Project description:Pathologic differentiation of tissue of origin in tumors found in the lung can be challenging, with differentiation of mesothelioma and lung adenocarcinoma emblematic of this problem. Indeed, proper classification is essential for determination of treatment regimen for these diseases, making accurate and early diagnosis critical. Here we investigate the potential of epigenetic profiles of lung adenocarcinoma, mesothelioma, and non-malignant pulmonary tissues (n=285) as differentiation markers in an analysis of DNA methylation at 1413 autosomal CpG loci associated with 773 cancer-related genes. Using an unsupervised recursively-partitioned mixture modeling technique for all samples, the derived methylation profile classes were significantly associated with sample type (P < 0.0001). In a similar analysis restricted to tumors, methylation profile classes significantly predicted tumor type (P < 0.0001). Random forests classification of CpG methylation of tumors - which splits the data into training and test sets - accurately differentiated MPM from lung adenocarcinoma over 99% of the time (P < 0.0001). In a locus-by-locus comparison of CpG methylation between tumor types, 1266 CpG loci had significantly different methylation between tumors following correction for multiple comparisons (Q < 0.05); 61% had higher methylation in adenocarcinoma. Using the CpG loci with significant differential methylation in a pathways analysis revealed significant enrichment of methylated gene-loci in Cell Cycle Regulation, DNA Damage Response, PTEN Signaling, and Apoptosis Signaling pathways in lung adenocarcinoma when compared to mesothelioma. Methylation-profile-based differentiation of lung adenocarcinoma and mesothelioma is highly accurate, informs on the distinct etiologies of these diseases, and holds promise for clinical application. Mesotheliomas (n=158) and grossly non-tumorigenic parietal pleura (n=18) were obtained following surgical resection at Brigham and Womenâ??s Hospital through the International Mesothelioma Program from a pilot study conducted in 2002 (n=70) and an incident case series beginning in 2005 (n=88) with a participation rate of 85%. We used biopsy specimens from patients treated for NSCLC at the Massachusetts General Hospital from 1992 â?? 1996 (18) including lung adenocarcinomas (n=57) and non-malignant pulmonary tissues (n=48) (of which 22 (39%) were taken from the adenocarcinoma patients) (18). Additional normal lung tissues were obtained from the National Disease Research Interchange from donors free of lung malignancy (n=4).

