NEO212, temozolomide conjugated to NEO100, exerts superior therapeutic activity over temozolomide in preclinical chemoradiation models of glioblastoma.

Background: The chemotherapeutic standard of care for patients with glioblastoma (GB) is radiation therapy (RT) combined with temozolomide (TMZ). However, during the twenty years since its introduction, this so-called Stupp protocol has revealed major drawbacks, because nearly half of all GBs harbor intrinsic treatment resistance mechanisms. Prime among these are the increased expression of the DNA repair protein O6-guanine-DNA methyltransferase (MGMT) and cellular deficiency in DNA mismatch repair (MMR). Patients with such tumors receive very little, if any, benefit from TMZ. We are developing a novel molecule, NEO212 (TMZ conjugated to NEO100), that harbors the potential to overcome these limitations. Methods: We used mouse models that were orthotopically implanted with GB cell lines or primary, radioresistant human GB stem cells, representing different treatment resistance mechanisms. Animals received NEO212 (or TMZ for comparison) without or with RT. Overall survival was recorded, and histology studies quantified DNA damage, apoptosis, microvessel density, and impact on bone marrow. Results: In all tumor models, replacing TMZ with NEO212 in a schedule designed to mimic the Stupp protocol achieved a strikingly superior extension of survival, especially in TMZ-resistant and RT-resistant models. While NEO212 displayed pronounced radiation-sensitizing, DNA-damaging, pro-apoptotic, and anti-angiogenic effects in tumor tissue, it did not cause bone marrow toxicity. Conclusions: NEO212 is a candidate drug to potentially replace TMZ within the standard Stupp protocol. It has the potential to become the first chemotherapeutic agent to significantly extend overall survival in TMZ-resistant patients when combined with radiation.

Neurotropism of SARS-CoV-2: A Pathological Examination of Neurosurgical Specimens.

BACKGROUND AND OBJECTIVES: Neurological manifestations may occur in more than 80% of patients hospitalized with COVID-19 infection, including severe disruptions of the central nervous system (CNS), such as strokes, encephalitis, or seizures. Although the primary pathophysiological mechanism for the effects of COVID-19 in CNS remains unknown, evidence exists for both direct injury from neuroinvasion and indirect effects from disruptions in systemic inflammatory and coagulation pathways. In this study, we analyzed CNS tissue from living patients to better understand these processes. METHODS: With institutional review board approval and patient consent, samples that would be otherwise discarded from patients with active or recent (within 6 days of surgery) COVID-19 infection undergoing neurosurgical intervention were collected and tested for the presence of SARS-CoV-2 using immunohistochemistry, in situ hybridization, electron microscopy, and reverse transcription polymerase chain reaction. RESULTS: Five patients with perioperative mild-to-moderate COVID-19 infection met inclusion criteria (2 male, 3 female; mean age 38.8 ± 13.5 years). Neurosurgical diagnoses included a glioblastoma, a ruptured arteriovenous malformation, a ruptured posterior inferior cerebellar artery aneurysm, a middle cerebral artery occlusion, and a hemorrhagic pontine cavernous malformation. Samples analyzed included the frontal lobe cortex, olfactory nerve, arteriovenous malformation/temporal lobe parenchyma, middle cerebral artery, cerebellum, and cavernous malformation/brainstem parenchyma. Testing for the presence of SARS-CoV-2 was negative in all samples. CONCLUSION: The CNS is likely not a significant viral reservoir during mild-to-moderate COVID-19 infection, although direct neuroinvasion is not definitively excluded. Additional testing to help elucidate the relative contributions of direct and indirect pathways for CNS injury from COVID is warranted.

SARS-CoV-2 viral variants can rapidly be identified for clinical decision making and population surveillance using a high-throughput digital droplet PCR assay.

Epidemiologic surveillance of circulating SARS-CoV-2 variants is essential to assess impact on clinical outcomes and vaccine efficacy. Whole genome sequencing (WGS), the gold-standard to identify variants, requires significant infrastructure and expertise. We developed a digital droplet polymerase chain reaction (ddPCR) assay that can rapidly identify circulating variants of concern/interest (VOC/VOI) using variant-specific mutation combinations in the Spike gene. To validate the assay, 800 saliva samples known to be SARS-CoV-2 positive by RT-PCR were used. During the study (July 2020-March 2022) the assay was easily adaptable to identify not only existing circulating VAC/VOI, but all new variants as they evolved. The assay can discriminate nine variants (Alpha, Beta, Gamma, Delta, Eta, Epsilon, Lambda, Mu, and Omicron) and sub-lineages (Delta 417N, Omicron BA.1, BA.2). Sequence analyses confirmed variant type for 124/124 samples tested. This ddPCR assay is an inexpensive, sensitive, high-throughput assay that can easily be adapted as new variants are identified.

