Unsupervised Hierarchical Clustering Analysis Research Articles

NMR-based metabonomics study on the effect of Gancao in the attenuation of toxicity in rats induced by Fuzi

Ethnopharmacological relevanceFuzi, the processed lateral root of Aconitum carmichaelii Debeaux, is a traditional Chinese medicine used for its analgesic, antipyretic, anti-rheumatoid arthritis and anti-inflammation effects; however, it is also well known for its toxicity. Gancao, the root of Glycyrrhiza uralensis Fisch., is often used concurrently with Fuzi to alleviate its toxicity. However, the mechanism of detoxication is still not well clear. Aim of the studyIn this study, the effect of Gancao on the metabolic changes induced by Fuzi was investigated by NMR-based metabonomic approaches. Materials and methodsFifty male Wistar rats were randomly divided into five groups (group A: control, group B: Fuzi decoction alone, group C: Gancao decoction alone, group D: Fuzi decoction and Gancao decoction simultaneously, group E: Fuzi decoction 5h after Gancao decoction) and urine samples were collected for NMR-based metabolic profiling analysis. Statistical analyses such as unsupervised PCA, t-test, hierarchical cluster, and pathway analysis were used to detect the effects of Gancao on the metabolic changes induced by Fuzi. ResultsThe behavioral and biochemical characteristics showed that Fuzi exhibited toxic effects on treated rats (group B) and statistical analyses showed that their metabolic profiles were in contrast to those in groups A and C. However, when Fuzi was administered with Gancao, the metabolic profiles became similar to controls, whereby Gancao reduced the levels of trimethylamine N-oxide, betaine, dimethylglycine, valine, acetoacetate, citrate, fumarate, 2-ketoglutarate and hippurate, and regulated the concentrations of taurine and 3-hydroxybutyrate, resulting in a decrease in toxicity. Furthermore, important pathways that are known to be involved in the effect of Gancao on Fuzi, including phenylalanine, tyrosine and tryptophan biosynthesis, the synthesis and degradation of ketone bodies, and the TCA cycle, were altered in co-treated rats. ConclusionsGancao treatment mitigated the metabolic changes altered by Fuzi administration in rats, demonstrating that dosing with Gancao could reduce the toxicity of Fuzi at the metabolic level. Fuzi and Gancao administered simultaneously resulted in improved toxicity reduction than when Gancao was administrated 5h prior to Fuzi. In summary, co-administration of Gancao with Fuzi reduces toxicity at the metabolic level.

On the Identification of Rayon/Viscose as a Major Fraction of Microplastics in the Marine Environment: Discrimination between Natural and Manmade Cellulosic Fibers Using Fourier Transform Infrared Spectroscopy.

This work was sparked by the reported identification of man-made cellulosic fibers (rayon/viscose) in the marine environment as a major fraction of plastic litter by Fourier transform infrared (FT-IR) transmission spectroscopy and library search. To assess the plausibility of such findings, both natural and man-made fibers were examined using FT-IR spectroscopy. Spectra acquired by transmission microscopy, attenuated total reflection (ATR) microscopy, and ATR spectroscopy were compared. Library search was employed and results show significant differences in the identification rate depending on the acquisition method of the spectra. Careful selection of search parameters and the choice of spectra acquisition method were found to be essential for optimization of the library search results. When using transmission spectra of fibers and ATR libraries it was not possible to differentiate between man-made and natural fibers. Successful differentiation of natural and man-made cellulosic fibers has been achieved for FT-IR spectra acquired by ATR microscopy and ATR spectroscopy, and application of ATR libraries. As an alternative, chemometric methods such as unsupervised hierarchical cluster analysis, principal component analysis, and partial least squares-discriminant analysis were employed to facilitate identification based on intrinsic relationships of sample spectra and successful discrimination of the fiber type could be achieved. Differences in the ATR spectra depending on the internal reflection element (Ge versus diamond) were observed as expected; however, these did not impair correct classification by chemometric analysis. Moreover, the effects of different levels of humidity on the IR spectra of natural and man-made fibers were investigated, too. It has been found that drying and re-humidification leads to intensity changes of absorption bands of the carbohydrate backbone, but does not impair the identification of the fiber type by library search or cluster analysis.

MicroRNA Gene Expression Signature Driven by miR-9 Overexpression in Ovarian Clear Cell Carcinoma.

Previous studies have identified microRNA (miRNA) involvement in human cancers. This study aimed to elucidate potential clinical and biological associations of ovarian cancer-related miRNA gene expression profiles in high-grade serous carcinoma (HGSC) and ovarian clear cell carcinoma (OCCC). Accordingly, we investigated 27 patients with ovarian cancer (12 HGSC and 15 OCCC cases) using quantitative real-time reverse transcription polymerase chain reaction to determine the cancer-related miRNA expressions. Gene Cluster 3.0 was used for hierarchical clustering analysis, and differentially expressed miRNAs between HGSC and OCCC were identified by the class comparison analysis using BRB-ArrayTools. An unsupervised hierarchical clustering analysis identified two distinct miRNA expression clusters, with histological subtype-related significant differences in the associations between clusters and clinicopathological features. A comparison of miRNA expression in HGSCs and OCCCs identified five miRNAs (miR-132, miR-9, miR-126, miR-34a, and miR-21), with OCCCs demonstrating a statistically higher expression. Further investigation of the biological significance of miR-9 overexpression in OCCC revealed that miR-9 inhibition reduced the cell invasion ability and upregulated E-cadherin expression. Using a luciferase reporter assay, we further demonstrated the direct binding of miR-9 to E-cadherin. Global cancer-related miRNA expression analysis identified statistically unique profiles that could discriminate ovarian cancer histotypes. In OCCC, miR-9 overexpression may affect pathogenesis by targeting E-cadherin, thereby inducing an epithelial–mesenchymal transition. Therefore, miR-9 may be a promising therapeutic target strategy for OCCC.

