Imprints and DPPA3 are bypassed during pluripotency- and differentiation-coupled methylation reprogramming in testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) share germline ancestry but diverge phenotypically and clinically as seminoma (SE) and nonseminoma (NSE), the latter including the pluripotent embryonal carcinoma (EC) and its differentiated derivatives, teratoma (TE), yolk sac tumor (YST), and choriocarcinoma. Epigenomes from TGCTs may illuminate reprogramming in both normal development and testicular tumorigenesis. Herein we investigate pure-histological forms of 130 TGCTs for conserved and subtype-specific DNA methylation, including analysis of relatedness to pluripotent stem cell (ESC, iPSC), primordial germ cell (PGC), and differentiated somatic references. Most generally, TGCTs conserve PGC-lineage erasure of maternal and paternal genomic imprints and DPPA3 (also known as STELLA); however, like ESCs, TGCTs show focal recurrent imprinted domain hypermethylation. In this setting of shared physiologic erasure, NSEs harbor a malignancy-associated hypermethylation core, akin to that of a diverse cancer compendium. Beyond these concordances, we found subtype epigenetic homology with pluripotent versus differentiated states. ECs demonstrate a striking convergence of both CpG and CpH (non-CpG) methylation with pluripotent states; the pluripotential methyl-CpH signature crosses species boundaries and is distinct from neuronal methyl-CpH. EC differentiation to TE and YST entails reprogramming toward the somatic state, with loss of methyl-CpH but de novo methylation of pluripotency loci such as NANOG Extreme methyl-depletion among SE reflects the PGC methylation nadir. Adjacent to TGCTs, benign testis methylation profiles are determined by spermatogenetic proficiency measured by Johnsen score. In sum, TGCTs share collective entrapment in a PGC-like state of genomic-imprint and DPPA3 erasure, recurrent hypermethylation of cancer-associated targets, and subtype-dependent pluripotent, germline, or somatic methylation.

ONCOGENE PANEL SEQUENCING ANALYSIS IDENTIFIES CANDIDATE ACTIONABLE GENES IN ADVANCED WELL-DIFFERENTIATED GASTROENTEROPANCREATIC NEUROENDOCRINE TUMORS.

Objective: To report the rate of candidate actionable somatic mutations in patients with locally advanced and metastatic gastro-enteropancreatic (GEP) neuroendocrine tumors (NET) and of other genetic alterations that may be associated with tumorigenesis. Methods: A phase II mutation targeted therapy trial was conducted in patients with advanced well-differentiated G1/G2 GEP-NET. Mutations found in the mTOR pathway-associated genes led to treatment with the mTOR inhibitor everolimus, and were defined as actionable. Tumor deoxyribonucleic acid (DNA) from GEP-NET were sequenced and compared with germline DNA, using the OncoVAR-NET assay, designed for hybrid capture sequencing of 500 tumor suppressor genes and oncogenes. Somatic variants were called and copy-number (CN) variant analysis was performed. Results: Thirty patients (14 small-intestine, 8 pancreatic, 3 unknown primary NET, and 5 of other primary sites) harbored 37 lesions (4 patients had DNA of multiple lesions sequenced). Only 2 patients with sporadic NET (n = 26) had an actionable mutation leading to treatment with everolimus. Driver somatic mutations were detected in 18 of 30 patients (21/37 lesions sequenced). In the remaining samples without a driver mutation, CN alterations were found in 11/16 tumors (10/12 patients), including CN loss of chromosome (Chr) 18 (P<.05), CN gain of Chr 5, and loss of Chr 13. CN losses in Chr 18 were more common in patients without driver mutations detected. Pronounced genetic heterogeneity was detected in patients with multiple lesions sequenced. Conclusion: Genome-wide DNA sequencing may identify candidate actionable genes and lead to the identification of novel target genes for advanced well-differentiated GEP-NET. Abbreviations: Chr = chromosome; CN = copy number; DNA = deoxyribonucleic acid; FDA = Food and Drug Administration; GEP = gastro-enteropancreatic; MEN-1 = multiple endocrine neoplasia syndrome type 1; mTOR = mammalian target of rapamycin; NET = neuroendocrine tumor; PFS = progression-free survival; PNET = pancreatic neuroendocrine tumors; SINET = small-intestine neuroendocrine tumor.

Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3-FOXO1 or PAX7-FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from 'aberrant' hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3-FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3-FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2'-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.

Genome-wide DNA methylation profiling in chronic lymphocytic leukaemia.

Background: DNA methylation aberrations are widespread among the malignant B lymphocytes of patients with chronic lymphocytic leukaemia (CLL), suggesting that DNA methylation might contribute to the pathogenesis of CLL. Aim: We aimed to explore the differentially methylated positions (DMPs) associated with CLL and screen the differentially methylated and expressed genes (DMEGs) by combining public databases. We aimed to observe the direction of each DMEG in CLL based on the DMPs in the promoter and the body region respectively to narrow down DMEGs. We also aimed to explore the methylation heterogeneity of CLL subgroups and the effect of B cells maturation on CLL. Methods: In this population-based case control study, we reported a genome-wide DNA methylation association study using the Infinium HumanMethylation450 BeadChip, profiling the DNA methylation of CD19+ B Cells from 48 CLL cases and 28 healthy controls. By integrating methylation data and expression data from public databases, gene sets were jointly screened, and then the relationship between methylation sites in promoter and body region and expression of each gene was explored. In addition, support vector machine (SVM) classification algorithm was used to identify subgroups of CLL cases based on methylation pattern, and the effect of B-cell differentiation related methylation sites on CLL-related sites was observed. Results: We identified 34,797 DMPs related to CLL across the genome, most of which were hypomethylated; the majority were located in gene body regions. By combining these DMPs with published DNA methylation and RNA sequencing data, we detected 26,244 replicated DMPs associated with 1,130 genes whose expression were significantly different in CLL cases. Among these DMEGs, nine low expressed DMEGs were selected with hypermethylated in promoter and hypomethylated in body region, and 83 high expressed DMEGs were selected with both hypomethylated in promoter and body region. The 48 CLL cases were divided into 3 subgroups based on methylation site by SVM algorithm. Over 92% of CpGs associated with B cell subtypes were found in CLL-related DMPs. Conclusion: The DNA methylation pattern was altered across the genome in CLL patients. The methylation of ZAP70, FMOD, and ADAMTS17 was significantly different between CLL cases and controls. Further studies are warranted to confirm our findings and identify the underlying mechanisms through which these methylation markers are associated with CLL.

Author Correction: Recurrent PTPRT/JAK2 mutations in lung adenocarcinoma among African Americans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs in vivo. Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.

Recurrent PTPRT/JAK2 mutations in lung adenocarcinoma among African Americans.

Reducing or eliminating persistent disparities in lung cancer incidence and survival has been challenging because our current understanding of lung cancer biology is derived primarily from populations of European descent. Here we show results from a targeted sequencing panel using NCI-MD Case Control Study patient samples and reveal a significantly higher prevalence of PTPRT and JAK2 mutations in lung adenocarcinomas among African Americans compared with European Americans. This increase in mutation frequency was validated with independent WES data from the NCI-MD Case Control Study and TCGA. We find that patients carrying these mutations have a concomitant increase in IL-6/STAT3 signaling and miR-21 expression. Together, these findings suggest the identification of these potentially actionable mutations could have clinical significance for targeted therapy and the enrollment of minority populations in clinical trials.

Dental case management: increasing access to oral health care for families and children with low incomes.

BACKGROUND: Medicaid beneficiaries have lower rates of dental visits and higher rates of dental disease compared with the rest of the population. Beneficiaries ascribe their low use of services to difficulties finding dentists who treat patients with Medicaid. Dentists cite low reimbursement rates, excessive paperwork, and patients' not keeping appointments and poor oral health literacy as reasons for not accepting patients with Medicaid. The authors pilot-tested the effectiveness of a dental case management program (DCMP) in increasing dentists' participation in Medicaid and Medicaid beneficiaries' use of services. METHODS: A dental case manager recruits dentists to participate in the Medicaid program, arranges training in billing procedures, resolves billing and payment problems, educates clients about the use of dental services and keeping appointments, links clients to dental offices, identifies potential barriers to care and helps clients obtain transportation to appointments. The authors evaluated the levels of participation of dentists in the DCMP in Medicaid and Medicaid beneficiaries' use of services. RESULTS: Dentists accepting new Medicaid patients increased from two to 28, with 145 dental visits a month provided to Medicaid beneficiaries. The percentage of Medicaid beneficiaries receiving dental services increased from 9 to 41 percent after the DCMP was implemented. CONCLUSIONS: The authors found that the DCMP was effective in increasing Medicaid beneficiaries' use of services, increasing dentists' participation in Medicaid, minimizing administrative burdens related to Medicaid participation, and increasing oral health literacy and treatment compliance among clients with low incomes.