The Sulfotransferase SULT1C2 Is Epigenetically Activated and Transcriptionally Induced by Tobacco Exposure and Is Associated with Patient Outcome in Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer-related death. Tobacco exposure is associated with 80-90% of lung cancer cases. The SULT1C2 sulfotransferase modifies xenobiotic compounds to enhance secretion but can also render these compounds carcinogenic. To determine if SULT1C2 contributes to tobacco-related carcinogenesis in the lung, we analyzed the expression and epigenetic state of SULT1C2 in human lung adenocarcinoma (LUAD) samples and in LUAD cell lines exposed to cigarette smoke condensate (CSC). SULT1C2 expression was significantly positively correlated to overall LUAD patient survival in smokers, was elevated in LUAD tumors compared to adjacent non-tumor lung, and was significantly correlated with levels of patient exposure to tobacco smoke. SULT1C2 promoter DNA methylation was inversely correlated with expression in LUAD, and hypomethylation of the SULT1C2 promoter was observed in Asian patients, as compared to Caucasians. In vitro analysis of LUAD cell lines indicates that CSC stimulates expression of SULT1C2 in a dose-dependent and cell-line-specific manner. In vitro methylation of the SULT1C2 promoter significantly decreased transcriptional activity of a reporter plasmid, and SULT1C2 expression was activated by the DNA demethylating agent 5-Aza-2'-deoxycytidine in a cell line in which the SULT1C2 promoter was hypermethylated. An aryl hydrocarbon receptor (AHR) binding site was detected spanning critical methylation sites upstream of SULT1C2. CSC exposure significantly increased AHR binding to this predicted binding site in the SULT1C2 promoter in multiple lung cell lines. Our data suggest that CSC exposure leads to activation of the AHR transcription factor, increased binding to the SULT1C2 promoter, and upregulation of SULT1C2 expression and that this process is inhibited by DNA methylation at the SULT1C2 locus. Additionally, our results suggest that the level of SULT1C2 promoter methylation and gene expression in normal lung varies depending on the race of the patient, which could in part reflect the molecular mechanisms of racial disparities seen in lung cellular responses to cigarette smoke exposure.

Differences in Genome-wide DNA Methylation Profiles in Breast Milk by Race and Lactation Duration.

Black women in the United States are disproportionately affected by early-onset, triple-negative breast cancer. DNA methylation has shown differences by race in healthy and tumor breast tissues. We examined associations between genome-wide DNA methylation levels in breast milk and breast cancer risk factors, including race, to explain how this reproductive stage influences a woman's risk for, and potentially contributes to racial disparities in, breast cancer. Breast milk samples and demographic, behavioral, and reproductive data, were obtained from cancer-free, uniparous, and lactating U.S. black (n = 57) and white (n = 82) women, ages 19-44. Genome-wide DNA methylation analysis was performed on extracted breast milk DNA using the Infinium HumanMethylation450 BeadChip. Statistically significant associations between breast cancer risk factors and DNA methylation beta values, adjusting for potential confounders, were determined using linear regression followed by Bonferroni Correction (P < 1.63 × 10-7). Epigenetic analysis in breast milk revealed statistically significant associations with race and lactation duration. Of the 284 CpG sites associated with race, 242 were hypermethylated in black women. All 227 CpG sites associated with lactation duration were hypomethylated in women who lactated longer. Ingenuity Pathway Analysis of differentially methylated promoter region CpGs by race and lactation duration revealed enrichment for networks implicated in carcinogenesis. Associations between DNA methylation and lactation duration may offer insight on its role in lowering breast cancer risk. Epigenetic associations with race may mediate social, behavioral, or other factors related to breast cancer and may provide insight into potential mechanisms underlying racial disparities in breast cancer incidence.

Gain of TP53 Mutation in Imatinib-treated SDH-Deficient Gastrointestinal Stromal Tumor and Clinical Utilization of Targeted Next-generation Sequencing Panel for Therapeutic Decision Support.

