage disequilibrium; OR, odds ratio; PC, principal component; SNP, single nucleotide polymorphism; TCGA, The Cancer Genome Atlas
2.  Rare variants of large effect in BRCA2 and CHEK2 affect risk of lung cancer 
Wang, Yufei | McKay, James D. | Rafnar, Thorunn | Wang, Zhaoming | Timofeeva, Maria | Broderick, Peter | Zong, Xuchen | Laplana, Marina | Wei, Yongyue | Han, Younghun | Lloyd, Amy | Delahaye-Sourdeix, Manon | Chubb, Daniel | Gaborieau, Valerie | Wheeler, William | Chatterjee, Nilanjan | Thorleifsson, Gudmar | Sulem, Patrick | Liu, Geoffrey | Kaaks, Rudolf | Henrion, Marc | Kinnersley, Ben | Vallée, Maxime | LeCalvez-Kelm, Florence | Stevens, Victoria L. | Gapstur, Susan M. | Chen, Wei V. | Zaridze, David | Szeszenia-Dabrowska, Neonilia | Lissowska, Jolanta | Rudnai, Peter | Fabianova, Eleonora | Mates, Dana | Bencko, Vladimir | Foretova, Lenka | Janout, Vladimir | Krokan, Hans E. | Gabrielsen, Maiken Elvestad | Skorpen, Frank | Vatten, Lars | Njølstad, Inger | Chen, Chu | Goodman, Gary | Benhamou, Simone | Vooder, Tonu | Valk, Kristjan | Nelis, Mari | Metspalu, Andres | Lener, Marcin | Lubiński, Jan | Johansson, Mattias | Vineis, Paolo | Agudo, Antonio | Clavel-Chapelon, Francoise | Bueno-de-Mesquita, H.Bas | Trichopoulos, Dimitrios | Khaw, Kay-Tee | Johansson, Mikael | Weiderpass, Elisabete | Tjønneland, Anne | Riboli, Elio | Lathrop, Mark | Scelo, Ghislaine | Albanes, Demetrius | Caporaso, Neil E. | Ye, Yuanqing | Gu, Jian | Wu, Xifeng | Spitz, Margaret R. | Dienemann, Hendrik | Rosenberger, Albert | Su, Li | Matakidou, Athena | Eisen, Timothy | Stefansson, Kari | Risch, Angela | Chanock, Stephen J. | Christiani, David C. | Hung, Rayjean J. | Brennan, Paul | Landi, Maria Teresa | Houlston, Richard S. | Amos, Christopher I.
Nature genetics  2014;46(7):736-741.
We conducted imputation to the 1000 Genomes Project of four genome-wide association studies of lung cancer in populations of European ancestry (11,348 cases and 15,861 controls) and genotyped an additional 10,246 cases and 38,295 controls for follow-up. We identified large-effect genome-wide associations for squamous lung cancer with the rare variants of BRCA2-K3326X (rs11571833; odds ratio [OR]=2.47, P=4.74×10−20) and of CHEK2-I157T (rs17879961; OR=0.38 P=1.27×10−13). We also showed an association between common variation at 3q28 (TP63; rs13314271; OR=1.13, P=7.22×10−10) and lung adenocarcinoma previously only reported in Asians. These findings provide further evidence for inherited genetic susceptibility to lung cancer and its biological basis. Additionally, our analysis demonstrates that imputation can identify rare disease-causing variants having substantive effects on cancer risk from pre-existing GWAS data.
PMCID: PMC4074058  PMID: 24880342
3.  MiR-145 Expression Accelerates Esophageal Adenocarcinoma Progression by Enhancing Cell Invasion and Anoikis Resistance 
PLoS ONE  2014;9(12):e115589.
Carcinoma of the esophagus has a high case fatality ratio and is now the 6th most common cause of cancer deaths in the world. We previously conducted a study to profile the expression of miRNAs in esophageal adenocarcinoma (EAC) pre and post induction therapy. Of the miRNAs differentially expressed post induction chemoradiation, miR-145, a known tumor suppressor miRNA, was upregulated 8-fold following induction therapy, however, its expression was associated with shorter disease-free survival. This unexpected result was explored in this current study.
In order to study the role of miR-145 in EAC, miRNA-145 was overexpressed in 3 EAC cell lines (OE33, FLO-1, SK-GT-4) and one ESCC cell line (KYSE-410). After validation of the expression of miR-145, hallmarks of cancer such as cell proliferation, resistance to chemotherapy drugs or anoikis, and cell invasion were analyzed.