Genetic Diversity and Structure of Tobacco in Greece on the Basis of Morphological and Microsatellite Markers

Oriental tobacco varieties have greater aroma value, and consumer demand for these varieties is now greater than ever. Yet our knowledge of morphological and genetic diversity within diverse gene pools of cultivated oriental tobacco (Nicotiana tabacum L.) varieties is limited. Phenotypic and genetic analysis of these genotypes is important for the improvement of tobacco varieties. Information on morphology and genetics could help toward future germplasm maintenance and selection of suitable materials for breeding elite varieties. This study presents an assessment of morphology and genetic diversity for 34 tobacco cultivars maintained in an ex situ GeneBank collection. Data of 36 traits, describing phenology, plant morphology, and leaf quality, were recorded over 3 yr and analyzed using principal component analysis. Euclidean distance metric and the Ward's agglomeration method were used in an unsupervised hierarchical cluster analysis of the 34 different cultivars used. The tobacco cultivars were classified into three main clusters, suggesting that the characterized tobacco collection has high potential for specific breeding goals. Furthermore, the 34 tobacco cultivars were genotyped with 10 microsatellite markers. High levels of polymorphism were detected, and 63 distinctive alleles were identified; on average 6.3 alleles per primer. Both the principal component analysis, derived from the morphological data, and the unweighted pair group method, with arithmetic mean from the molecular analysis, clustered the tobacco cultivars according to their type (oriental and flue‐cured tobacco). Furthermore, a Bayesian clustering approach analysis allowed a more detailed grouping of subpopulations.

Aberrant Endometrial DNA Methylome and Associated Gene Expression in Women with Endometriosis.

Endometriosis is an estrogen-dependent, progesterone-resistant disorder largely derived from retrograde transplantation of menstrual tissue/cells into the pelvis, eliciting an inflammatory response, pelvic pain, and infertility. Eutopic endometrium (within the uterus), giving rise to pelvic disease, displays cycle-dependent transcriptomic, proteomic, and signaling abnormalities, and although its DNA methylation profiles dynamically change across the cycle in healthy women, studies in endometriosis are limited. Herein, we investigated the DNA methylome and associated gene expression in three phases of the cycle in eutopic endometrium of women with severe endometriosis versus controls, matched for ethnicity, medications, smoking, and no recent contraceptive steroid use. Genome-wide DNA methylation and gene expression were coassessed in each sample. Cycle phase was determined by histology, serum hormone levels, and unsupervised principal component and hierarchical cluster analyses of microarray data. Altered endometrial DNA methylation in endometriosis was most prominent in the midsecretory phase (peak progesterone), with disruption of the normal pattern of cycle-dependent DNA methylation changes, including a bias toward methylation of CpG islands, suggesting wide-range abnormalities of the chromatin remodeling machinery in endometriosis. DNA methylation changes were associated with altered gene expression relevant to endometrial function/dysfunction, including cell proliferation, inflammation/immune response, angiogenesis, and steroid hormone response. The data provide insight into epigenetic reprogramming and steroid hormone actions in endometrium contributing to the pathogenesis and pathophysiology of endometriosis.

Abstract 2780: Genome-wide methylation analysis reveals methylator subtypes of Barrett's esophagus and esophageal adenocarcinoma