Patients with succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumor (GIST) have few therapeutic options. Despite lack of KIT or platelet-derived growth factor receptor A (PDGFRA) driver mutations, SDH-deficient GISTs display strong expression of KIT by immunohistochemistry and these patients are often treated with tyrosine kinase inhibitors, including imatinib as a first-line therapy. Using a targeted next-generation sequencing panel of mutation hotspots of 50-clinically relevant genes, we investigated (1) concurrence of somatic/actionable mutations and (2) tumor molecular evolution by comparing 2 resection specimens 1.5 years apart while the patient was on imatinib adjuvant therapy. We found the tumors did not harbor KIT, PDGFRA, or any other clinically actionable mutations. However, a TP53 mutation (c.422G>A; p.C141Y) was detected in the second recurrent lesion. This represents the first study to monitor the molecular evolution of a SDH-deficient GIST during adjuvant treatment. These findings emphasize the critical need for next-generation sequencing testing before initiating targeted therapy.

MethyLight and Digital MethyLight.

MethyLight is a quantitative, fluorescence-based, real-time PCR method to sensitively detect and quantify DNA methylation of candidate regions of the genome. MethyLight is uniquely suited for detecting low-frequency methylated DNA regions against a high background of unmethylated DNA, as it combines methylation-specific priming with methylation-specific fluorescent probing. The quantitative accuracy of real-time PCR and the ability to design bisulfite-dependent, DNA methylation-independent control reactions together allow for a quantitative assessment of these low frequency methylation events. Here we describe the experimental steps of MethyLight analysis in detail. Furthermore, we present principles and design examples for three types of quality control reactions. QC-1 reactions are methylation-independent reactions to monitor sample quantity and integrity. QC-2 reactions are bisulfite-independent reactions to monitor recovery efficiencies of the bisulfite conversion methodology used. QC-3 reactions are bisulfite-independently primed reactions with variable bisulfite-dependent probing to monitor completeness of the sodium bisulfite treatment. We show that these control reactions perform as expected in a time course experiment interrupting sodium bisulfite conversion at various timepoints. Finally, we describe Digital MethyLight, in which MethyLight is combined with Digital PCR, for the highly sensitive detection of individual methylated molecules, with use in disease detection and screening.

Association of high microvessel αvß3 and low PTEN with poor outcome in stage 3 neuroblastoma: rationale for using first in class dual PI3K/BRD4 inhibitor, SF1126.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvß3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvß3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvß3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvß3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvß3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvß3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma.

Characterizing the genetic basis of methylome diversity in histologically normal human lung tissue.

The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34,304 cis- and 585 trans-meQTLs, a genetic-epigenetic interaction of surprising magnitude, including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes, promoters and CpG islands (CGIs), while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF-binding sites, DNaseI hypersensitivity regions and histone marks. Importantly, four of the five established lung cancer risk loci in European ancestry are cis-meQTLs and, in aggregate, cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11,587 subjects. Thus, inherited genetic variation may affect lung carcinogenesis by regulating the human methylome.

Genome-scale discovery of DNA-methylation biomarkers for blood-based detection of colorectal cancer.

BACKGROUND: There is an increasing demand for accurate biomarkers for early non-invasive colorectal cancer detection. We employed a genome-scale marker discovery method to identify and verify candidate DNA methylation biomarkers for blood-based detection of colorectal cancer. METHODOLOGY/PRINCIPAL FINDINGS: We used DNA methylation data from 711 colorectal tumors, 53 matched adjacent-normal colonic tissue samples, 286 healthy blood samples and 4,201 tumor samples of 15 different cancer types. DNA methylation data were generated by the Illumina Infinium HumanMethylation27 and the HumanMethylation450 platforms, which determine the methylation status of 27,578 and 482,421 CpG sites respectively. We first performed a multistep marker selection to identify candidate markers with high methylation across all colorectal tumors while harboring low methylation in healthy samples and other cancer types. We then used pre-therapeutic plasma and serum samples from 107 colorectal cancer patients and 98 controls without colorectal cancer, confirmed by colonoscopy, to verify candidate markers. We selected two markers for further evaluation: methylated THBD (THBD-M) and methylated C9orf50 (C9orf50-M). When tested on clinical plasma and serum samples these markers outperformed carcinoembryonic antigen (CEA) serum measurement and resulted in a high sensitive and specific test performance for early colorectal cancer detection. CONCLUSIONS/SIGNIFICANCE: Our systematic marker discovery and verification study for blood-based DNA methylation markers resulted in two novel colorectal cancer biomarkers, THBD-M and C9orf50-M. THBD-M in particular showed promising performance in clinical samples, justifying its further optimization and clinical testing.