There were no differences in cell proliferation and 5 FU resistance between miR145 cell lines and the control cell lines. miR-145 expression also had no effect on cisplatin resistance in two of three cell lines (OE33 and FLO-1), but miR-145 appeared to protect SK-GT-4 cells against cisplatin treatment. However, there was a significant difference in cell invasion, cell adhesion and resistance to anoikis. All three EAC miR-145 cell lines invaded more than their respective controls. Similarly, OE33 and SK-GT-4 miR-145 cell lines were able to survive longer in a suspension state.
While expression of miR-145 in ESCC stopped proliferation and invasion, expression of miR-145 in EAC cells enhanced invasion and anoikis resistance. Although more work is required to understand how miR-145 conveys these effects, expression of miR-145 appears to promote EAC progression by enhancing invasion and protection against anoikis, which could in turn facilitate distant metastasis.
PMCID: PMC4281214  PMID: 25551563
4.  Germline Genetic Contributions to Risk for Esophageal Adenocarcinoma, Barrett’s Esophagus, and Gastroesophageal Reflux 
Esophageal adenocarcinoma (EA) is an increasingly common cancer with poor survival. Barrett’s esophagus (BE) is the main precursor to EA, and every year 0.12% to 0.5% of BE patients progress to EA. BE typically arises on a background of chronic gastroesophageal reflux (GERD), one of the risk factors for EA.
We used genome-wide association data to investigate the genetic architecture underlying GERD, BE, and EA. We applied a method to estimate the variance explained (array heritability, h2 g) and the genetic correlation (rg) between GERD, BE, and EA by considering all single nucleotide polymorphisms (SNPs) simultaneously. We also estimated the polygenic overlap between GERD, BE, and EA using a prediction approach. All tests were two-sided, except in the case of variance-explained estimation where one-sided tests were used.
We estimated a statistically significant genetic variance explained for BE (h2 g = 35%; standard error [SE] = 6%; one-sided P = 1 × 10−9) and for EA (h2 g = 25 %; SE = 5%; one-sided P = 2 × 10−7). The genetic correlation between BE and EA was found to be high (rg = 1.0; SE = 0.37). We also estimated a statistically significant polygenic overlap between BE and EA (one-sided P = 1 × 10−6), which suggests, together with the high genetic correlation, that shared genes underlie the development of BE and EA. Conversely, no statistically significant results were obtained for GERD.
We have demonstrated that risk to BE and EA is influenced by many germline genetic variants of small effect and that shared polygenic effects contribute to risk of these two diseases.
PMCID: PMC3833931  PMID: 24168968
5.  Ceritinib in ALK-Rearranged Non–Small-Cell Lung Cancer 
The New England journal of medicine  2014;370(13):1189-1197.
Non–small-cell lung cancer (NSCLC) harboring the anaplastic lymphoma kinase gene (ALK) rearrangement is sensitive to the ALK inhibitor crizotinib, but resistance invariably develops. Ceritinib (LDK378) is a new ALK inhibitor that has shown greater antitumor potency than crizotinib in preclinical studies.
In this phase 1 study, we administered oral ceritinib in doses of 50 to 750 mg once daily to patients with advanced cancers harboring genetic alterations in ALK. In an expansion phase of the study, patients received the maximum tolerated dose. Patients were assessed to determine the safety, pharmacokinetic properties, and antitumor activity of ceritinib. Tumor biopsies were performed before ceritinib treatment to identify resistance mutations in ALK in a group of patients with NSCLC who had had disease progression during treatment with crizotinib.
A total of 59 patients were enrolled in the dose-escalation phase. The maximum tolerated dose of ceritinib was 750 mg once daily; dose-limiting toxic events included diarrhea, vomiting, dehydration, elevated aminotransferase levels, and hypophosphatemia. This phase was followed by an expansion phase, in which an additional 71 patients were treated, for a total of 130 patients overall. Among 114 patients with NSCLC who received at least 400 mg of ceritinib per day, the overall response rate was 58% (95% confidence interval [CI], 48 to 67). Among 80 patients who had received crizotinib previously, the response rate was 56% (95% CI, 45 to 67). Responses were observed in patients with various resistance mutations in ALK and in patients without detectable mutations. Among patients with NSCLC who received at least 400 mg of ceritinib per day, the median progression-free survival was 7.0 months (95% CI, 5.6 to 9.5).
Ceritinib was highly active in patients with advanced, ALK-rearranged NSCLC, including those who had had disease progression during crizotinib treatment, regardless of the presence of resistance mutations in ALK. (Funded by Novartis Pharmaceuticals and others; number, NCT01283516.)