Abstract Esophageal adenocarcinoma (EAC) arises from Barrett's Esophagus (BE) through a progression sequence driven by the accumulation of epi/genetic alterations. Genetic alterations and aberrantly methylated genes/loci, the best-characterized epigenetic alteration, vary among individual BE and EAC cases. To better understand the molecular heterogeneity in BE/ EAC, we analyzed genome-wide DNA methylation patterns in BE (N = 98), EAC (N = 23), and normal esophagus and stomach (N = 78) collected via Esophagus Translational Research Network (BETRNet) using Human Methylation 450K (HM450K) arrays. We identified methylation-based subtypes using a model-based recursively partitioned mixture model and unsupervised resampling-based hierarchical cluster analysis with the most variable probes based on standard deviation. All analyses were performed in R. Four methylator subtypes were identified in the BE/EAC dataset using our clustering approach: high (HM); intermediate (IM); low (LM); and minimal (MM). The clusters showed widespread methylation differences in CpGs primarily located in CpG islands (72% of 414 total probes). Each methylation subtype showed distinct genetic and clinical features suggesting underlying biological differences (Table 1). HM exhibited methylation patterns reminiscent of a CpG Island Methylator Phenotype (CIMP). Among BE/EAC cases with known TP53 mutation status, the majority of mutants were among HM and IM clusters (15/19, 78.9%), Finally, identification of methylation subtypes in an independent HM450K dataset of EAC samples (N = 89) from the Cancer Genome Atlas validated the existence of the BE/EAC methylation subtypes. In summary, our data suggest genome wide alterations in DNA methylation occur in the BE stage of the BE-to-EAC progression. We also identified four distinct methylation subtypes of BE/EAC, including a CIMP-like HM subtype, and found more TP53 mutants in the HM and IM subtypes, revealing molecular heterogeneity in different methylation subtypes. Table 1.Characteristics of primary BE/EAC dataset and subtypes from cluster analyses.Cluster CharacteristicSubtypesHigh N (%)Intermediate N (%)Low N (%)Minimal N (%)Total N (%)Case CountsTotal31 (26%)44 (36%)28 (23%)18 (23%)121 (100%)BE23 (23%)34 (35%)24 (24%)17 (17%)98 (100%)EAC8 (35%)10 (43%)4 (17%)1 (4%)23 (100%)Demographic VariablesGenderM28 (28%)35 (35%)23 (23%)14 (14%)100 (100%)GenderF3 (14%)9 (43%)5 (24%)4 (19%)21 (100%)Age (mean ± sd)68 ± 1163 ± 1361 ± 1461 ± 1064 ± 13TP53 Mutations***Mutation*6 (32%)9 (47%)4 (21%)0 (0%)19 (100%)WT6 (30%)8 (40%)5 (25%)1 (5%)20 (100%)Unknown1927191782*** Mutation categories included: Frame Shift Deletion; In Frame Insertion; In Frame Deletion; Missense Mutation; Nonsense Mutation; and Splice Site Citation Format: Ming Yu, Sean Maden, Andrew Kaz, Tai Heinzerling, Rachele O’Leary, Matthew Stachler, Adam Bass, Amitabh Chak, Joseph Willis, Sanford Markowitz, William Grady. Genome-wide methylation analysis reveals methylator subtypes of Barrett's esophagus and esophageal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2780.

Abstract 3981: In situ mass spectrometry using desorption electrospray ionization mass spectrometry (DESI-MS) can detect specific lipid profiles across breast cancer molecular subtypes

Abstract INTRODUCTION Lipid profiling as a source for biomarkers identification is an evolving field with some enthusiastic results in cancer. Alterations on lipid metabolism have been increasingly recognized as a hallmark of cancer cells and may help to understand breast cancer carcinogenesis. Analytical chemistry and Pathology have been recently combined in the development of chemical microscopy using in situ mass spectrometry (MS) and may help to decipher breast cancer molecular initiation and progression. OBJECTIVE We aimed to use in situ Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) to investigate lipid profiles across invasive breast cancer (IBC) molecular subtypes and related normal parenchyma (NBP) and ductal carcinoma in situ (DCIS). METHODS: We selected frozen samples from 9 IBC with various ER/PR/HER2 status (ER 0 to 100%, PR 0 to 90%, HER2 0 and 3+, Ki67 10 to 70%). Unstained slides obtained from frozen tissue at 10μm were submitted to DESI-MS analysis with no pretreatment and spatial resolution of 100μm. Molecular profiles (mass to charge [m/z] ratio versus signal intensity) were generated in triplicate directly from tissues to detect changes from areas of interest: IBC (and adjacent normal breast parenchyma and DCIS whenever present) regions were identified by a breast pathologist on HE-stained sequential slides. Experiment was conducted in full-scan mode and m/z range of 100-1200. DESI mass spectra were obtained using a Thermo ScientificTM Q ExactiveTM Hybrid Quadrupole Orbitrap Mass Spectrometer in the positive ion mode. The Biomap software was used to visualize the images and to export the spectrums to metaboanalyst, where the data were statiscally analyzed. RESULTS: The images obtained by lipid profiling presented a direct correlation with HE stained slides, with some ions m/z clearly overlaying areas of normal parenchyma, DCIS and invasive breast cancer. The unsupervised hierarchical clustering analysis showed all technical triplicates remained grouped. All NBP but one clustered together. Triple negative IBC and its related NBP formed a distinct cluster from all other ER+ samples. The ER+/HER2 + tumor segregated from other ER+/HER2- tumors. DCIS and IBC from ER+/PR+/HER2- tumor clustered together. The Ions with m/z 186.9 and m/z 303.2 were consistently related to NBP and IBC respectively. CONCLUSIONS: Chemical microscopy using DESI-MS is a powerful tool to identify specific lipid profiles across molecular subtypes of IBC and can contribute to understand breast cancer carcinogenesis. Citation Format: Victor P. Andrade, Adriana L. Santoro, Sabrina T. Moretti, Nicolas V. Schwab, Marcos N. Eberlin. In situ mass spectrometry using desorption electrospray ionization mass spectrometry (DESI-MS) can detect specific lipid profiles across breast cancer molecular subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3981.