Genome-scale analysis of DNA methylation in lung adenocarcinoma and integration with mRNA expression.

Lung cancer is the leading cause of cancer death worldwide, and adenocarcinoma is its most common histological subtype. Clinical and molecular evidence indicates that lung adenocarcinoma is a heterogeneous disease, which has important implications for treatment. Here we performed genome-scale DNA methylation profiling using the Illumina Infinium HumanMethylation27 platform on 59 matched lung adenocarcinoma/non-tumor lung pairs, with genome-scale verification on an independent set of tissues. We identified 766 genes showing altered DNA methylation between tumors and non-tumor lung. By integrating DNA methylation and mRNA expression data, we identified 164 hypermethylated genes showing concurrent down-regulation, and 57 hypomethylated genes showing increased expression. Integrated pathways analysis indicates that these genes are involved in cell differentiation, epithelial to mesenchymal transition, RAS and WNT signaling pathways, and cell cycle regulation, among others. Comparison of DNA methylation profiles between lung adenocarcinomas of current and never-smokers showed modest differences, identifying only LGALS4 as significantly hypermethylated and down-regulated in smokers. LGALS4, encoding a galactoside-binding protein involved in cell-cell and cell-matrix interactions, was recently shown to be a tumor suppressor in colorectal cancer. Unsupervised analysis of the DNA methylation data identified two tumor subgroups, one of which showed increased DNA methylation and was significantly associated with KRAS mutation and to a lesser extent, with smoking. Our analysis lays the groundwork for further molecular studies of lung adenocarcinoma by identifying novel epigenetically deregulated genes potentially involved in lung adenocarcinoma development/progression, and by describing an epigenetic subgroup of lung adenocarcinoma associated with characteristic molecular alterations.

Genome-scale screen for DNA methylation-based detection markers for ovarian cancer.

BACKGROUND: The identification of sensitive biomarkers for the detection of ovarian cancer is of high clinical relevance for early detection and/or monitoring of disease recurrence. We developed a systematic multi-step biomarker discovery and verification strategy to identify candidate DNA methylation markers for the blood-based detection of ovarian cancer. METHODOLOGY/PRINCIPAL FINDINGS: We used the Illumina Infinium platform to analyze the DNA methylation status of 27,578 CpG sites in 41 ovarian tumors. We employed a marker selection strategy that emphasized sensitivity by requiring consistency of methylation across tumors, while achieving specificity by excluding markers with methylation in control leukocyte or serum DNA. Our verification strategy involved testing the ability of identified markers to monitor disease burden in serially collected serum samples from ovarian cancer patients who had undergone surgical tumor resection compared to CA-125 levels. We identified one marker, IFFO1 promoter methylation (IFFO1-M), that is frequently methylated in ovarian tumors and that is rarely detected in the blood of normal controls. When tested in 127 serially collected sera from ovarian cancer patients, IFFO1-M showed post-resection kinetics significantly correlated with serum CA-125 measurements in six out of 16 patients. CONCLUSIONS/SIGNIFICANCE: We implemented an effective marker screening and verification strategy, leading to the identification of IFFO1-M as a blood-based candidate marker for sensitive detection of ovarian cancer. Serum levels of IFFO1-M displayed post-resection kinetics consistent with a reflection of disease burden. We anticipate that IFFO1-M and other candidate markers emerging from this marker development pipeline may provide disease detection capabilities that complement existing biomarkers.

Epigenetic subgroups of esophageal and gastric adenocarcinoma with differential GATA5 DNA methylation associated with clinical and lifestyle factors.