PMCID: PMC4079055  PMID: 24670165
6.  Haplotype approach for association analysis on hypertension 
BMC Proceedings  2014;8(Suppl 1):S57.
We applied a gene-based haplotype approach for the genome-wide association analysis on hypertension using Genetic Analysis Workshop 18 data for unrelated individuals. Association of single-nucleotide polymorphisms and clinical outcome were first assessed and haplotypes were then constructed based on the gene information and the linkage disequilibrium plot. Extensive haplotype analysis was also conducted for the whole chromosome 3. We found 1 block from the ULK4 gene and 2 blocks from the LOC64690 gene that were significantly associated with hypertension.
PMCID: PMC4143719  PMID: 25519392
7.  Genetic association analysis for common variants in the Genetic Analysis Workshop 18 data: a Dirichlet regression approach 
BMC Proceedings  2014;8(Suppl 1):S70.
We propose a genetic association analysis using Dirichlet regression to analyze the Genetic Analysis Workshop 18 data. Clinical variables, arranged in a longitudinal data structure, are employed to fit a multistate transition model in which the transition probabilities are served as a response in the proposed analysis. Furthermore, a gene-based association analysis via penalized regression is implemented using the markers at a single-nucleotide polymorphism level that we previously identified via nonpenalized Dirichlet regression.
PMCID: PMC4143809  PMID: 25519401
8.  Genetic association analysis using weighted false discovery rate approach on Genetic Analysis Workshop 18 data 
BMC Proceedings  2014;8(Suppl 1):S76.
In a genome-wide association study, association between disease trait and hundreds of thousands of genetic markers are tested. Several methods have been proposed to control the false discovery rate in such high-throughput data to adjust for multiple hypotheses testing. For Genetic Analysis Workshop 18, we applied the method of false discovery rate control with p value weighting on family-based association tests on quantitative trait to detect association between single-nucleotide polymorphisms (SNPs) and mean arterial pressure. This method can improve statistical power by incorporating independent but relevant information about the research objective. Using the real genetic and phenotype data of chromosome 3 from Genetic Analysis Workshop 18, 1 SNP from gene CACNA2D3 was found to have significant association with mean arterial pressure.
PMCID: PMC4143671  PMID: 25519404
10.  Two BRM promoter insertion polymorphisms increase the risk of early-stage upper aerodigestive tract cancers 
Cancer Medicine  2014;3(2):426-433.
Brahma (BRM) has a key function in chromatin remodeling. Two germline BRM promoter insertion–deletion polymorphisms, BRM-741 and BRM-1321, have been previously associated with an increased risk of lung cancer in smokers and head and neck cancer. To further evaluate their role in cancer susceptibility particularly in early disease, we conducted a preplanned case–control study to investigate the association between the BRM promoter variants and stage I/II upper aerodigestive tract (UADT) cancers (i.e., lung, esophageal, head and neck), a group of early-stage malignancies in which molecular and genetic etiologic factors are poorly understood. The effects of various clinical factors on this association were also studied. We analyzed 562 cases of early-stage UADT cancers and 993 matched healthy controls. The double homozygous BRM promoter variants were associated with a significantly increased risk of early stage UADT cancers (adjusted odds ratio [aOR], 2.46; 95% confidence interval [CI], 1.7–3.8). This association was observed in lung (aOR, 2.61; 95% CI, 1.5–4.9) and head and neck (aOR, 2.75; 95% CI, 1.4–5.6) cancers, but not significantly in esophageal cancer (aOR, 1.66; 95% CI, 0.7–5.8). There was a nonsignificant trend for increased risk in the heterozygotes or single homozygotes. The relationship between the BRM polymorphisms and early-stage UADT cancers was independent of age, sex, smoking status, histology, and clinical stage. These findings suggest that the BRM promoter double insertion homozygotes may be associated with an increased risk of early-stage UADT cancers independent of smoking status and histology, which must be further validated in other populations.
PMCID: PMC3987092  PMID: 24519853
BRM; cancer risk; case–control study; esophageal cancer; genetic polymorphisms; head and neck cancer; lung cancer; upper aerodigestive tract cancers
11.  Probability of Cancer in Pulmonary Nodules Detected on First Screening CT 
The New England journal of medicine  2013;369(10):910-919.
Major issues in the implementation of screening for lung cancer by means of low-dose computed tomography (CT) are the definition of a positive result and the management of lung nodules detected on the scans. We conducted a population-based prospective study to determine factors predicting the probability that lung nodules detected on the first screening low-dose CT scans are malignant or will be found to be malignant on follow-up.
