Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway–mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH -mutant GIST with isocitrate dehydrogenase -mutant glioma, another Krebs cycle–defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance. SIGNIFICANCE This study shows that SDH deficiency underlies pervasive DNA hypermethylation in multiple tumor lineages, generally defining the Krebs cycle as mitochondrial custodian of the methylome. We propose that this phenomenon may result from a failure of maintenance CpG demethylation, secondary to inhibition of the TET 5-methylcytosine dioxgenase demethylation pathway, by inhibitory metabolites that accumulate in tumors with Krebs cycle dysfunction.

Limited information is available regarding epigenomic events mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to examine epigenomic effects of cigarette smoke in respiratory epithelia. Normal human small airway epithelial cells and cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media with or without cigarette smoke condensate (CSC) for up to 9 months under potentially relevant exposure conditions. Western blot analysis showed that CSC mediated dose-and timedependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNA methyltransferase 1 (DNMT1) and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3; activation of Wnt signaling; and hypermethylation of tumor suppressor genes such as RASSF1A and RAR-b, which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft-agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft-agar clonogenicity. Collectively, these data indicate that cigarette smoke induces 'cancer-associated' epigenomic alterations in cultured respiratory epithelia. This in vitro model may prove useful for delineating early epigenetic mechanisms regulating gene expression during pulmonary carcinogenesis.

A subset (7–10%) of gastric GISTs is notable for the immunohistochemical loss of succinate dehydrogenase (SDH) subunit B (SDHB), which signals the loss of function of the SDH-complex consisting of mitochondrial inner membrane proteins. These SDH-deficient GISTs are known to be KIT/PDGFRA wild type, and most patients are young. Some of these patients have germline mutations of SDH-subunits B, C, or D, known as Carney-Stratakis syndrome when combined with paraganglioma. More recently, germline mutations in SDH-subunit A (SDHA) have been also reported in few patients with KIT/PDGFRA wild type GISTs. In this study we examined immunohistochemically 127 SDHB-negative and 556 SDHB-positive gastric GISTs and 261 SDHB-positive intestinal GISTs for SDHA expression using a mouse monoclonal antibody 2E3 (Abcam). Cases with available DNA were tested for SDHA, B, C, and D gene mutations using a hybridization-based custom capture next-generation sequencing assay. A total of 36 SDHA-negative GISTs (28%) were found among 127 SDHB-negative gastric GISTs. No SDHB-positive GIST was SDHA-negative. Among 7 SDHA-negative tumors analyzed, there were 7 SDHA mutants, most germline. A second hit indicating biallelic inactivation of SDHA was present in 6 of those cases. These patients had no other SDH subunit mutations. Among the 25 SDHA-positive, SDHB-negative GISTs analyzed, we identified 3 SDHA mutations (one germline), and 11 SDHB, SDHC or SDHD mutations (mostly germline), and 11 patients with no SDH mutations. Compared with patients with SDHA-positive GISTs, those with SDHA-negative GISTs had an older median age (34 vs. 21 years), lower female to male ratio (1.8 vs. 3.1) but similar mitotic counts and median tumor sizes, with a slow course of disease in most cases, despite a slightly higher rate of liver metastases. SDHA-negative GISTs comprise approximately 30% of SDHB-negative/SDH-deficient GISTs, and SDHA loss generally correlates with SDHA mutations.

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.

The goal of this study was to characterize and classify pulmonary neuroendocrine tumors based on array comparative genomic hybridization (aCGH). Using aCGH, we performed karyotype analysis of 33 small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial carcinoids, and 9 gastrointestinal carcinoids. In contrast to the relatively conserved karyotypes of carcinoid tumors, the karyotypes of SCLC tumors and cell lines were highly aberrant. High copy number (CN) gains were detected in SCLC tumors and cell lines in cytogenetic bands encoding JAK2, FGFR1, and MYC family members. In some of those samples, the CN of these genes exceeded 100, suggesting that they could represent driver alterations and potential drug targets in subgroups of SCLC patients. In SCLC tumors, as well as bronchial carcinoids and carcinoids of gastrointestinal origin, recurrent CN alterations were observed in 203 genes, including the RB1 gene and 59 microRNAs of which 51 locate in the DLK1-DIO3 domain. These findings suggest the existence of partially shared CN alterations in these tumor types. In contrast, CN alterations of the TP53 gene and the MYC family members were predominantly observed in SCLC. Furthermore, we demonstrated that the aCGH profile of SCLC cell lines highly resembles that of clinical SCLC specimens. Finally, by analyzing potential drug targets, we provide a genomics-based rationale for targeting the AKT-mTOR and apoptosis pathways in SCLC.carcinoid | cell line | gene dosage | small cell lung carcinoma | therapy

Malignant adrenocortical tumors are globally hypomethylated as compared with normal and benign tumors. Methylation profile differences may accurately distinguish between primary benign and malignant adrenocortical tumors. Several differentially methylated sites are associated with genes known to be dysregulated in malignant adrenocortical tumors.

The value and suitability of cytology specimens for molecular diagnosis has been demonstrated by numerous studies. In practice, however, the success rates vary widely across institutions depending on the disease setting, institutional practices of acquisition, handling/processing, and testing methodologies. As the number of clinically relevant biomarkers continues to increase, more laboratories are turning to next-generation sequencing platforms for testing. Although amplicon-based next-generation sequencing assays, interrogating a limited genomic territory, can be performed with minimal input material, broader-based next-generation sequencing assays have higher DNA input requirements that may not be met if the small tissue samples are not acquired and handled appropriately. We briefly describe some of the process changes we have instituted in our laboratories when handling cytologic material to maximize the tissue available for broad hybrid-capture-based next-generation sequencing assays. Among the key changes established were the consolidation and preservation of previously discarded supernatant material in cytologic samples, the introduction of mineral oil for deparaffinization of cell blocks, and adjustments in the molecular laboratory process and bioinformatics pipelines. We emphasize that even minimal changes can have broad implications for test performance, highlighting the importance of a cohesive group-based approach among clinical, cytopathology, surgical pathology, molecular, and bioinformatics teams.

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