A protein and mRNA expression-based classification of gastric cancer

The overall survival of gastric carcinoma patients remains poor despite improved control over known risk factors and surveillance. This highlights the need for new classifications, driven towards identification of potential therapeutic targets. Using sophisticated molecular technologies and analysis, three groups recently provided genetic and epigenetic molecular classifications of gastric cancer (The Cancer Genome Atlas, ‘Singapore-Duke' study, and Asian Cancer Research Group). Suggested by these classifications, here, we examined the expression of 14 biomarkers in a cohort of 146 gastric adenocarcinomas and performed unsupervised hierarchical clustering analysis using less expensive and widely available immunohistochemistry and in situ hybridization. Ultimately, we identified five groups of gastric cancers based on Epstein–Barr virus (EBV) positivity, microsatellite instability, aberrant E-cadherin, and p53 expression; the remaining cases constituted a group characterized by normal p53 expression. In addition, the five categories correspond to the reported molecular subgroups by virtue of clinicopathologic features. Furthermore, evaluation between these clusters and survival using the Cox proportional hazards model showed a trend for superior survival in the EBV and microsatellite-instable related adenocarcinomas. In conclusion, we offer as a proposal a simplified algorithm that is able to reproduce the recently proposed molecular subgroups of gastric adenocarcinoma, using immunohistochemical and in situ hybridization techniques.

Morpho-physiological diversity in the collection of sour cherry (Prunus cerasus) cultivars of the Fruit Genebank in Naoussa, Greece using multivariate analysis

The phenotypic characterization of sour cherry cultivars provides important information on their attributes, which is of paramount importance for their breeding and germplasm management. This study examined the Hellenic Sour Cherry Genebank, which consists of 27 sour cherry cultivars, located in the Institute of Pomology Fruit Genebank, Naoussa, Greece. The data analyzed in this study were obtained after a ten-year consecutive monitoring of 38 morpho-physiological descriptors of phenology, plant morphology, yield and fruit quality. For the statistical analysis were applied hierarchical cluster analysis and principal component analysis (PCA). According to the analysis, the descriptors demonstrated a high degree of variability, especially, yield, ratio of titratable acidity and soluble solids, number of flower buds, and pit weight. Moreover, the variables fruit equatorial diameter, fruit width, fruit weight, fruit size, fruit shape and lamella shape characters exhibited high discriminating power. Significant positive and negative correlations were detected as well among the studied quantitative traits. The highest significant positive correlation was found between the fruit equatorial diameter and the fruit width (0.974). Whereas, the highest significant negative correlation was found between the ratio of pit volume to fruit volume and the fruit weight (−0.737). An unsupervised hierarchical cluster analysis was performed using the Euclidean distance and the Ward's agglomeration method. The sour cherry cultivars were classified into three main clusters, suggesting that the characterized sour cherry collection has a high potential for specific breeding goals. We discuss the usefulness of the identified correlations among the traits, for potential breeding projects regarding fruit size and quality.

Stress transgenerationally programs metabolic pathways linked to altered mental health.

Stress is among the primary causes of mental health disorders, which are the most common reason for disability worldwide. The ubiquity of these disorders, and the costs associated with them, lends a sense of urgency to the efforts to improve prediction and prevention. Down-stream metabolic changes are highly feasible and accessible indicators of pathophysiological processes underlying mental health disorders. Here, we show that remote and cumulative ancestral stress programs central metabolic pathways linked to mental health disorders. The studies used a rat model consisting of a multigenerational stress lineage (the great-great-grandmother and each subsequent generation experienced stress during pregnancy) and a transgenerational stress lineage (only the great-great-grandmother was stressed during pregnancy). Urine samples were collected from adult male F4 offspring and analyzed using 1H NMR spectroscopy. The results of variable importance analysis based on random variable combination were used for unsupervised multivariate principal component analysis and hierarchical clustering analysis, as well as metabolite set enrichment analysis (MSEA) and pathway analysis. We identified distinct metabolic profiles associated with the multigenerational and transgenerational stress phenotype, with consistent upregulation of hippurate and downregulation of tyrosine, threonine, and histamine. MSEA and pathway analysis showed that these metabolites are involved in catecholamine biosynthesis, immune responses, and microbial host interactions. The identification of metabolic signatures linked to ancestral programming assists in the discovery of gene targets for future studies of epigenetic regulation in pathogenic processes. Ultimately, this research can lead to biomarker discovery for better prediction and prevention of mental health disorders.

Identification of Differentially Expressed Genes in Kawasaki Disease Patients as Potential Biomarkers for IVIG Sensitivity by Bioinformatics Analysis.

Kawasaki disease (KD) is a leading cause of acquired heart disease predominantly affecting infants and young children. Intravenous immunoglobulin (IVIG) is applied as the most favorable treatment against KD, but IVIG resistant remains exist. Although several clinical scoring systems have been developed to identify children at highest risk of IVIG resistance, there is a need to identify sufficiently sensitive biomarkers for IVIG treatment. Some differentially expressed genes (DEGs) could be the promising potential biomarkers for IVIG-related sensitivity diagnosis. We employed a systematic and integrative bioinformatics framework to identify such kind of genes. The performance of the candidate genes was evaluated by hierarchical clustering, ROC analysis and literature mining. By analyzing three datasets of KD patients, 34 DEGs of the three groups have been found to be associated with IVIG-related sensitivity. A module of 12 genes could predict resistant group patients with high accuracy, and a module of ten genes could predict responsive group patients effectively with accuracy of 96%. And three of them are most likely to serve as drug targets or diagnostic biomarkers in the future. Compared with unsupervised hierarchical clustering analysis, our modules could distinct IVIG-resistant patients efficiently. Two groups of DEGs could predict IVIG-related sensitivity with high accuracy, which are potential biomarkers for the clinical diagnosis and prediction of IVIG treatment response in KD patients, improving the prognosis of patients.