BACKGROUND: Adenocarcinomas located near the gastroesophageal junction have unclear etiology and are difficult to classify. We used DNA methylation analysis to identify subtype-specific markers and new subgroups of gastroesophageal adenocarcinomas, and studied their association with epidemiological risk factors and clinical outcomes. METHODOLOGY/PRINCIPAL FINDINGS: We used logistic regression models and unsupervised hierarchical cluster analysis of 74 DNA methylation markers on 45 tumor samples (44 patients) of esophageal and gastric adenocarcinomas obtained from a population-based case-control study to uncover epigenetic markers and cluster groups of gastroesophageal adenocarcinomas. No distinct epigenetic differences were evident between subtypes of gastric and esophageal cancers. However, we identified two gastroesophageal adenocarcinoma subclusters based on DNA methylation profiles. Group membership was best predicted by GATA5 DNA methylation status. We analyzed the associations between these two epigenetic groups and exposure using logistic regression, and the associations with survival time using Cox regression in a larger set of 317 tumor samples (278 patients). There were more males with esophageal and gastric cardia cancers in Cluster Group 1 characterized by higher GATA5 DNA methylation values (all p<0.05). This group also showed associations of borderline statistical significance with having ever smoked (p-value = 0.07), high body mass index (p-value = 0.06), and symptoms of gastroesophageal reflux (p-value = 0.07). Subjects in cluster Group 1 showed better survival than those in Group 2 after adjusting for tumor differentiation grade, but this was not found to be independent of tumor stage. CONCLUSIONS/SIGNIFICANCE: DNA methylation profiling can be used in population-based studies to identify epigenetic subclasses of gastroesophageal adenocarcinomas and class-specific DNA methylation markers that can be linked to epidemiological data and clinical outcome. Two new epigenetic subgroups of gastroesophageal adenocarcinomas were identified that differ to some extent in their survival rates, risk factors of exposure, and GATA5 DNA methylation.

DNA methylation changes in atypical adenomatous hyperplasia, adenocarcinoma in situ, and lung adenocarcinoma.

BACKGROUND: Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation. METHODOLOGY/PRINCIPAL FINDINGS: We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma), and invasive lung adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML, RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer. CONCLUSIONS/SIGNIFICANCE: The gradual increase in DNA methylation seen for numerous loci in progressively more transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to lay out a possible roadmap for aberrant DNA methylation events in tumor development. In addition, it identifies which DNA methylation changes might be used as molecular markers for the detection of preinvasive lesions.

Age-dependent DNA methylation of genes that are suppressed in stem cells is a hallmark of cancer.

Polycomb group proteins (PCGs) are involved in repression of genes that are required for stem cell differentiation. Recently, it was shown that promoters of PCG target genes (PCGTs) are 12-fold more likely to be methylated in cancer than non-PCGTs. Age is the most important demographic risk factor for cancer, and we hypothesized that its carcinogenic potential may be referred by irreversibly stabilizing stem cell features. To test this, we analyzed the methylation status of over 27,000 CpGs mapping to promoters of approximately 14,000 genes in whole blood samples from 261 postmenopausal women. We demonstrate that stem cell PCGTs are far more likely to become methylated with age than non-targets (odds ratio = 5.3 [3.8-7.4], P < 10(-10)), independently of sex, tissue type, disease state, and methylation platform. We identified a specific subset of 69 PCGT CpGs that undergo hypermethylation with age and validated this methylation signature in seven independent data sets encompassing over 900 samples, including normal and cancer solid tissues and a population of bone marrow mesenchymal stem/stromal cells (P < 10(-5)). We find that the age-PCGT methylation signature is present in preneoplastic conditions and may drive gene expression changes associated with carcinogenesis. These findings shed substantial novel insights into the epigenetic effects of aging and support the view that age may predispose to malignant transformation by irreversibly stabilizing stem cell features.

DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.

BACKGROUND: Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. METHODOLOGY/PRINCIPAL FINDINGS: We obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited 'subtype-specific' DNA methylation patterns (FDR<1%) among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene. SIGNIFICANCE: The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of potential tumor progenitor cells, which may help illuminate the etiology and natural history of these cancers.

Analysis of the association between CIMP and BRAF in colorectal cancer by DNA methylation profiling.