BP1 transcriptionally activates bcl-2 and inhibits TNFalpha-induced cell death in MCF7 breast cancer cells.

INTRODUCTION: We have previously shown that the Beta Protein 1 (BP1) homeodomain protein is expressed in 81% of invasive ductal breast carcinomas, and that increased BP1 expression correlates with tumor progression. The purpose of our current investigation was to determine whether elevated levels of BP1 in breast cancer cells are associated with increased cell survival. METHODS: Effects on cell viability and apoptosis of MCF7 cells stably overexpressing BP1 were determined using MTT and Annexin V assays, and through examination of caspase activation. TNFalpha was used to induce apoptosis. The potential regulation of apoptosis-associated genes by BP1 was studied using real-time PCR and western blot analyses. Electrophoretic mobility shift assays, site-directed mutagenesis, and transient assays were performed to specifically characterize the interaction of BP1 with the promoter of the bcl-2 gene. RESULTS: Stable overexpression of BP1 led to inhibition of apoptosis in MCF7 breast cancer cells challenged with TNFalpha. Increased BP1 resulted in reduced processing and activation of caspase-7, caspase-8, and caspase-9, and inactivation of the caspase substrate Poly(ADP-Ribose) Polymerase (PARP). Increased levels of full-length PARP and a decrease in procaspase-8 were also associated with BP1 overexpression. The bcl-2 gene is a direct target of BP1 since: (i) BP1 protein bound to a consensus binding sequence upstream of the bcl-2 P1 promoter in vitro. (ii) MCF7 cells overexpressing BP1 showed increased levels of bcl-2 mRNA and protein. (iii) Transient assays indicated that increased bcl-2 promoter activity is due to direct binding and modulation by BP1 protein. BP1 expression also prevented TNFalpha-mediated downregulation of bcl-2 mRNA and protein. CONCLUSION: These findings suggest mechanisms by which increased BP1 may impart a survival advantage to breast cancer cells, which could lead to increased resistance to therapeutic agents in patients.

BP1, a homeodomain-containing isoform of DLX4, represses the beta-globin gene.

In earlier studies we identified a putative repressor of the human beta-globin gene, termed beta protein 1 (BP1), which binds to two silencer DNA sequences upstream of the adult human beta-globin gene and to a negative control region upstream of the adult delta-globin gene. Further studies demonstrated an inverse correlation between the binding affinity of the BP1 protein for the distal beta-globin silencer sequence and the severity of sickle cell anemia, suggesting a possible role for BP1 in determining the production of hemoglobin S. We have now cloned a cDNA expressing the BP1 protein. Sequencing revealed that BP1 is a member of the homeobox gene family and belongs to the subfamily called Distal-less (DLX), genes important in early development. Further analysis showed that BP1 is an isoform of DLX4. BP1 protein has repressor function towards the beta-globin promoter, acting through the two beta-globin DNA silencers, demonstrated in transient transfection assays. Strong BP1 expression is restricted to placenta and kidney tissue, with no expression in 48 other human tissues. BP1 exhibits regulated expression in the human erythroid cell line MB-02, where its expression decreases upon induction of the beta-globin gene. BP1 is thus the first member of the DLX family with known DNA binding sites and a function in globin gene regulation.

The NCI-60 Methylome and Its Integration into CellMiner.

