ABSTRACT: Histologic classification of thymomas has significant limitations since complete surgical excision can be curative. In order to better understand the biology of the disease processes, we performed whole genome gene expression analysis. RNA was extracted from fresh frozen tumors from 36 patients with thymomas and follow-up data was available. Gene expression data was correlated with clinicopathological data. Using the Illumina BeadStudio® platform and Human Ref-8 Beadchip, gene expression data was analyzed by Partek®, and Ingenuity Pathways Analysis (IPA). Validation of the chosen genes was performed using quantitative real-time RT-PCR (qRT-PCR). Unsupervised clustering resulted in identification of four clusters of tumors (C1-C4). Using IPA, the top significant biological functions and pathways displayed cell cycle related category and genes in C1 and C2. Carbohydrate metabolism and cellular growth and proliferation were among the most significant for C3 and C4, respectively. On the other hand, cancer and metabolism related functions and pathways were prominent in clinical outcome including metastasis and stage. Our gene expression analysis representing one of the largest series in literature, revealed at least four distinct clusters of thymic tumors. The study identified number of metastasis-associated genes that are potential candidates for therapeutics 41 Samples, 3 Cell Line samples with 1 duplicate (total = 4). 37 Patient samples including 1 duplicate (total = 37).
Project description:Histologic classification of thymomas has significant limitations with respect to both subtype definitions and consistency. In order to better understand the biology of the disease processes, we performed whole genome gene expression analysis. RNA was extracted from fresh frozen tumors from 34 patients with thymomas and followup data was available. Using the Illumina BeadStudio® platform and Human Ref-8 Beadchip, gene expression data was analyzed with Partek Genomics Suite®, and Ingenuity Pathways Analysis (IPA). Unsupervised clustering of gene expression data, representing one of the largest series in literature, resulted in identification of four molecular clusters of tumors (C1-C4), which correlated with histology (P = 0.002). However, neither histology nor clusters correlated with clinical outcomes. Correlation of gene expression data with clinical data showed that a number of genes were associated with either advanced stage at diagnosis or development of recurrence or metastases. The top pathways associated with metastases were amino acid metabolisms, biosynthesis of steroids and glycosphingolipids, cell cycle checkpoint proteins and Notch signaling. The differential expression of some of the top genes related to both metastases and stage was confirmed by RT-PCR in all cases of metastases and matched nonmetastatic cases. A number of potential candidates for therapeutics were also identified.
Project description:We applied whole-genome single nucleotide polymorphism (SNP) arrays to define a comprehensive genetic profile of 23 esophageal adenocarcinoma (EAC) primary tumor biopsies based on loss of heterozygosity (LOH) and DNA copy number changes. Alterations were common, averaging 97 (range 23-208) per tumor. LOH and gains averaged 33 (range 3-83) and 31 (range 11-73) per tumor, respectively. Copy neutral LOH events averaged 27 (range 7-57) per EAC. We noted 126 homozygous deletions (HDs) across the EAC panel (range 0-11 in individual tumors). Frequent HDs within FHIT (17/23), WWOX (8/23) and DMD (6/23) suggest a role for common fragile sites or genomic instability in EAC etiology. HDs were also noted for known tumor suppressor genes (TSGs) including: CDKN2A, CDKN2B, SMAD4 and GALR1, and identified PDE4D and MGC48628 as potentially novel TSGs. All tumors showed LOH for most of chromosome 17p, suggesting that TSGs other than TP53 maybe targeted. Frequent gains were noted around MYC (13/23), BCL9 (12/23), CTAGE1 (14/23) and ZNF217 (12/23). Thus, we have confirmed previous reports indicating frequent changes to FHIT, CDKN2A, TP53 and MYC in EAC and identified additional genes of interest. Meta analysis of previous genome-wide EAC studies together with the data presented here, highlighted consistent regions of gain on 8q, 18q and 20q, and multiple LOH regions on 4q, 5q, 17p and 18q, suggesting that more than one gene may be targeted on each of these chromosome arms. The focal gains and deletions documented here are a step towards identifying the key genes involved in EAC development. Keywords: High Density SNP array Here we use Illumina 317K whole-genome single-nucleotide polymorphism arrays to define a comprehensive allelotype of melanoma based on loss of heterozygosity (LOH) and copy number changes in a panel of 23 esophageal adenocarcinoma (EAC) primary tumor biopsies. Each EAC was paired to normal squamous biopsy from 40328 (GSM266078) to generate the log_R_ratio.