We analyzed data from two cohorts of participants undergoing low-dose CT screening. The development data set included participants in the Pan-Canadian Early Detection of Lung Cancer Study (PanCan). The validation data set included participants involved in chemoprevention trials at the British Columbia Cancer Agency (BCCA), sponsored by the U.S. National Cancer Institute. The final outcomes of all nodules of any size that were detected on baseline low-dose CT scans were tracked. Parsimonious and fuller multivariable logistic-regression models were prepared to estimate the probability of lung cancer.
In the PanCan data set, 1871 persons had 7008 nodules, of which 102 were malignant, and in the BCCA data set, 1090 persons had 5021 nodules, of which 42 were malignant. Among persons with nodules, the rates of cancer in the two data sets were 5.5% and 3.7%, respectively. Predictors of cancer in the model included older age, female sex, family history of lung cancer, emphysema, larger nodule size, location of the nodule in the upper lobe, part-solid nodule type, lower nodule count, and spiculation. Our final parsimonious and full models showed excellent discrimination and calibration, with areas under the receiver-operating-characteristic curve of more than 0.90, even for nodules that were 10 mm or smaller in the validation set.
Predictive tools based on patient and nodule characteristics can be used to accurately estimate the probability that lung nodules detected on baseline screening low-dose CT scans are malignant. (Funded by the Terry Fox Research Institute and others; number, NCT00751660.)
PMCID: PMC3951177  PMID: 24004118
12.  Single nucleotide polymorphisms in the matrix metalloproteinase gene family and the frequency and duration of gastroesophageal reflux disease influence the risk of esophageal adenocarcinoma 
The matrix metalloproteinase (MMP) family of proteins mediates various cellular pathways, including apoptosis and angiogenesis. Polymorphisms of MMP genes are associated with increased esophageal adenocarcinoma (EAC) risk. Gastroesophageal reflux disease (GERD) is an established EAC risk factor. We examined whether MMP polymorphism-EAC risk is modified by GERD. In total, 309 EAC patients and 279 frequency-matched healthy controls underwent MMP1 1G/2G, MMP3 6A/5A, MMP12 −82A/G and MMP12 1082A/G genotyping. Questionnaires collected GERD history. EAC risk was analyzed using logistic regression, adjusted for key covariates and stratified by GERD. Joint effects models explored GERD severity and duration, whereas additional models explored genotype–GERD interactions in EAC risk. We determined that each MMP1 and MMP3 minor (variant) allele was independently associated with increased EAC risk (adjusted odds ratio (AOR) 3.2, 95% confidence interval (CI) 2.0–5.1, p < 0.001 and AOR 1.8, 95% CI 1.1–2.7, p = 0.01, respectively) only among those with GERD but not in GERD-free individuals (all p = nonsignificant). There were significant interactions between the MMP1 variants and the presence of GERD (p = 0.002) and between MMP3 variants and GERD (p = 0.04). There was an equally strong interaction between cumulative GERD severity and MMP1 (p = 0.002). The AOR of each variant allele was 14.9 (95% CI 1.6–136) for individuals with severe GERD, 1.7 (95% CI 1.0–2.7) for mild-moderate GERD and 0.98 (95% CI 0.7–1.4) for those without GERD. This was further reflected in separate analyses of frequency and duration of GERD. In conclusion, MMP1 1G/2G (and possibly MMP3 6A/5A) polymorphisms alter EAC risk differentially for GERD and GERD-free individuals.
PMCID: PMC3908453  PMID: 22422400
matrix metalloproteinase; gene polymorphism; gastroesophageal reflux disease; esophageal cancer
13.  Resistance to Bleomycin in Cancer Cell Lines Is Characterized by Prolonged Doubling Time, Reduced DNA Damage and Evasion of G2/M Arrest and Apoptosis 
PLoS ONE  2013;8(12):e82363.
To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines.
Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05).
Bleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.
PMCID: PMC3857806  PMID: 24349265
14.  Previous Lung Diseases and Lung Cancer Risk: A Pooled Analysis From the International Lung Cancer Consortium 
American Journal of Epidemiology  2012;176(7):573-585.