A CpG island methylator phenotype (CIMP) is displayed by a distinct subset of colorectal cancers with a high frequency of DNA hypermethylation in a specific group of CpG islands. Recent studies have shown that an activating mutation of BRAF (BRAF(V600E)) is tightly associated with CIMP, raising the question of whether BRAF(V600E) plays a causal role in the development of CIMP or whether CIMP provides a favorable environment for the acquisition of BRAF(V600E). We employed Illumina GoldenGate DNA methylation technology, which interrogates 1,505 CpG sites in 807 different genes, to further study this association. We first examined whether expression of BRAF(V600E) causes DNA hypermethylation by stably expressing BRAF(V600E) in the CIMP-negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We determined 100 CIMP-associated CpG sites and examined changes in DNA methylation in eight stably transfected clones over multiple passages. We found that BRAF(V600E) is not sufficient to induce CIMP in our system. Secondly, considering the alternative possibility, we identified genes whose DNA hypermethylation was closely linked to BRAF(V600E) and CIMP in 235 primary colorectal tumors. Interestingly, genes that showed the most significant link include those that mediate various signaling pathways implicated in colorectal tumorigenesis, such as BMP3 and BMP6 (BMP signaling), EPHA3, KIT, and FLT1 (receptor tyrosine kinases) and SMO (Hedgehog signaling). Furthermore, we identified CIMP-dependent DNA hypermethylation of IGFBP7, which has been shown to mediate BRAF(V600E)-induced cellular senescence and apoptosis. Promoter DNA hypermethylation of IGFBP7 was associated with silencing of the gene. CIMP-specific inactivation of BRAF(V600E)-induced senescence and apoptosis pathways by IGFBP7 DNA hypermethylation might create a favorable context for the acquisition of BRAF(V600E) in CIMP+ colorectal cancer. Our data will be useful for future investigations toward understanding CIMP in colorectal cancer and gaining insights into the role of aberrant DNA hypermethylation in colorectal tumorigenesis.

DNA hypermethylation of ESR1 and PGR in breast cancer: pathologic and epidemiologic associations.

Improved understanding of the etiology of estrogen receptor-alpha (ERalpha)-negative and progesterone receptor (PR)-negative breast cancers may permit improved risk prediction. In vitro studies implicate DNA hypermethylation of the ERalpha and PR promoters in the pathogenesis of ERalpha-negative and PR-negative tumors, but results are not definitive. We evaluated 200 invasive breast cancers selected from a population-based case-control study. DNA extracted from fixed tumor tissue cores was tested using MethyLight to assess DNA methylation at four CpG islands: ESR1 promoters A and B; PGR promoters A and B; and a CpG shore, ESR1 promoter C. DNA methylation results were compared with levels of ERalpha and PR, tumor characteristics, and breast cancer risk factors. We observed mild to moderate DNA methylation levels in most tumors for ESR1 promoters A and B and PGR promoter B, and a few tumors showed mild methylation in PGR promoter A. In contrast, ESR1 promoter C showed a wide range of methylation and was weakly correlated with lower expression levels of ERalpha (beta = -0.26; P < 0.0001) and PR (beta = -0.25; P < 0.0001). The percentage of tumors with methylated PGR promoters A and B was significantly higher for tumors with low ERalpha (A, Fisher's test P = 0.0001; B, P = 0.033) and PR levels (A, P = 0.0006; B, P = 0.001). Our data suggest that the relationships between DNA methylation of ESR1 and PGR promoters and protein expression are weak and unlikely to represent a predominant mechanism of receptor silencing. In contrast to CpG islands, ESR1 promoter C showed a wider range of methylation levels and inverse associations with ERalpha and PR expression.

The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: a possible molecular basis for the site-specific cancer risk modification.

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

MethyLight.

MethyLight is a sodium-bisulfite-dependent, quantitative, fluorescence-based, real-time PCR method to sensitively detect and quantify DNA methylation in genomic DNA. MethyLight relies on methylation-specific priming combined with methylation-specific fluorescent probing. This combination of methylation-specific detection principles results in a highly methylation-specific detection technology, with an accompanying ability to sensitively detect very low frequencies of hypermethylated alleles. The high sensitivity and specificity of MethyLight make it uniquely well suited for detection of low-frequency DNA methylation biomarkers as evidence of disease. At the same time, the quantitative accuracy of real-time PCR and the flexibility to design bisulfite-dependent, methylation-independent control reactions allows for a quantitative assessment of these low-frequency methylation events. We describe the experimental steps of MethyLight analysis in detail. Furthermore, we present here principles and design examples for three types of quality-control reactions. QC-1 reactions are methylation-independent reactions to monitor sample quantity and integrity. QC-2 reactions are bisulfite-independent reactions to monitor recovery efficiencies of the bisulfite-conversion methodology used. QC-3 reactions are bisulfite-independent primed reactions with variable bisulfite-dependent probing to monitor completeness of the sodium bisulfite treatment. We show that these control reactions perform as expected in a time-course experiment interrupting sodium bisulfite conversion at various timepoints.