Innate correlates of influenza clinical outcomes in a multicenter, longitudinal study of critically ill children

Abstract Paradoxically, influenza infection can trigger pro- and anti-inflammatory responses, both of which can lead to significant morbidity and mortality. Inflammation can lead to septic shock and acute lung injury (ALI), while suppression can slow viral clearance and increase susceptibility to bacterial coinfection (BCo). We collected and analyzed lung and peripheral blood samples from a multi-center cohort of critically ill children with influenza infection (N = 171) to better understand innate signatures of disease. Samples were collected at admission to the PICU and assayed for levels of 42 cytokines and chemokines. We found intra-compartment analyte correlations were high, while those between compartments were comparatively weak. To increase statistical power and identify groups of co-signaling cytokines, we pre-specified an unsupervised hierarchical clustering analysis to form influenza-specific modules. Some modules were consistent with the literature; e.g. IL4, IL5, IL9 and IL13 (Th2-like) clustered within both compartments. We hypothesized that modules would associate with shock, ALI and death or near death (ECMO). A pro-inflammatory PB module including IL6, IL8, IL10, IP10, GCSF and MCP1 was associated with shock (FWER p<1e-4; OR 3.4) and ALI (FWER p=0.003; OR 2.2), validating previous studies. Inverse associations were also identified with lung and serum modules, only after adjusting for patients’ mean analyte levels, suggesting that relative levels are important. An integrative analysis of lung and PB analytes showed that BCo has a distinct signature from shock that can be used as a biomarker to distinguish the two. Significant modules also generated hypotheses about analytes that could be targeted by novel therapies.

MicroRNA expression profiling of sputum for the detection of early and locally advanced non-small-cell lung cancer: a prospective case-control study.

Non-small-cell lung cancer (nsclc) is associated with very poor overall survival because 70% of patients present with locally advanced or metastatic disease at the time of diagnosis. Micrornas (mirnas) are a class of short, noncoding rna molecules whose presence in samples of biologic fluids such as sputum has demonstrated promise as a potential means of detecting nsclc. We investigated the stage-specific nsclc detection potential of an efficient panel of 3 mirnas (mir-21, mir-210, mir-372) using a single sputum sample. A single spontaneously expectorated sputum sample was prospectively collected from 21 early nsclc (≤stage ii) patients, 22 advanced nsclc (≥stage iii) patients, and 10 control subjects. Mirna expression profiles were determined by quantitative real-time polymerase chain reaction and were analyzed by unsupervised hierarchical cluster analysis. Mean tumour size (±95% confidence interval) in the early and advanced nsclc patients was 3.3 cm ± 0.9 cm and 4.8 cm ± 0.7 cm respectively. Adenocarcinoma constituted 61.9% of the early and 45.5% of the advanced nsclc cases respectively. In comparing the early nsclc group with the control group, the mirna panel yielded a diagnostic sensitivity of 67% and a specificity of 90.0%. For the advanced nsclc group, the mirna panel detected nsclc with a sensitivity and specificity of 64% and 100% respectively. A sputum mir-21, mir-210, and mir-372 expression profile might provide a sensitive and highly specific means for detecting nsclc. Sputum mirna analysis demonstrates promise as a potential complementary screening tool.

A selective screening platform reveals unique global expression patterns of microRNAs in a cohort of human soft-tissue sarcomas

Sarcomas are malignant heterogenous tumors of mesenchymal derivation. Emerging data suggest that miRNA might have a causal role in sarcomagenesis. Herein, we used a selective miRNA screening platform to study the comparative global miRNA expression signatures in a cohort of human sarcomas with the caveat that comparisons between tumor and non-tumor cells were performed from the same patients using formalin-fixed paraffin-embedded tissue. Five histologic types were examined that included: myxoid liposarcoma, well-differentiated liposarcoma, dedifferentiated liposarcoma, pleomorphic rhabdomyosarcoma, and synovial sarcoma. In addition, soft-tissue lipomas and normal fat were included as a separate set of controls for the lipogenic tumors. Clustering analysis showed a distinct global difference in expression patterns between the normal and sarcoma tissues. Expression signatures in an unsupervised hierarchical clustering analysis revealed tight clustering in synovial and myxoid liposarcomas, and the least clustering was observed in the pleomorphic rhabdomyosarcoma subtype. MiR-145 showed underexpression in pleomorphic rhabdomyosarcoma, well-differentiated liposarcoma, and synovial sarcoma. Unexpectedly, we found that a set of muscle-specific microRNAs (miRNAs; myomiRs): miR-133, miR-1, and miR-206 was significantly underexpressed in well-differentiated liposarcoma and synovial sarcoma, suggesting that they may function as tumor suppressors as described in muscle-relevant rhabdomyosarcomas. In addition, a tight linear progression of miRNA expression was identified from normal fat to dedifferentiated liposarcoma. These results suggest that miRNA expression profiles could elucidate classes of miRNAs that may elicit tumor-relevant activities in specific sarcoma subtypes.