To clarify the role of previous lung diseases (chronic bronchitis, emphysema, pneumonia, and tuberculosis) in the development of lung cancer, the authors conducted a pooled analysis of studies in the International Lung Cancer Consortium. Seventeen studies including 24,607 cases and 81,829 controls (noncases), mainly conducted in Europe and North America, were included (1984–2011). Using self-reported data on previous diagnoses of lung diseases, the authors derived study-specific effect estimates by means of logistic regression models or Cox proportional hazards models adjusted for age, sex, and cumulative tobacco smoking. Estimates were pooled using random-effects models. Analyses stratified by smoking status and histology were also conducted. A history of emphysema conferred a 2.44-fold increased risk of lung cancer (95% confidence interval (CI): 1.64, 3.62 (16 studies)). A history of chronic bronchitis conferred a relative risk of 1.47 (95% CI: 1.29, 1.68 (13 studies)). Tuberculosis (relative risk = 1.48, 95% CI: 1.17, 1.87 (16 studies)) and pneumonia (relative risk = 1.57, 95% CI: 1.22, 2.01 (12 studies)) were also associated with lung cancer risk. Among never smokers, elevated risks were observed for emphysema, pneumonia, and tuberculosis. These results suggest that previous lung diseases influence lung cancer risk independently of tobacco use and that these diseases are important for assessing individual risk.
PMCID: PMC3530374  PMID: 22986146
bronchitis; chronic; emphysema; lung diseases; lung neoplasms; meta-analysis; pneumonia; pulmonary disease; chronic obstructive; tuberculosis
15.  Fibulin-3 as a Blood and Effusion Biomarker for Pleural Mesothelioma 
The New England journal of medicine  2012;367(15):1417-1427.
New biomarkers are needed to detect pleural mesothelioma at an earlier stage and to individualize treatment strategies. We investigated whether fibulin-3 in plasma and pleural effusions could meet sensitivity and specificity criteria for a robust biomarker.
We measured fibulin-3 levels in plasma (from 92 patients with mesothelioma, 136 asbestos-exposed persons without cancer, 93 patients with effusions not due to mesothelioma, and 43 healthy controls), effusions (from 74 patients with mesothelioma, 39 with benign effusions, and 54 with malignant effusions not due to mesothelioma), or both. A blinded validation was subsequently performed. Tumor tissue was examined for fibulin-3 by immunohistochemical analysis, and levels of fibulin-3 in plasma and effusions were measured with an enzyme-linked immunosorbent assay.
Plasma fibulin-3 levels did not vary according to age, sex, duration of asbestos exposure, or degree of radiographic changes and were significantly higher in patients with pleural mesothelioma (105±7 ng per milliliter in the Detroit cohort and 113±8 ng per milliliter in the New York cohort) than in asbestos-exposed persons without mesothelioma (14±1 ng per milliliter and 24±1 ng per milliliter, respectively; P<0.001). Effusion fibulin-3 levels were significantly higher in patients with pleural mesothelioma (694±37 ng per milliliter in the Detroit cohort and 636±92 ng per milliliter in the New York cohort) than in patients with effusions not due to mesothelioma (212±25 and 151±23 ng per milliliter, respectively; P<0.001). Fibulin-3 preferentially stained tumor cells in 26 of 26 samples. In an overall comparison of patients with and those without mesothelioma, the receiver-operating-characteristic curve for plasma fibulin-3 levels had a sensitivity of 96.7% and a specificity of 95.5% at a cutoff value of 52.8 ng of fibulin-3 per milliliter. In a comparison of patients with early-stage mesothelioma with asbestos-exposed persons, the sensitivity was 100% and the specificity was 94.1% at a cutoff value of 46.0 ng of fibulin-3 per milliliter. Blinded validation revealed an area under the curve of 0.87 for plasma specimens from 96 asbestos-exposed persons as compared with 48 patients with mesothelioma.
Plasma fibulin-3 levels can distinguish healthy persons with exposure to asbestos from patients with mesothelioma. In conjunction with effusion fibulin-3 levels, plasma fibulin-3 levels can further differentiate mesothelioma effusions from other malignant and benign effusions. (Funded by the Early Detection Research Network, National Institutes of Health, and others.)
PMCID: PMC3761217  PMID: 23050525
16.  Special considerations in prognostic research in cancer involving genetic polymorphisms 
BMC Medicine  2013;11:149.
Analysis of genetic polymorphisms may help identify putative prognostic markers and determine the biological basis of variable prognosis in patients. However, in contrast to other variables commonly used in the prognostic studies, there are special considerations when studying genetic polymorphisms. For example, variable inheritance patterns (recessive, dominant, codominant, and additive genetic models) need to be explored to identify the specific genotypes associated with the outcome. In addition, several characteristics of genetic polymorphisms, such as their minor allele frequency and linkage disequilibrium among multiple polymorphisms, and the population substructure of the cohort investigated need to be accounted for in the analyses. In addition, in cancer research due to the genomic differences between the tumor and non-tumor DNA, differences in the genetic information obtained using these tissues need to be carefully assessed in prognostic studies. In this article, we review these and other considerations specific to genetic polymorphism by focusing on genetic prognostic studies in cancer.