Project description:Thymomas are rare mediastinal tumors that are difficult to treat and pose a major public health concern. Identifying mutations in target genes is vital for the development of novel therapeutic strategies. Type A thymomas possess a missense mutation in GTF2I (chromosome 7 c.74146970T>A) with high frequency. However, the molecular pathways underlying the tumorigenesis of other thymomas remain to be elucidated. We aimed to detect this missense mutation in GTF2I in other thymoma subtypes (types B). This study involved 22 patients who underwent surgery for thymomas between January 2014 and August 2019. We isolated tumor cells from formalin-fixed paraffin-embedded tissues from the primary lesions using laser-capture microdissection. Subsequently, we performed targeted sequencing to detect mutant GTF2I coupled with molecular barcoding. We used PyClone analysis to determine the fraction of tumor cells harboring mutant GTF2I. We detected the missense mutation (chromosome 7 c.74146970T>A) in GTF2I in 14 thymomas among the 22 samples (64%). This mutation was harbored in many type B thymomas as well as type A and AB thymomas. The allele fraction for the tumors containing the mutations was variable, primarily owing to the coexistence of normal lymphocytes in the tumors, especially in type B thymomas. PyClone analysis revealed a high cellular prevalence of mutant GTF2I in tumor cells. Mutant GTF2I was not detected in other carcinomas (lung, gastric, colorectal, or hepatocellular carcinoma) or lymphomas. In conclusion, the majority of thymomas harbor mutations in GTF2I that can be potentially used as a novel therapeutic target in patients with thymomas.
Project description:Background:Polysaccharide monooxygenases (PMOs) of the auxiliary activity 9 (AA9) family have been reported to oxidize C1, C4, and C6 positions in cellulose. However, currently no direct evidence exists that PMOs oxidize C6 positions in cellulose, and molecular mechanism of C1, C4 and C6 oxidation is unclear. Results:In this study, a PMO gene (Ctpmo1) belonging to AA9 was isolated from Chaetomium thermophilum and successfully expressed and correctly processed in Pichia pastoris. A simple and effective chemical method of using Br2 to oxidize CtPMO1 reaction products was developed to directly identify C4- and C6-oxidized products by matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF-MS). The PMO (CtPMO1) cleaves phosphoric acid-swollen cellulose (PASC) and celloheptaose, resulting in the formation of oxidized and nonoxidized oligosaccharides. Product identification shows that the enzyme can oxidize C1, C4, and C6 in PASC and cello-oligosaccharides. Mutagenesis of the aromatic residues Tyr27, His64, His157 and residue Tyr206 on the flat surface of CtPMO1 was carried out using site-directed mutagenesis to form the mutated enzymes Y27A, H64A, H157A, and Y206A. It was demonstrated that Y27A retained complete activity of C1, C4, and C6 oxidation on cellulose; Y206A retained partial activity of C1 and C4 oxidation but completely lost activity of C6 oxidation on cellulose; H64A almost completely lost activity of C1, C4, and C6 oxidation on cellulose; and H157A completely lost activity of C1, C4, and C6 oxidation on cellulose. Conclusions:This finding provides direct and molecular evidence for C1, C4, especially C6 oxidation by lytic polysaccharide monooxygenase. CtPMO1 oxidizes not only C1 and C4 but also C6 positions in cellulose. The aromatic acid residues His64, His157 and residue Tyr206 on CtPMO1 flat surface are involved in activity of C1, C4, C6 oxidation.