Abstract PR01: A novel MLL-AF4 in vivo model phenocopies t(4;11) pro-B ALL and reveals a lymphoid lineage bias of the fusion protein in human cells

Abstract The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is fused by reciprocal translocation to a diverse group of partner genes in both acute myeloid and acute lymphoid leukemia (AML, ALL). As a result of the t(4;11)(q21;q23), MLL fuses to AF4 (AFF1), one of the most common MLL fusion partner proteins. Unlike several other MLL fusions that are frequently found in both AML and ALL, MLL-AF4 is almost exclusively associated with B-cell ALL with a pro-B immunophenotype. It accounts for 10-15% of ALL cases and confers a poor prognosis. Although many MLL-fusion leukemia models have been established, it has not been possible to generate a t(4;11) pro-B leukemia model that accurately recapitulates the human disease, hampering research into the molecular mechanisms that underlie the development of this subtype of leukemia. Here we present a faithful model of t(4;11) pro-B-ALL that fully recapitulates the immunophenotypic and molecular aspects of the human disease. Human hematopoietic CD34+ cells transduced with a modified MLL-AF4 fusion gene successfully initiate ALL in NSG mice with high penetrance. The leukemia cells have a CD19+CD34+ pro-B immunophenotype and are CD10(-), a common feature in MLL-AF4 patients. The effect of the oncogene is species-specific, as retroviral transduction and transplantation of murine hematopoietic cells results in only AML using either lymphoid or myeloid conditions. An MLL-AF4 specific gene signature derived from patients is significantly enriched in our model cells, as shown by RNA-Seq, and the model samples group tightly with MLL-AF4 patients, even when compared with other MLL-fusion leukemia samples in unsupervised hierarchical clustering analysis. Interestingly, using gene profiles of normal pro-B and pre-B cells as reference, our MLL-AF4 leukemia cells show strong enrichment for pro-B genes, while instead, pre-B but not pro-B genes are overrepresented in our MLL-AF9 B-ALL leukemia cells. This differential developmental stage blockage of MLL-fusions is also reflected in patient samples. More strikingly, in accordance with the distinct lineage bias of MLL-fusions observed in the clinic, human cells expressing MLL-AF4 have a strong predilection for the lymphoid lineage and a demonstrated resistance to lineage redirection in response to myeloid signals compared to human cells expressing MLL-AF9. This difference in lineage predisposition of MLL-AF4 compared to MLL-AF9 can be attributed to differential effects on lineage-specific gene expression. Under myeloid-priming conditions, phenotypically (CD33+CD19-) and morphologically myeloid MLL-AF4 cells are still able to initiate pro-B ALL in immunodeficient mice, while only AML is generated by MLL-AF9 myeloid cells. Accordingly, an active lymphoid molecular program with lower expression of critical myeloid genes is observed in MLL-AF4 myeloid cells compared to MLL-AF9 myeloid cells. Interestingly, we find that the polycomb gene BMI1, which was reported to be critical to prevent lymphoid priming in normal hematopoiesis, is expressed at significantly lower levels in MLL-AF4 than in MLL-AF9 myeloid cells. Reintroduction of BMI1 into MLL-AF4 cells enables AML generation with variable penetrance, while control vector transduced cells always result in B-ALL. Our results demonstrate that lineage fate in response to MLL-fusion protein expression involves a complex interplay of oncogene, intra- and extra-cellular microenvironmental cues. In addition, our data clearly demonstrate the species specificity associated with the t(4;11). This in vivo model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis. This abstract is also presented as Poster A06. Citation Format: Shan Lin, Roger T. Luo, Mark Wunderlich, Joseph J. Kaberlein, Ahmad Rayes, John Anastasi, Maureen M. O'Brien, James C. Mulloy, Michael J. Thirman. A novel MLL-AF4 in vivo model phenocopies t(4;11) pro-B ALL and reveals a lymphoid lineage bias of the fusion protein in human cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR01.

Similar protein expression profiles of ovarian and endometrial high-grade serous carcinomas.

Background:Ovarian and endometrial high-grade serous carcinomas (HGSCs) have similar clinical and pathological characteristics; however, exhaustive protein expression profiling of these cancers has yet to be reported.Methods:We performed protein expression profiling on 14 cases of HGSCs (7 ovarian and 7 endometrial) and 18 endometrioid carcinomas (9 ovarian and 9 endometrial) using iTRAQ-based exhaustive and quantitative protein analysis.Results:We identified 828 tumour-expressed proteins and evaluated the statistical similarity of protein expression profiles between ovarian and endometrial HGSCs using unsupervised hierarchical cluster analysis (P<0.01). Using 45 statistically highly expressed proteins in HGSCs, protein ontology analysis detected two enriched terms and proteins composing each term: IMP2 and MCM2. Immunohistochemical analyses confirmed the higher expression of IMP2 and MCM2 in ovarian and endometrial HGSCs as well as in tubal and peritoneal HGSCs than in endometrioid carcinomas (P<0.01). The knockdown of either IMP2 or MCM2 by siRNA interference significantly decreased the proliferation rate of ovarian HGSC cell line (P<0.01).Conclusions:We demonstrated the statistical similarity of the protein expression profiles of ovarian and endometrial HGSC beyond the organs. We suggest that increased IMP2 and MCM2 expression may underlie some of the rapid HGSC growth observed clinically.