PMCID: PMC3729672  PMID: 23773794
Genetic models; Genetic polymorphisms; Genetic prognostic factors; Genotypes; Prognostic research; Tumor DNA
17.  Association between UGT1A1*28 Polymorphisms and Clinical Outcomes of Irinotecan-Based Chemotherapies in Colorectal Cancer: A Meta-Analysis in Caucasians 
PLoS ONE  2013;8(3):e58489.
Whether UGT1A1*28 genotype is associated with clinical outcomes of irinotecan (IRI)-based chemotherapy in Colorectal cancer (CRC) is an important gap in existing knowledge to inform clinical utility. Published data on the association between UGT1A1*28 gene polymorphisms and clinical outcomes of IRI-based chemotherapy in CRC were inconsistent.
Methodology/Principal Findings
Literature retrieval, trials selection and assessment, data collection, and statistical analysis were performed according to the PRISMA guidelines. Primary outcomes included therapeutic response (TR), progression-free survival (PFS) and overall survival (OS). We calculated odds ratios (OR) and hazard ratios (HR) with 95% confidence intervals (CI). Twelve clinical trials were included. No statistical heterogeneity was detected in analyses of all studies and for each subgroup. Differences in TR, PFS and OS for any genotype comparison, UGT1A1*28/*28 versus (vs) UGT1A1*1/*1 (homozygous model), UGT1A1*1/*28 vs UGT1A1*1/*1 (heterozygous model), and UGT1A1*28/*28 vs all others (recessive model, only for TR) were not statistically significant. IRI dose also did not impact upon TR and PFS differences between UGT1A1 genotype groups. A statistically significant increase in the hazard of death was found in Low IRI subgroup of the homozygous model (HR = 1.48, 95% CI = 1.06–2.07; P = 0.02). The UGT1A1*28 allele was associated with a trend of increase in the hazard of death in two models (homozygous model: HR = 1.22, 95% CI = 0.99–1.51; heterozygous model: HR = 1.13, 95% CI = 0.96–1.32). These latter findings were driven primarily by one single large study (Shulman et al. 2011).
UGT1A1*28 polymorphism cannot be considered as a reliable predictor of TR and PFS in CRC patients treated with IRI-based chemotherapy. The OS relationship with UGT1A1*28 in the patients with lower-dose IRI chemotherapy requires further validation.
PMCID: PMC3597733  PMID: 23516488
18.  Interactions between Environmental Factors and Polymorphisms in Angiogenesis Pathway Genes in Esophageal Adenocarcinoma Risk: A Case-Only Study 
Cancer  2011;118(3):804-811.
Gastroesophageal reflux symptoms (GERD), higher body mass index (BMI), smoking, and genetic variants in angiogenic pathway genes have been individually associated with increased risk of esophageal adenocarcinoma (EA). However, how angiogenic gene polymorphisms and environmental factors jointly affect EA development remains unclear.
Using a case-only design (n = 335), we examined interaction between 141 functional/tagging angiogenic SNPs and environmental factors (GERD, BMI, smoking) in modulating EA risk. Gene-environment interactions were assessed by a two-step approach. First, we applied random forest (RF) to screen for important SNPs that had either main or interaction effects. Second, we used case-only logistic regression (LR) to assess the effects of gene-environment interactions on EA risk, adjusting for covariates and false-discovery rate (FDR).
RF analyses identified three sets of SNPs (17 SNPs-GERD, 26 SNPs-smoking, and 34 SNPs-BMI) that had the highest importance scores. In subsequent LR analyses, interactions between 3 SNPs (rs2295778 of HIF1AN, rs133376 of TSC2, and rs2519757 of TSC1) and GERD, 2 SNPs (rs2295778 of HIF1AN, rs2296188 (VEGFR1) and smoking, and 7 SNPs (rs2114039 of PDGRFA, rs2296188 of VEGFR1, rs11941492 of VEGFR1, rs3756309 of PDGFRB, rs7324547 of VEGFR1, rs17619601 of VEGFR1, and rs17625898 of VEGFR1) and BMI were significantly associated with EA development (all FDR ≤0.10). Moreover, these interactions tended to have a SNP dose-response effects for increased EA risk with increasing number of combined risk genotypes.
These findings suggest that genetic variations in angiogenic genes may modify EA susceptibility through interactions with environmental factors in a SNP dose-response manner.
PMCID: PMC3193872  PMID: 21751195
Esophageal adenocarcinoma; angiogenesis pathway genes; gene-environment interaction; case-only analysis
19.  Kernel Machine SNP-set Analysis for Censored Survival Outcomes in Genome-wide Association Studies 
Genetic Epidemiology  2011;35(7):620-631.
