William I. Smith

Succinate Dehydrogenase Mutation Underlies Global Epigenomic Divergence in Gastrointestinal Stromal Tumor

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.

Development and Independent Validation of a Prognostic Assay for Stage II Colon Cancer Using Formalin-Fixed Paraffin-Embedded Tissue

PURPOSE Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. PATIENTS AND METHODS A gene signature was developed from a balanced set of 73 patients with recurrent disease (high risk) and 142 patients with no recurrence (low risk) within 5 years of surgery. RESULTS The 634-probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P < .001) during cross validation of the training set. In an independent validation set of 144 samples, the signature identified high-risk patients with an HR of 2.53 (P < .001) for recurrence and an HR of 2.21 (P = .0084) for cancer-related death. Additionally, the signature was shown to perform independently from known prognostic factors (P < .001). CONCLUSION This gene signature represents a novel prognostic biomarker for patients with stage II colon cancer that can be applied to FFPE tumor samples.

Array comparative genomic hybridization-based characterization of genetic alterations in pulmonary neuroendocrine tumors

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.

Recurrent epimutation of SDHC in gastrointestinal stromal tumors

Methylation of the SDH gene explains the loss of SDH gene expression in SDH wild-type gastrointestinal stromal tumors. All Roads Lead to Loss of Expression Gastrointestinal stromal tumors are the most common mesenchymal tumors in the gastrointestinal tract, and they can occur in isolation or as part of a constellation of cancers known as Carney triad. A subtype of this cancer, characterized by lack of expression in a gene called SDH, is not well understood and lacks a specific treatment, and this is the type that most commonly occurs in children. Now, Killian et al. have identified methylation of the SDH gene in patients with SDH-deficient gastrointestinal stromal tumors who lack mutations in the SDH gene. This finding provides a common link explaining the pathogenesis of these SDH-deficient tumors, including many of the ones associated with Carney triad. 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.

Archival Fine-Needle Aspiration Cytopathology (FNAC) Samples: Untapped Resource for Clinical Molecular Profiling

Microarray technologies provide high-resolution maps of chromosome imbalances and epigenomic aberrations in the cancer cell genome. Such assays are often sensitive to sample DNA integrity, voiding the utility of many archival pathology specimens and necessitating the special handling of prospective clinical specimens. We have identified the remarkable preservation of higher-molecular weight DNA in archival fine-needle aspiration cytopathology specimens from patients greater than 10 years of age. We further demonstrate the outstanding technical performance of 57 fine-needle aspiration cytopathology samples for aberration detection on high-resolution comparative genomic hybridization array, DNA methylation, and single nucleotide polymorphism genotyping platforms. Forty-four of 46 malignant aspirates in this study manifested unequivocal genomic aberrations. Importantly, matched Papanicolaou and Diff-Quik fine-needle aspiration cytopathology samples showed critical differences in DNA preservation and DNA integrity. Overall, this study identifies a largely untapped reserve of human pathology specimens for molecular profiling studies, with ramifications for the prospective collection of clinical biospecimens.

Large-Scale Profiling of Archival Lymph Nodes Reveals Pervasive Remodeling of the Follicular Lymphoma Methylome

Emerging technologies allow broad profiling of the cancer genome for differential DNA methylation relative to benign cells. Herein, bisulfite-modified DNA from lymph nodes with either reactive hyperplasia or follicular lymphoma (FL) were analyzed using a commercial C/UpG genotyping assay. Two hundred fifty-nine differentially methylated targets (DMT) distributed among 183 unique genes were identified in FL. Comparison of matched formalin-fixed, paraffin-embedded and frozen surgical pathology replicates showed the complete preservation of the cancer methylome among differently archived tissue specimens. Analysis of the DMT profile is consistent with a pervasive epigenomic remodeling process in FL that affects predominantly nonlymphoid genes.

A flexible reporter system for direct observation and isolation of cancer stem cells.

Summary Many tumors are hierarchically organized with a minority cell population that has stem-like properties and enhanced ability to initiate tumorigenesis and drive therapeutic relapse. These cancer stem cells (CSCs) are typically identified by complex combinations of cell-surface markers that differ among tumor types. Here, we developed a flexible lentiviral-based reporter system that allows direct visualization of CSCs based on functional properties. The reporter responds to the core stem cell transcription factors OCT4 and SOX2, with further selectivity and kinetic resolution coming from use of a proteasome-targeting degron. Cancer cells marked by this reporter have the expected properties of self-renewal, generation of heterogeneous offspring, high tumor- and metastasis-initiating activity, and resistance to chemotherapeutics. With this approach, the spatial distribution of CSCs can be assessed in settings that retain microenvironmental and structural cues, and CSC plasticity and response to therapeutics can be monitored in real time.