A unique resource for systems pharmacology and genomic studies is the NCI-60 cancer cell line panel, which provides data for the largest publicly available library of compounds with cytotoxic activity (∼21,000 compounds), including 108 FDA-approved and 70 clinical trial drugs as well as genomic data, including whole-exome sequencing, gene and miRNA transcripts, DNA copy number, and protein levels. Here, we provide the first readily usable genome-wide DNA methylation database for the NCI-60, including 485,577 probes from the Infinium HumanMethylation450k BeadChip array, which yielded DNA methylation signatures for 17,559 genes integrated into our open access CellMiner version 2.0 (https://discover.nci.nih.gov/cellminer). Among new insights, transcript versus DNA methylation correlations revealed the epithelial/mesenchymal gene functional category as being influenced most heavily by methylation. DNA methylation and copy number integration with transcript levels yielded an assessment of their relative influence for 15,798 genes, including tumor suppressor, mitochondrial, and mismatch repair genes. Four forms of molecular data were combined, providing rationale for microsatellite instability for 8 of the 9 cell lines in which it occurred. Individual cell line analyses showed global methylome patterns with overall methylation levels ranging from 17% to 84%. A six-gene model, including PARP1, EP300, KDM5C, SMARCB1, and UHRF1 matched this pattern. In addition, promoter methylation of two translationally relevant genes, Schlafen 11 (SLFN11) and methylguanine methyltransferase (MGMT), served as indicators of therapeutic resistance or susceptibility, respectively. Overall, our database provides a resource of pharmacologic data that can reinforce known therapeutic strategies and identify novel drugs and drug targets across multiple cancer types. Cancer Res; 77(3); 601-12. ©2016 AACR.

ASXL3 Is a Novel Pluripotency Factor in Human Respiratory Epithelial Cells and a Potential Therapeutic Target in Small Cell Lung Cancer.

In this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSCs (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAECs. Of particular novelty, we identified the PRC2-associated protein, ASXL3, which was markedly upregulated in Lu-iPSCs and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity, and teratoma formation by Lu-iPSCs, and diminished clonogenicity and malignant growth of SCLC cells in vivo Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and highlight ASXL3 as a novel candidate target for SCLC therapy. Cancer Res; 77(22); 6267-81. ©2017 AACR.

Common Molecular Subtypes Among Asian Hepatocellular Carcinoma and Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are clinically disparate primary liver cancers with etiological and biological heterogeneity. We identified common molecular subtypes linked to similar prognosis among 199 Thai ICC and HCC patients through systems integration of genomics, transcriptomics, and metabolomics. While ICC and HCC share recurrently mutated genes, including TP53, ARID1A, and ARID2, mitotic checkpoint anomalies distinguish the C1 subtype with key drivers PLK1 and ECT2, whereas the C2 subtype is linked to obesity, T cell infiltration, and bile acid metabolism. These molecular subtypes are found in 582 Asian, but less so in 265 Caucasian patients. Thus, Asian ICC and HCC, while clinically treated as separate entities, share common molecular subtypes with similar actionable drivers to improve precision therapy.

Long-term stability of total RNA in RNAstable® as evaluated by expression microarray.

Storage of labile RNA in laboratories is accomplished through ultra-low freezing of the nucleic acids. This however requires expensive freezers, convenient storage, reliable electrical power, and increased shipping costs, thereby making it a less viable option. Biomatrica (San Diego, CA) has created RNAstable(®), a stabilization reagent that is used to store RNA in a dehydrated state at room temperature (RT) and protects the RNA from degradation. Our objective was to investigate the sequence integrity and suitability of RNA when stored in RNAstable at extended time periods and at varying temperatures through use of Illumina and Agilent RNA expression microarrays. We observed in Bioanalyzer electropherograms that total RNA extracted from 293 cells stored at RT in RNAstable for 4.5 and 11.5 months is similar in quality to RNA stored at -80°C. Illumina mRNA expression array QC metrics and gene expression patterns from RNAstable-protected RNA, in contrast to RNA stored without RNAstable, correlated well with those of freezer controls. Significantly, when RNA was stored in RNAstable at 45°C for 4.5 months, equivalent to 22 months RT storage, RNA quality, microarray probe signal intensities, probe detection rates, and expression profiles remained similar between RNAstable-protected RNA at RT and the -80°C controls. At 10.5 months, miRNA levels were compared among the storage conditions using miRNA expression arrays. Here too we found strong concordance between miRNA expression patterns when total RNA was stored in RNAstable or at -80°C. Further, Bioanalyzer electrophoresis of RNAstable-protected samples stored at RT for a relative total of 33 months or 50.5 months showed comparable integrity scores to those of -80°C controls. We conclude that use of RNAstable holds promise as an effective stabilization reagent for total RNA and should be useful in situations where shipping and storage options are limited resources.