In this paper, we develop a powerful test for identifying SNP-sets that are predictive of survival with data from genome-wide association studies (GWAS). We first group typed SNPs into SNP-sets based on genomic features and then apply a score test to assess the overall effect of each SNP-set on the survival outcome through a kernel machine Cox regression framework. This approach uses genetic information from all SNPs in the SNP-set simultaneously and accounts for linkage disequilibrium (LD), leading to a powerful test with reduced degrees of freedom when the typed SNPs are in LD with each other. This type of test also has the advantage of capturing the potentially non-linear effects of the SNPs, SNP-SNP interactions (epistasis), and the joint effects of multiple causal variants. By simulating SNP data based on the LD structure of real genes from the HapMap project, we demonstrate that our proposed test is more powerful than the standard single SNP minimum p-value based test for association studies with censored survival outcomes. We illustrate the proposed test with a real data application.
PMCID: PMC3373190  PMID: 21818772
cox model; genetic studies; gene-based analysis; kernel machine; multi-locus test; score test; single nucleotide polymorphism
20.  Genetic associations with sporadic neuroendocrine tumor risk 
Carcinogenesis  2011;32(8):1216-1222.
Genetic risk factors for sporadic neuroendocrine tumors (NET) are poorly understood. We tested risk associations in patients with sporadic NET and non-cancer controls, using a custom array containing 1536 single-nucleotide polymorphisms (SNPs) in 355 candidate genes. We identified 18 SNPs associated with NET risk at a P-value <0.01 in a discovery set of 261 cases and 319 controls. Two of these SNPs were found to be significantly associated with NET risk in an independent replication set of 235 cases and 113 controls, at a P value ≤0.05. An SNP in interleukin 12A (IL12A rs2243123), a gene implicated in inflammatory response, replicated with an adjusted odds ratio (95% confidence interval) (aOR) = 1.47 (1.03, 2.11) P-trend = 0.04. A second SNP in defender against cell death, (DAD1 rs8005354), a gene that modulates apoptosis, replicated at aOR = 1.43 (1.02, 2.02) P-trend = 0.04. Consistent with our observations, a pathway analysis, performed in the discovery set, suggested that genetic variation in inflammatory pathways or apoptosis pathways is associated with NET risk. Our findings support further investigation of the potential role of IL12A and DAD1 in the etiology of NET.
PMCID: PMC3149206  PMID: 21606320
21.  Interactions between genetic polymorphisms in the apoptotic pathway and environmental factors on esophageal adenocarcinoma risk 
Carcinogenesis  2011;32(4):502-506.
How genetic variations in apoptosis pathway interact with environmental factors to contribute to esophageal adenocarcinoma (EA) risk has not been comprehensively investigated. We conducted a case-only analysis in 335 Caucasian EA patients that were genotyped for 242 single nucleotide polymorphisms (SNPs) in 43 apoptotic genes. Gene–environment interactions were assessed using a two-step approach. First, random forest algorithm was used to screen for the potential interacting markers. Next, we used case-only logistic regression model to estimate the effects of gene–environment interactions on EA risk. Four SNPs (PERP rs648802; PIK3CA rs4855094, rs7644468 and TNFRSF1A rs4149579) had significant interaction with gastroesophageal reflux disease (GERD). The presence of variant alleles in TP53BP1 rs560191, CASP7 rs7907519 or BCL2 rs12454712 enhanced the risk of smoking by 2.08–2.58 times [interaction odds ratio (ORi) = 2.08–2.58, adjusted P-value (Padj) = 0.02–0.04]. Compared with patients carrying ≤1 risk genotype, the risk of GERD on EA was increased in persons with two (ORi = 1.89, Padj = 0.016) or ≥3 (ORi = 4.30, Padj < 0.0001) risk genotypes. Compared with cases with ≤1 risk genotype, smoking-associated EA risk increased by 3.15 times when ≥2 risk genotypes were present (ORi = 3.15, Padj < 0.0001). In conclusion, interactions among apoptotic SNPs and GERD or smoking play an important role in EA development.
PMCID: PMC3066416  PMID: 21212151
22.  Dietary Vitamin D Intake and Serum 25-Hydroxyvitamin D Level in Relation to Disease Outcomes in Head and Neck Cancer Patients 
Low pre-treatment vitamin D status has been associated with worsened disease outcomes in patients with cancer at various sites. Its prognostic significance in head and neck cancer (HNC) patients has not been studied.