A methyl-deviator epigenotype of estrogen receptor-positive breast carcinoma is associated with malignant biology.

We broadly profiled DNA methylation in breast cancers (n = 351) and benign parenchyma (n = 47) for correspondence with disease phenotype, using FFPE diagnostic surgical pathology specimens. Exploratory analysis revealed a distinctive primary invasive carcinoma subclass featuring extreme global methylation deviation. Subsequently, we tested the correlation between methylation remodeling pervasiveness and malignant biological features. A methyl deviation index (MDI) was calculated for each lesion relative to terminal ductal-lobular unit baseline, and group comparisons revealed that high-grade and short-survival estrogen receptor-positive (ER(+)) cancers manifest a significantly higher MDI than low-grade and long-survival ER(+) cancers. In contrast, ER(-) cancers display a significantly lower MDI, revealing a striking epigenomic distinction between cancer hormone receptor subtypes. Kaplan-Meier survival curves of MDI-based risk classes showed significant divergence between low- and high-risk groups. MDI showed superior prognostic performance to crude methylation levels, and MDI retained prognostic significance (P < 0.01) in Cox multivariate analysis, including clinical stage and pathological grade. Most MDI targets individually are significant markers of ER(+) cancer survival. Lymphoid and mesenchymal indexes were not substantially different between ER(+) and ER(-) groups and do not explain MDI dichotomy. However, the mesenchymal index was associated with ER(+) cancer survival, and a high lymphoid index was associated with medullary carcinoma. Finally, a comparison between metastases and primary tumors suggests methylation patterns are established early and maintained through disease progression for both ER(+) and ER(-) tumors.

Genome-wide methylation profiling in archival formalin-fixed paraffin-embedded tissue samples

New technologies allow for genome-scale measurement of DNA methylation. In an effort to increase the clinical utility of DNA methylation as a biomarker, we have adapted a commercial bisulfite epigenotyping assay for genome-wide methylation profiling in archival formalin-fixed paraffin-embedded pathology specimens. This chapter takes the reader step by step through a biomarker discovery experiment to identify phenotype-correlated DNA methylation signatures in routine pathology specimens.

Abstract B45: A novel reporter system with potential for in situ assessment of tumor microenvironmental effects on cancer stem cells

Many tumors consist of a hierarchy of cells with different proliferative and developmental potential. A small number of cancer stem cells (CSCs) give rise to a larger population of highly proliferative, committed progenitor cells, which may then undergo limited differentiation. Importantly, CSCs are uniquely capable of initiating and sustaining tumorigenesis, and they have been implicated in driving disease recurrence after cancer therapy. Thus understanding CSC biology will be critical to the development of more effective therapies. CSCs are most commonly identified by FACS analysis but the optimal marker combinations are very dependent on the tissue and specific cell-of-origin of the tumor, and they cannot be used to monitor the CSCs in situ, with all the microenvironmental cues intact. Such markers cannot readily be used for real-time assessment of stem cell behavior at a single cell rather than a population level. To address this problem, we have developed and validated a novel lentiviral-based reporter system for direct visualization, quantitation and isolation of the cells with CSC properties. The construct consists of a tandemly repeated composite Sox2-Oct4 response element (SORE6) driving expression of a destabilized green fluorescent protein reporter. The reporter responds to the presence of the core stem cell transcription factors Oct4 and Sox2, with further stem cell selectivity and kinetic resolution coming from the use of a proteosome-targeting degron on the fluorescent protein. Using the human MCF10CA1h breast cancer cell line, we have shown that SORE6-GFP+ cells within the cell population are undifferentiated and enriched for stem cell markers. These cells can self-renew and regenerate GFP- cells, show enhanced asymmetric division, and are enriched for tumorsphere formation in vitro. Most importantly the SORE6-GFP+ cells are enriched for tumor-initiating and metastasis-initiating ability in vivo and they are relatively resistant to chemotherapeutics both in vitro and in vivo. Thus by a number of criteria, the reporter is marking a cell population that is substantially enriched for CSCs. The reporter works in primary human breast cancer cultures and patient-derived xenografts in addition to established cell line models. Our novel imaging approach opens up the possibility of assessing the spatial distribution of CSCs and temporal changes in CSC properties in experimental settings that retain the complex microenvironmental and structural cues of the tumor bed. Citation Format: Binwu Tang, Asaf Raviv, Dominic Esposito, Catherine Daniel, Bao Tram Nghiem, Susan Garfield, Langston Lim, Poonam Mannan, Ana Robles, William Smith, Joshua Zimmerberg, Rea Ravin, Lalage Wakefield. A novel reporter system with potential for in situ assessment of tumor microenvironmental effects on cancer stem cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B45. doi:10.1158/1538-7445.CHTME14-B45