Gene expression profiling of prostate tissue based on variable expression of 5-alpha reductase 2.

143 Background: Androgen signaling plays a central role in the pathophysiology of both benign and malignant prostatic disease. We previously showed that some men carry epigenetic modifications of the 5-alpha reductase (AR) promoter that modulate gene expression, raising the hypothesis that there may be molecular heterogeneity underlying the variability in clinical response to 5-AR inhibitors for management of benign and malignant prostatic diseases. In this study, we performed whole-genome expression profiling of prostatic tissue based on variable expression of 5-AR2 to determine whether molecular subtypes of clinical relevance could be identified. Methods: Prostatic tissue specimens were obtained from 22 patients with symptomatic BPH undergoing prostate debulking surgery. 5-AR2 protein expression was measured by immunohistochemistry, and 5-AR2 promoter methylation determined by PCR. RNA was extracted from each specimen and whole-genome expression profiling was performed using Illumina Human BeadChip Arrays. Gene expression data was analyzed by unsupervised hierarchical clustering and Gene Set Enrichment Analysis. Results: Unsupervised hierarchical clustering identified two subtypes of patients with distinct gene expression signatures. Among the most differentially expressed genes were several notable androgen-regulated genes, including TMPRSS2, NKX3-1, AZGP1, KLK3 (encoding the PSA protein) and multiple other members of the kallikrein family of serine proteases (all P&lt; 0.001, FDR &lt; 0.02). Multiple gene sets composed of androgen-regulated genes and androgen receptor target genes were found to be significantly enriched in one of the two subtypes. Interestingly, patients from this Androgen-Up subtype were also noted to have a higher frequency of 5-AR2 promoter methylation (though not statistically significant). Conclusions: Our findings suggest that there are distinct molecular subtypes among prostatic tissues with variable expression of 5-AR2, driven by differences in androgen gene regulation. These differences may partially explain the variable response to 5-AR inhibitors and thereby inform clinical strategies for devising therapies for benign and malignant prostatic disease.

Lymphoid Lineage Preference of MLL-AF4 Is Revealed in a Species-Specific Model

The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is fused by reciprocal translocation to a diverse group of partner genes that drive both acute myeloid and acute lymphoid leukemia (AML and ALL). As a result of the t(4;11)(q21;q23), MLL fuses to AF4 (also referred to as AFF1), one of the most common MLL fusion partner proteins. Unlike several other MLL fusions that are frequently identified in AML, for example MLL-AF9 caused by t(9;11)(p22;q23), MLL-AF4 is almost exclusively associated with B-cell ALL with a pro-B immunophenotype. It is the most frequent MLL fusion in ALL and accounts for 10-15% of ALL cases. Patients with t(4;11) have a poor prognosis compared with other cytogenetically defined subsets. Although many MLL-fusion leukemia models have successfully been established, it has not been possible to generate a t(4;11) pro-B leukemia model that accurately recapitulates the human disease, hampering research into the molecular mechanisms that underlie the development of this subtype of leukemia. Here we present a faithful human cell based model of t(4;11) pro-B-ALL that fully recapitulates the immunophenotypic and molecular aspects of the human disease. Transduced with a modified MLL-AF4 fusion gene, human hematopoietic CD34+ cells successfully initiate ALL in a xenograft system with high penetrance. The leukemia cells have a CD19+CD34+ pro-B immunophenotype and are CD10(-), a common feature in MLL-AF4 patients. The effect of the oncogene is species-specific, as retroviral transduction and transplantation of murine hematopoietic cells with MLL-AF4 results in only AML using either lymphoid or myeloid conditions. An MLL-AF4 specific gene signature derived from patients is significantly enriched in our model cells, as shown by RNAseq, and the model samples group tightly with MLL-AF4 patients, even when compared with other MLL-fusions in unsupervised hierarchical clustering analysis. Interestingly, using gene profiles of normal pro-B and pre-B cells as reference, our MLL-AF4 leukemia cells show strong enrichment for pro-B genes, while instead, pre-B but not pro-B genes are overrepresented in our MLL-AF9 B-ALL leukemia cells. This differential developmental stage blockage of MLL-fusions is also reflected in patient samples. More strikingly, in accordance with the distinct lineage bias of MLL-fusions observed in the clinic, human cells expressing MLL-AF4 have a strong predilection for the lymphoid lineage and a demonstrated resistance to reprogramming in response to myeloid signals compared to human cells expressing MLL-AF9. This difference in lineage predisposition of MLL-AF4 compared to MLL-AF9 can be attributed to differential effects on lineage-specific gene expression. Under myeloid-priming conditions, phenotypically (CD33+CD19-) and morphologically myeloid MLL-AF4 cells are still able to initiate pro-B ALL in immunodeficient mice, while only AML is generated by MLL-AF9 myeloid cells. Accordingly, an active lymphoid molecular program with lower expression of critical myeloid genes is observed in MLL-AF4 myeloid cells compared to MLL-AF9 myeloid cells. Interestingly, we find that the polycomb gene BMI1, which was reported to be critical to prevent lymphoid priming in normal hematopoiesis, is expressed at significantly lower levels in MLL-AF4 than in MLL-AF9 myeloid cells. Strikingly, this decreased BMI1 expression is evident in primary B-ALL patient samples as well, with MLL-AF9 B-ALL samples demonstrating increased BMI1 relative to MLL-AF4. Reintroduction of BMI1 into MLL-AF4 cells enables AML generation with variable penetrance, while control vector transduced cells always result in B-ALL. Our results demonstrate that lineage fate in response to MLL-fusion protein expression involves a complex interplay of oncogene, intra- and extra-cellular microenvironmental cues. In addition, our data clearly demonstrate the species specificity associated with the t(4;11) oncogene and highlight the limitations of using murine cells in human disease modeling. The model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis. DisclosuresThirman:AbbVie: Research Funding; Pharmacyclics: Research Funding; Gilead: Research Funding.