Project description:This study aimed to evaluate the role of pretreatment SUVmax and volumetric FDG positron emission tomography (PET) parameters in the differentiation between benign and malignant mediastinal tumors. In addition, we investigated whether pretreatment SUVmax and volumetric FDG-PET parameters could distinguish thymomas from thymic carcinomas, and low-risk from high-risk thymomas.This study was conducted on 52 patients with mediastinal tumors undergoing FDG-PET/CT. Histological examination indicated that 29 mediastinal tumors were benign, and 23 cases were malignant. To obtain quantitative PET/CT parameters, we determined the maximum standardized uptake value (SUVmax), volumetric parameters, metabolic tumor volume (MTV), and total lesion glycolysis (TLG) for primary tumors using SUVmax cut-off value of 2.5. SUVmax, MTV and TLG of benign and malignant tumors were compared using the Mann-Whitney U test. Moreover, receiver-operating curve (ROC) analysis was applied to identify the cut-off values of SUVmax, MTV and TLG for the accurate differentiation of benign and malignant tumors. SUVmax, MTV and TLG were compared between thymomas and thymic carcinomas, as well as low-risk and high-risk thymomas.Mean SUVmax, MTV and TLG of malignant mediastinal tumors were significantly higher compared to benign tumors (P<0.001). Sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of SUVmax were 78.2%, 86.2%, 82.6%, 81.8%, and 83.3%, respectively. These values were estimated at 82.6%, 96.6%, 90.4%, 95%, and 87.5% for MTV and TLG, respectively. Additionally, optimal cut-off values for the differentiation of benign and malignant mediastinal tumors were determined at 4.2 and 22.3 mL and 79.7 g for SUVmax, MTV and TLG, respectively. Mean SUVmax, MTV and TLG of thymic carcinomas were significantly higher compared to thymomas (P<0.01), while no significant differences were observed in the mean quantitative parameters between low-risk and high-risk thymomas.Although SUVmax, MTV and TLG could not distinguish between low-risk and high-risk thymomas, these parameters might be able to differentiate benign tumors from malignant mediastinal tumors noninvasively. These parameters could be used to distinguish between thymomas and thymic carcinomas as well. Therefore, FDG-PET/CT parameters seem to be accurate indices for the detection of malignant mediastinal tumors.
Project description:To retrospectively investigate the added value of quantitative 3D shape analysis in differentiating encapsulated from invasive thymomas.From February 2002 to October 2013, 53 patients (25 men and 28 women; mean age, 53.94 ± 13.13 years) with 53 pathologically-confirmed thymomas underwent preoperative chest CT scans (slice thicknesses ? 2.5 mm). Twenty-three tumors were encapsulated thymomas and 30 were invasive thymomas. Their clinical and CT characteristics were evaluated. In addition, each thymoma was manually-segmented from surrounding structures, and their 3D shape features were assessed using an in-house developed software program. To evaluate the added value of 3D shape features in differentiating encapsulated from invasive thymomas, logistic regression analysis and receiver-operating characteristics curve (ROC) analysis were performed.Significant differences were observed between encapsulated and invasive thymomas, in terms of cystic changes (p=0.004), sphericity (p=0.016), and discrete compactness (p=0.001). Subsequent binary logistic regression analysis revealed that absence of cystic change (adjusted odds ratio (OR) = 6.636; p=0.015) and higher discrete compactness (OR = 77.775; p=0.012) were significant differentiators of encapsulated from invasive thymomas. ROC analyses revealed that the addition of 3D shape analysis to clinical and CT features (AUC, 0.955; 95% CI, 0.935-0.975) provided significantly higher performance in differentiating encapsulated from invasive thymomas than clinical and CT features (AUC, 0.666; 95% CI, 0.626-0.707) (p<0.001).Addition of 3D shape analysis, particularly discrete compactness, can improve differentiation of encapsulated thymomas from invasive thymomas.