Project description:Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variation in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1413 autosomal CpG loci associated with 773 genes to investigate tissue-specific differences in DNA methylation, and to discern how aging and exposures contribute to normal variation in methylation. Methylation profile classes derived from unsupervised modeling were significantly associated with age (P < 0.0001), and were significant predictors of tissue origin (P < 0.0001). In solid tissues (n=119) we found striking, highly significant CpG island dependent correlations between age and methylation; loci in CpG islands gained methylation with age, loci not in CpG islands lost methylation with age (P < 0.001), and this pattern was consistent across tissues and in an analysis of blood-derived DNA. Our data clearly demonstrate age and exposure related differences in tissue-specific methylation, and significant age associated methylation patterns which are CpG island context dependent. This work provides novel insight into the role of aging and the environment in susceptibility to diseases such as cancer, and critically informs the field of epigenomics by providing evidence of epigenetic dysregulation by age-related methylation alterations. Collectively we reveal key issues to consider both in the construction of reference. The causes and extent of tissue-specific interindividual variation in human epigenomes are underappreciated and hence, poorly characterized. We surveyed over 200 carefully annotated human tissue samples from ten anatomic sites at 1413 CpGs for methylation alterations to appraise the nature of phenotypically, and hence potentially clinically important epigenomic alterations. Within tissue types, across individuals, we found variation in methylation that was significantly related to aging and environmental exposures such as tobacco smoking. Individual variation in age and exposure-related methylation may significantly contribute to increased susceptibility to several diseases. As the NIH-funded HapMap project is critically contributing to annotating the human reference genome defining normal genetic variability, our work raises key issues to consider in the construction of reference epigenomes. It is well recognized that understanding genetic variation is essential to understanding disease. Our work, and the known interplay of epigenetics and genetics, makes it equally clear that a more complete characterization of epigenetic variation and its sources must be accomplished to reach the goal of a complete understanding of disease. Additional research is absolutely necessary to define the mechanisms controlling epigenomic variation. We have begun to lay the foundations for essential normal tissue controls for comparison to diseased tissue, which will allow the identification of the most crucial disease-related alterations and provide more robust targets for novel treatments. Normal human tissues were assembled by a collaborative, multi-institutional network of investigators conducting molecular epidemiologic studies of human cancer. Tissues were obtained through Institutional Review Board approved studies already underway at these institutions, or were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA). Briefly, normal brain tissues (n=12) were contributed by the Wiencke lab at UCSF through the San Francisco Adult Glioma Study (Wiemels JL, Int J Ca 2002). Normal lung tissues (n=49) were obtained from adjacent non-tumor portions of lung in patients treated for NSCLC (Wiencke JK, Ca Res, 1995) or from the NDRI from non-diseased individuals at autopsy (n=4). Peripheral blood DNA was obtained from controls enrolled in a study of bladder cancer (n=15) (Karagas MR, Env Health Persp 1998), controls enrolled in a study of head and neck cancer (n=15) (Peters ES, CEBP 2005, 14:476-82), and newborn infants (n=55) (Urayama KY, CEBP, 2007, 16:1172). Non-tumorigenic pleural samples (n=18) were obtained from grossly disease-uninvolved regions of incident mesothelioma (Christensen BC, Carcinogenesis 2008). Head and neck anatomic sites (n=11), bladder (n=5), kidney (n=6), and small intestine (n=5) were obtained from the NDRI, all from individuals with no gross diseases or tumors of the obtained tissues. Non-pathologic placenta samples (n=19) were obtained as residual tissues from control infant term births as part of an ongoing hospital-based case-control study of intrauterine growth restriction at Women and Infants Hospital in Providence RI. All tissues obtained from patients with disease (lung, pleura) were histologically confirmed as normal by independent study pathologist review of tissue samples prior to DNA extraction.

Project description:Integrated profiling of somatic molecular alterations present in tumors is necessary to further our understanding of the tumorigenic process. We investigated the potential relationships between gene copy number alterations and DNA methylation profiles in a case series of pleural mesotheliomas (n=23). Gene copy number (CN) alterations profiled with 500K SNP arrays and DNA methylation measured at over 750 cancer-related genes with methylation bead-arrays were examined concomitantly. Considering each probed locus, there were no instances of significantly correlated CN alteration and methylation (no loci with Q < 0.05) and averaging loci over their associated genes revealed only two genes with significantly correlated CN and methylation alterations (Q < 0.04). In contrast to the lack of discrete correlations, the overall extent of tumor CN alteration was significantly associated with DNA methylation profile when comparing CN alteration extent among methylation profile classes (P < 0.02), and there was evidence that this association was partially attributable to prevalent allele loss observed at the maintenance DNA methyltransferase DNMT1. Taken together, this work indicates a strong association between global genetic and global epigenetic dysregulation in mesothelioma rather than a discrete, local coordination of gene inactivation, and further highlights the utility and necessity of integrative genomics approaches in cancer biology. Mesotheliomas were obtained following surgical resection at Brigham and Women’s Hospital through the International Mesothelioma Program from a pilot study conducted in 2002 and an incident case series beginning in 2005 as previously described (PMIDs: 19118007 and 19638575). All patients provided informed consent under the approval of the appropriate Institutional Review Boards. Clinical information, including histologic diagnosis was obtained from pathology reports. The study pathologist confirmed the histologic diagnoses and further assessed the percent tumor from resected specimens (mean >60%). DNA extraction and methylation analysis: DNA from fresh frozen tissue and matched whole blood was isolated with QIAamp DNA mini kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. Tumor DNA was modified with sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research, Orange, CA). Illumina GoldenGate® methylation bead arrays interrogated 1505 CpG loci associated with 803 cancer-related genes processed at the UCSF Institute for Human Genetics, Genomics Core Facility using methods described in (28). The GoldenGate methylation data used in the analysis has been previously described (PMIDs: 19118007 and 19638575).

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