Abstract 18802: Transcriptome Profiles of Chronically Closed or Cycling Aortic Valve Leaflets are Highly Similar Compared to Chronically Open Valves: Distention Drives Leaflet Homeostasis

Introduction: Throughout the valve cycle mechanical forces of multiple types and intensities are exerted on the leaflets, sinus and annulus. The mechanisms through which these forces regulate the homeostatic maintenance of valve leaflet architecture are still poorly understood. Ventricular mechanical support with a continuous flow device results in chronic closure of the outflow valve. Whether chronic valve closure affects valve leaflet homeostasis is a significant but unsettled clinical concern. Hypothesis: We asked if chronic closure of the aortic valve (AV) impairs leaflet homeostasis by comparing the transcriptome profiles of AV cultured under different mechanical force conditions: Normally cycling (Flow); Not cycling but open (Static); or Not cycling but closed (Static-Closed, SC). Methods: Ex vivo culture of native rat aortic valves, (8 valves per bioreactor, 4 valves per condition) was conducted in a flow bioreactor for seven days at 37C in endothelial cell culture media under conditions approximating the normal stroke volume of the rat heart. In each of 4 independent experiments, flow-induced valve cycling (Flow) was compared with either Static or SC condition in paired groups. After culture, leaflets were dissected from 3 valves/condition and pooled, mRNA was extracted and expression was evaluated using whole genome microarrays. Results: Statistical-based unsupervised hierarchical clustering analysis of the leaflet transcriptome profiles revealed only two distinct patterns of leaflet gene expression between the three groups of AV. Flow and SC groups had nearly identical profiles whereas the Static valve group was distinctly different (p&lt;0.05). Conclusions: These results reveal that the stretching of valve leaflets by a filling volume inducing full coaptation is apparently sufficient to largely preserve their architecture and phenotypic gene expression in the seven day culture period tested. In contrast, the absence of valve closure produces a markedly different pattern of gene expression. Therefore, the absence of flow through the closed AV valve does not appear to be inherently destructive compared with conditions in which the valve remains open.

MPTH-26MOLECULAR REFINEMENT OF PEDIATRIC POSTERIOR FOSSA EPENDYMOMA

We have recently identified nine distinct molecular subgroups of ependymoma across all age groups, three in each major anatomical compartment of the CNS: spinal (SP), posterior fossa (PF), and supratentorial (ST). These nine molecular subgroups are genetically, epigenetically, transcriptionally, demographically, and clinically distinct. Pediatric intracranial ependymomas were either affiliated with one of two supratentorial subgroups characterized by RELA (ST-EPN-RELA) or YAP1 (ST-EPN-YAP1) fusion genes, or with the PF-EPN-A subgroup in the hindbrain. Observing distinct outcomes among children with PF-EPN-A tumors, the largest molecular subgroup, we tested the hypothesis that further molecular diversity exists among these tumors. The genome-wide DNA methylation profiles of 575 pediatric PF ependymomas were analyzed by unsupervised consensus hierarchical clustering and principal component analysis, which identified three distinct molecular subtypes of PF-EPN-A tumors: PFA-1, PFA-2, and PFA-3. PFA-1 and PFA-2 showed different chromosomal copy number alterations and comprised tumors almost exclusively from infants, while PFA-3 ependymomas were generally from older children. The PFA-3 subtype was significantly enriched for gain of 1q. Distinct developmental gene expression profiles and radiological characteristics on pre-operative MR imaging suggested separate anatomic origins for the infant subtypes, PFA-1 and PFA-2. Survival analyses identified PFA-3 as having the poorest outcome. Multivariate analysis revealed molecular subgroup, grade of resection and gain of 1q as independent prognostic markers among PF-EPN-A tumors. We conclude that further molecular refinement of pediatric PF-EPN-A tumors, with their division into three distinct molecular subtypes, has clinical utility and the potential for enhanced risk assessment or therapeutic stratification.