An Integrated Prognostic Classifier for Stage I Lung Adenocarcinoma Based on mRNA, microRNA, and DNA Methylation Biomarkers.

INTRODUCTION: Up to 30% stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers. METHODS: Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts. RESULTS: Promoters of genes marked by polycomb in embryonic stem cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in stage I tumors (p < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (hazard ratio [HR], 2.6; p = 0.02) and recurrence-free survival (HR, 3.0; p = 0.01), and identified high-risk patients in stratified analysis of stages IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1α, and DLC1), miR-21 expression, and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; p = 0.002; HR, 2.3; p = 0.01; and HR, 2.4; p = 0.005, respectively), and when combined, identified high-risk, therapy naive, stage I patients (HR, 10.2; p = 3 × 10). All associations were confirmed in two independently collected cohorts. CONCLUSION: A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of stage I lung cancer patients at high risk of recurrence.

Correlation of expression of BP1, a homeobox gene, with estrogen receptor status in breast cancer.

BACKGROUND: BP1 is a novel homeobox gene cloned in our laboratory. Our previous studies in leukemia demonstrated that BP1 has oncogenic properties, including as a modulator of cell survival. Here BP1 expression was examined in breast cancer, and the relationship between BP1 expression and clinicopathological data was determined. METHODS: Total RNA was isolated from cell lines, tumors, and matched normal adjacent tissue or tissue from autopsy. Reverse transcription polymerase chain reaction was performed to evaluate BP1 expression. Statistical analysis was accomplished with SAS. RESULTS: Analysis of 46 invasive ductal breast tumors demonstrated BP1 expression in 80% of them, compared with a lack of expression in six normal breast tissues and low-level expression in one normal breast tissue. Remarkably, 100% of tumors that were negative for the estrogen receptor (ER) were BP1-positive, whereas 73% of ER-positive tumors expressed BP1 (P = 0.03). BP1 expression was also associated with race: 89% of the tumors of African American women were BP1-positive, whereas 57% of those from Caucasian women expressed BP1 (P = 0.04). However, there was no significant difference in BP1 expression between grades I, II, and III tumors. Interestingly, BP1 mRNA expression was correlated with the ability of malignant cell lines to cause breast cancer in mice. CONCLUSION: Because BP1 is expressed abnormally in breast tumors, it could provide a useful target for therapy, particularly in patients with ER-negative tumors. The frequent expression of BP1 in all tumor grades suggests that activation of BP1 is an early event.

Micro-environment causes reversible changes in DNA methylation and mRNA expression profiles in patient-derived glioma stem cells.

In vitro and in vivo models are widely used in cancer research. Characterizing the similarities and differences between a patient's tumor and corresponding in vitro and in vivo models is important for understanding the potential clinical relevance of experimental data generated with these models. Towards this aim, we analyzed the genomic aberrations, DNA methylation and transcriptome profiles of five parental tumors and their matched in vitro isolated glioma stem cell (GSC) lines and xenografts generated from these same GSCs using high-resolution platforms. We observed that the methylation and transcriptome profiles of in vitro GSCs were significantly different from their corresponding xenografts, which were actually more similar to their original parental tumors. This points to the potentially critical role of the brain microenvironment in influencing methylation and transcriptional patterns of GSCs. Consistent with this possibility, ex vivo cultured GSCs isolated from xenografts showed a tendency to return to their initial in vitro states even after a short time in culture, supporting a rapid dynamic adaptation to the in vitro microenvironment. These results show that methylation and transcriptome profiles are highly dependent on the microenvironment and growth in orthotopic sites partially reverse the changes caused by in vitro culturing.

Genome-wide methylation patterns in papillary thyroid cancer are distinct based on histological subtype and tumor genotype.