Project description:BACKGROUND:Potato consumption has been hypothesized to be associated with higher risk of hypertension, diabetes, and colorectal cancer. OBJECTIVE:The aim of this study was to examine the association between potato consumption and the risk of overall and cause specific mortality in the large prospective National Institutes of Health-AARP (NIH-AARP) Study. DESIGN:The NIH-AARP study recruited 566,407 persons, aged 50-72 years in 1995-1996. We excluded subjects that reported a history of chronic disease at baseline. Potato consumption data from a validated food frequency questionnaire completed at baseline was used in Cox proportional hazard models to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) for overall and cause specific mortality. Final models were adjusted for potential risk factors for mortality. RESULTS:Among 410,701 participants included in this analysis, 76,921 persons died during the 15.6 years of follow-up. Eating baked, boiled, or mashed potatoes, French fries or potato salad seven or more times per week was associated with higher risk of overall mortality, in models adjusted only for age and sex (HR C4 vs C1 = 1.17, 95%CI = 1.13, 1.21). These results were attenuated in fully adjusted models (HR C4 vs C1 = 1.02, 95%CI = 0.97, 1.06). Potato consumption was not associated with risk of mortality caused by cancer (HR C4 vs C1 = 1.04, 95%CI = 0.97, 1.11), heart disease (HR C4 vs C1 = 1.00, 95%CI = 0.93, 1.09), respiratory disease (HR C4 vs C1 = 1.16, 95%CI = 0.99, 1.37), or diabetes (HR C4 vs C1 = 0.91, 95%CI = 0.71, 1.19). We tested for an association with different preparation methods and found limited evidence for differences by preparation method. The only statistically significant association was that for French fry consumption with cancer-related mortality (HR C4 vs C1 = 1.27, 95%CI = 1.02, 1.59), a finding for which uncontrolled confounding could not be ruled out. CONCLUSION:We find little evidence that potato consumption is associated with all-cause or cause-specific mortality.
Project description:Triple negative breast cancer (TNBC) is characterized by high proliferation, poor differentiation and a poor prognosis due to high rates of recurrence. Despite lower overall incidence African American (AA) patients suffer from higher breast cancer mortality in part due to the higher proportion of TNBC cases among AA patients compared to European Americans (EA). It was recently shown that the clinical heterogeneity of TNBC is reflected by distinct transcriptional programs with distinct drug response profiles in preclinical models. In this study, we used gene expression profiling and immunohistochemistry to eluicidate potential differences between TNBC tumors of EA and AA patients on a molecular level. WG-DASL experiment of 90 FFPE samples of ER, PR and HER2 (triple) negative breast cancer samples diagnosed between 1987 and 2007. Invasive disease was identified on H&E sections by the study pathologist and one to three 1.5 mm cores were punched from the top down in the designated tumor areas of each FFPE block. The cores were deparaffinized with xylene at 50°C for 3 minutes. RNA was extracted using the RecoverAll Total Nucleic Acid Isolation kit (Applied Biosystems) following the manufacturer's protocol. The isolated RNA was hybridized to Whole-Genome DASL (HumanRef8 V 3.0, Illumina) at the Yale Center for Genome Analysis. 90 primary tumor RNA samples from 90 patients were adequate for analysis and passed Quality control.
Project description:Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL-1β, IL-6, IL-8, IL-23p19, TNF-α, CXCL2 and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease. Gut specimens were derived from individuals undergoing resection for localized colon cancer. Microscopically normal colonic mucosa was dissected from the surgical specimen near the resection margin and immediately subjected to the experimental procedures. After extensive washing the mucus layer was removed by dithiothreitol (DTT) treatment. Subsequently, punches of defined surface area were prepared and denuded of epithelial cells by exposure to EDTA. Samples were collected prior to culturing and washing (control, t = 0 h, total mucosa, TM) as well as after loss of the epithelial layer (t = 5 h, mucosa after loss of epithelial layer, LEL-M) and snap frozen in liquid nitrogen. Lamina Propria (LP) was subsequently isolated via Laser Capture Microdissection (LMD) followed by RNA isolation. Microarray analysis of TM-LP (control) and LEL-LP samples was performed using the WG-DASL Human HT_12 V4 Expression BeadChip Assay from Illumina employing a minimum of 200 ng total RNA per sample. Four replicates from individual experiments were measured for each time point.
Project description:Analyses of gene expression profiling in sinonasal sarcoma (SNS) harboring PAX3-MAML3 fusion gene or PAX3 rearrangement and other types of tumors without having such fusion or rearrangement. The results provide important information for further investigations of the PAX3-MAML3 fusion functions in SNS. Total RNA was obtained from FFPE tissues of 41 tumors including 8 SNS (6 with PAX3-MAML3 fusion and 2 with PAX3 rearrangement only) and 33 cases from other 10 different types of tumors. Gene expression profiling of fusion group including 8 SNA vs.non-fusion group including 33 control tumors were analyszed.