Patients with HNC who participated in a randomized trial were evaluated for: (i) total intake of vitamin D from diet and supplements using a validated food frequency questionnaire (all trial participants, n=540) and (ii) pre-treatment serum 25-hydroxyvitamin D through a radioimmunoassay (n=522). The association of dietary/serum measures of vitamin D status with HNC recurrence, second primary cancer (SPC) incidence, and overall mortality was evaluated using multivariate Cox proportional hazard models.
There was no significant association between dietary or serum vitamin D measures and the three HNC outcomes. The hazard ratios (HR) comparing the highest to the lowest quartile of dietary/supplemental vitamin D intake were 1.10 (95% confidence interval (CI): 0.66-1.84) for recurrence, 1.05 (95% CI: 0.63-1.74) for SPC, and 1.27 (95% CI: 0.87-1.84) for overall mortality. HRs comparing the uppermost to the lowest quartile of serum 25-hydroxyvitamin D levels were 1.12 (95% CI: 0.65-1.93) for recurrence, 0.72 (95% CI: 0.40-1.30) for SPC, and 0.85 (95% CI: 0.57-1.28) for overall mortality. There was no effect modification by cancer stage, season of initial treatment, or trial arm.
Among patients with HNC, vitamin D status before treatment does not influence disease outcomes. Our results contrast with those from most published studies which suggest prognostic significance of vitamin D status in cancer patients at least in subgroups.
PMCID: PMC2990799  PMID: 20533282
23.  Comparison of Pathway Analysis Approaches Using Lung Cancer GWAS Data Sets 
PLoS ONE  2012;7(2):e31816.
Pathway analysis has been proposed as a complement to single SNP analyses in GWAS. This study compared pathway analysis methods using two lung cancer GWAS data sets based on four studies: one a combined data set from Central Europe and Toronto (CETO); the other a combined data set from Germany and MD Anderson (GRMD). We searched the literature for pathway analysis methods that were widely used, representative of other methods, and had available software for performing analysis. We selected the programs EASE, which uses a modified Fishers Exact calculation to test for pathway associations, GenGen (a version of Gene Set Enrichment Analysis (GSEA)), which uses a Kolmogorov-Smirnov-like running sum statistic as the test statistic, and SLAT, which uses a p-value combination approach. We also included a modified version of the SUMSTAT method (mSUMSTAT), which tests for association by averaging χ2 statistics from genotype association tests. There were nearly 18000 genes available for analysis, following mapping of more than 300,000 SNPs from each data set. These were mapped to 421 GO level 4 gene sets for pathway analysis. Among the methods designed to be robust to biases related to gene size and pathway SNP correlation (GenGen, mSUMSTAT and SLAT), the mSUMSTAT approach identified the most significant pathways (8 in CETO and 1 in GRMD). This included a highly plausible association for the acetylcholine receptor activity pathway in both CETO (FDR≤0.001) and GRMD (FDR = 0.009), although two strong association signals at a single gene cluster (CHRNA3-CHRNA5-CHRNB4) drive this result, complicating its interpretation. Few other replicated associations were found using any of these methods. Difficulty in replicating associations hindered our comparison, but results suggest mSUMSTAT has advantages over the other approaches, and may be a useful pathway analysis tool to use alongside other methods such as the commonly used GSEA (GenGen) approach.
PMCID: PMC3283683  PMID: 22363742
24.  Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus12 
Neoplasia (New York, N.Y.)  2012;14(1):29-33.
Aberrant DNA methylation (DNAm) is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm) profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA) and Barrett esophagus (BE, EA precursor). We performed genome-wide DNAm profiling in EA tissue DNA (n = 8) and matched serum DNA (n = 8), in serum DNA of BE (n = 10), and in healthy controls (n = 10) using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92) in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.
PMCID: PMC3281939  PMID: 22355271
25.  A pathway-based association analysis model using common and rare variants 
BMC Proceedings  2011;5(Suppl 9):S85.
How various genetic effects in combination affect susceptibility to certain disease states continues to be a major area of methodological research. Various rare variant models have been proposed, in response to a common failure to either identify or validate biologically driven causal genetic variants in genome-wide association studies. Adopting the idea that multiple rare variants may effectively produce a combined effect equal to a single common variant effect through common linkage with this variant, we construct a pathway-based genetic association analysis model using both common and rare variants. This genetic model is applied to the disease status of unrelated individuals in replication 1 from Genetic Analysis Workshop 17. In this simulated example, we were able to identify several pathways that were potentially associated with the disease status and found that common variants showed stronger genetic effect than rare variants.
PMCID: PMC3287926  PMID: 22373433

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