CONTEXT: Aberrant DNA methylation is known to be a major factor in oncogenesis and cancer progression, but effects of methylation in papillary thyroid cancer (PTC) are not well defined. OBJECTIVE: The objective of the study was to identify altered methylation patterns, which may be associated with PTC disease behavior. DESIGN: This study was a genome-wide methylation analysis of PTC. SETTING: The study was conducted at the National Institutes of Health Clinical Center. PATIENTS: PTC tissue from 51 patients were analyzed and compared with normal thyroid tissue from seven patients. INTERVENTIONS: CpG methylation status was assessed using advanced genome-wide methylation bead chips. OUTCOME MEASURES: Altered methylation patterns in PTC were analyzed by stage, recurrence, histological subtype of tumor, and tumor genotype. RESULTS: PTC is globally hypomethylated compared with normal thyroid with 2837 differentially methylated CpG sites. The follicular variant of PTC demonstrated less differential methylation with only 569 differentially methylated CpG sites. Tumors with mutations in BRAF, RET/PTC, and RAS demonstrated a 3.6-fold increase in the number of differentially methylated sites compared with wild-type tumors. The differentially methylated genes were associated with oncological pathways including cellular movement, growth, and proliferation. CONCLUSION: PTC is epigenetically distinct from the follicular variant of PTC and by gene mutation status (BRAF, RET/PTC, and RAS).

Recurrent epimutation of SDHC in gastrointestinal stromal tumors.

Succinate dehydrogenase (SDH) is a conserved effector of cellular metabolism and energy production, and loss of SDH function is a driver mechanism in several cancers. SDH-deficient gastrointestinal stromal tumors (dSDH GISTs) collectively manifest similar phenotypes, including hypermethylated epigenomic signatures, tendency to occur in pediatric patients, and lack of KIT/PDGFRA mutations. dSDH GISTs often harbor deleterious mutations in SDH subunit genes (SDHA, SDHB, SDHC, and SDHD, termed SDHx), but some are SDHx wild type (WT). To further elucidate mechanisms of SDH deactivation in SDHx-WT GIST, we performed targeted exome sequencing on 59 dSDH GISTs to identify 43 SDHx-mutant and 16 SDHx-WT cases. Genome-wide DNA methylation and expression profiling exposed SDHC promoter-specific CpG island hypermethylation and gene silencing in SDHx-WT dSDH GISTs [15 of 16 cases (94%)]. Six of 15 SDHC-epimutant GISTs occurred in the setting of the multitumor syndrome Carney triad. We observed neither SDHB promoter hypermethylation nor large deletions on chromosome 1q in any SDHx-WT cases. Deep genome sequencing of a 130-kbp (kilo-base pair) window around SDHC revealed no recognizable sequence anomalies in SDHC-epimutant tumors. More than 2000 benign and tumor reference tissues, including stem cells and malignancies with a hypermethylator epigenotype, exhibit solely a non-epimutant SDHC promoter. Mosaic constitutional SDHC promoter hypermethylation in blood and saliva from patients with SDHC-epimutant GIST implicates a postzygotic mechanism in the establishment and maintenance of SDHC epimutation. The discovery of SDHC epimutation provides a unifying explanation for the pathogenesis of dSDH GIST, whereby loss of SDH function stems from either SDHx mutation or SDHC epimutation.

MYC-driven accumulation of 2-hydroxyglutarate is associated with breast cancer prognosis.

Metabolic profiling of cancer cells has recently been established as a promising tool for the development of therapies and identification of cancer biomarkers. Here we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors using an untargeted discovery approach and validation of key metabolites. The oncometabolite 2-hydroxyglutarate (2HG) accumulated at high levels in a subset of tumors and human breast cancer cell lines. We discovered an association between increased 2HG levels and MYC pathway activation in breast cancer, and further corroborated this relationship using MYC overexpression and knockdown in human mammary epithelial and breast cancer cells. Further analyses revealed globally increased DNA methylation in 2HG-high tumors and identified a tumor subtype with high tissue 2HG and a distinct DNA methylation pattern that was associated with poor prognosis and occurred with higher frequency in African-American patients. Tumors of this subtype had a stem cell-like transcriptional signature and tended to overexpress glutaminase, suggestive of a functional relationship between glutamine and 2HG metabolism in breast cancer. Accordingly, 13C-labeled glutamine was incorporated into 2HG in cells with aberrant 2HG accumulation, whereas pharmacologic and siRNA-mediated glutaminase inhibition reduced 2HG levels. Our findings implicate 2HG as a candidate breast cancer oncometabolite associated with MYC activation and poor prognosis.