Helene Myrtue Nielsen

Epigenetics and cancer treatment

In addition to the genetic alterations, observed in cancer cells, are mitotically heritable changes in gene expression not encoded by the DNA sequences, which are referred to as epigenetic changes. DNA methylation is among the most studied epigenetic mechanisms together with various histone modifications involved in chromatin remodeling. As opposed to genetic lesions, the epigenetic changes are potentially reversible by a number of small molecules, known as epi-drugs. This review will focus on the biological mechanisms underlying the epigenetic silencing of tumor suppressor genes observed in cancer cells, and the targeted molecular strategies that have been investigated to reverse these aberrations. In particular, we will focus on DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) as epigenetic targets for cancer treatment. A synergistic effect of a combined use of DNMT and HDAC inhibitors has been observed. Moreover, epi-drugs sensitize multiple different cancer cells to a large variety of other treatment strategies. In particular, we have focused on the ability of DNMT and HDAC inhibitors to restore the estrogen receptor alpha (ERalpha) activity in breast cancer. Finally, we will discuss the potential of DNA methylation changes as biomarkers to be used in diverse areas of cancer treatment, especially for predicting response to treatment with DNMT and HDAC inhibitors.

DNA methylation based biomarkers: Practical considerations and applications

A biomarker is a molecular target analyzed in a qualitative or quantitative manner to detect and diagnose the presence of a disease, to predict the outcome and the response to a specific treatment allowing personalized tailoring of patient management. Biomarkers can belong to different types of biochemical molecules such as proteins, DNA, RNA or lipids, whereby protein biomarkers have been the most extensively studied and used, notably in blood-based protein quantification tests or immunohistochemistry. The rise of interest in epigenetic mechanisms has allowed the identification of a new type of biomarker, DNA methylation, which is of great potential for many applications. This stable and heritable covalent modification mostly affects cytosines in the context of a CpG dinucleotide in humans. It can be detected and quantified by a number of technologies including genome-wide screening methods as well as locus- or gene-specific high-resolution analysis in different types of samples such as frozen tissues and FFPE samples, but also in body fluids such as urine, plasma, and serum obtained through non-invasive procedures. In some cases, DNA methylation based biomarkers have proven to be more specific and sensitive than commonly used protein biomarkers, which could clearly justify their use in clinics. However, very few of them are at the moment used in clinics and even less commercial tests are currently available. The objective of this review is to discuss the advantages of DNA methylation as a biomarker, the practical considerations for their development, and their use in disease detection, prediction of outcome or treatment response, through multiple examples mainly focusing on cancer, but also to evoke their potential for complex diseases and prenatal diagnostics.

Sensitive Detection of KRAS Mutations Using Enhanced‐ice‐COLD‐PCR Mutation Enrichment and Direct Sequence Identification

A number of methods allowing the detection of low levels of KRAS mutations have been developed in the last years. However, although these methods have become increasingly sensitive, they can rarely identify the mutated base directly without prior knowledge on the mutated base and are often incompatible with a sequencing‐based read‐out desirable in clinical practice. Here, we present a modified version of the ice‐COLD‐PCR assay called Enhanced‐ice‐COLD‐PCR (E‐ice‐COLD‐PCR) for KRAS mutation detection and identification, which allows the enrichment of the six most frequent KRAS mutations. The method is based on a nonextendable chemically modified blocker sequence, complementary to the wild‐type (WT) sequence leading to the enrichment of mutated sequences. This assay permits the reliable detection of down to 0.1% mutated sequences in a WT background. A single genotyping assay of the amplification product by pyrosequencing directly following the E‐ice‐COLD‐PCR is performed to identify the mutated base. This developed two‐step method is rapid and cost‐effective, and requires only a small amount of starting material permitting the sensitive detection and sequence identification of KRAS mutations within 3 hr. This method is applied in the current study to clinical colorectal cancer samples and enables detection of mutations in samples, which appear as WT using standard detection technologies.

Methylation of MGMT in malignant pleural mesothelioma occurs in a subset of patients and is associated with the T allele of the rs16906252 MGMT promoter SNP

Silencing of the DNA repair gene O⁶-methylguanine DNA methyltransferase (MGMT) by promoter methylation is an early event in several human cancers. MGMT removes alkyl adducts from the O⁶ position of guanine thereby preventing G>A mutations in the genome. For this reason, MGMT promoter methylation predicts a favorable outcome for glioblastoma patients treated with alkylating agents. In this study, we investigated whether MGMT becomes silenced by promoter methylation in malignant pleural mesothelioma (MPM), an aggressive cancer of the pleura associated with a poor prognosis. Ninety-five samples from patients diagnosed with MPM were studied. These samples were genotyped for the MGMT rs16906252 promoter SNP using high-resolution melting, and methylation status was analyzed using SMART-MSP and confirmed by Sanger sequencing. The SMART-MSP assay was designed to provide information on the allelic methylation status in samples heterozygous for rs16906252. MGMT immunohistochemistry was performed on samples showing no methylation, monoallelic methylation, and biallelic methylation. Thirteen of the 95 MPM samples (13.7%) were methylation positive and a strong association with the T allele of the rs16906252 SNP (P<0.001) was observed. Detection of the protein was found to be dependent not only on the allelic methylation status but also on the methylation level, and complete silencing was observed in only one sample, showing biallelic methylation and a methylation level close to 100%. In conclusion, methylation of the MGMT promoter occurs in a subset of MPM patients and is associated with the T allele of the MGMT rs16906252 SNP. However, complete silencing of MGMT in MPM is a rare event.

Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing

The tumor suppressor genes MGMT and DAPK1 become methylated in several cancers including diffuse large B-cell lymphoma (DLBCL). However, allelic methylation patterns have not been investigated in DLBCL. We developed a fast and cost-efficient method for the analysis of allelic methylation based on pyrosequencing of methylation specific PCR (MSP) products including a SNP. Allelic methylation patterns were reliably analyzed in standards of known allelic methylation status even when diluted in unmethylated DNA to below 1% methylation. When studying 148 DLBCL patients MGMT and DAPK1 methylation was observed in 19% and 89%, respectively, and among methylated and heterozygous patients 29% and 55%, respectively, were biallelically methylated. An association between the T-allele of the rs16906252 SNP and MGMT methylation was observed (p-value = 0.04), and DAPK1 methylation of the A-allele was associated with shorter overall survival (p-value = 0.006). In future cancer research allelic MSP-pyrosequencing may be used to study a wide range of other loci.

Epigenetic changes in inflammatory and autoimmune diseases.

In higher eukaryotic organisms epigenetic modifications are crucial for proper chromatin folding and thereby proper regulation of gene expression. In the last years the involvement of aberrant epigenetic modifications in inflammatory and autoimmune diseases has been recognized and attracted significant interest. However, the epigenetic mechanisms underlying the different disease phenotypes are still poorly understood. As autoimmune and inflammatory diseases are at least partly T cell mediated, we will provide in this chapter an introduction to the epigenetics of T cell differentiation followed by a summary of the current knowledge on aberrant epigenetic modifications that dysfunctional T cells display in various diseases such as type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, and asthma.

Whole-exome sequencing and genome-wide methylation analyses identify novel disease associated mutations and methylation patterns in idiopathic hypereosinophilic syndrome

A thorough understanding of the idiopathic hypereosinophilic syndrome (IHES) and further optimization of diagnostic work-up procedures are warranted. We analyzed purified eosinophils from patients with IHES by next-generation whole-exome sequencing and compared DNA methylation profiles from reactive eosinophilic conditions to known clonal and suspected clonal eosinophilia. Somatic missense mutations in cancer-related genes were detected in three IHES patients. These included the spliceosome gene PUF60 and the cadherin gene CDH17. Furthermore, reactive eosinophilia samples could be differentiated from known- and suspected clonal eosinophilia samples based on 285 differentially methylated CpG sites corresponding to 128 differentially methylated genes. Using Ingenuity pathway analysis, we found that differentially methylated genes were highly enriched in functional pathways such as cancer, cell death and survival, and hematological disease. Our data show that a subset of IHES may be of clonal origin not related to the classical molecular aberrations of FGFR, PDGFRA/B, or T-cells, and that the initiating hits could be point mutations in a variety of genes, including spliceosome mutations or hypermethylated tumor suppressor genes. In addition, we identified a DNA methylation signature that is relevant for distinguishing clonal and suspected clonal eosinophilia from reactive eosinophilia per se, which may be useful in daily clinical work.

Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors.

Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conns adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof.

Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations

This is the first study to compare genome-wide DNA methylation profiles of sorted blood cells from myelofibrosis (MF) patients and healthy controls. We found that differentially methylated CpG sites located to genes involved in ‘cancer’ and ‘embryonic development’ in MF CD34+ cells, in ‘inflammatory disease’ in MF mononuclear cells, and in ‘immunological diseases’ in MF granulocytes. Only few differentially methylated CpG sites were common among the three cell populations. Mutations in the epigenetic regulators ASXL1 (47%) and TET2 (20%) were not associated with a specific DNA methylation pattern using an unsupervised approach. However, in a supervised analysis of ASXL1 mutated versus wild-type cases, differentially methylated CpG sites were enriched in regions marked by histone H3K4me1, histone H3K27me3, and the bivalent histone mark H3K27me3 + H3K4me3 in human CD34+ cells. Hypermethylation of selected CpG sites was confirmed in a separate validation cohort of 30 MF patients by pyrosequencing. Altogether, we show that individual MF cell populations have distinct differentially methylated genes relative to their normal counterparts, which likely contribute to the phenotypic characteristics of MF. Furthermore, differentially methylated CpG sites in ASXL1 mutated MF cases are found in regulatory regions that could be associated with aberrant gene expression of ASXL1 target genes.

Abstract B28: Investigation of MGMT methylation patterns in diffuse large B-cell lymphoma using a novel allelic methylation-specific PCR pyrosequencing assay

MGMT is a tumor suppressor gene, which becomes methylated in many different cancers including diffuse large B-cell lymphoma (DLBCL). Methylation of MGMT has been associated with the T-allele of the rs16906252 SNP found in the MGMT promoter. However, allelic MGMT methylation patterns and a possible association with the T-allele of this SNP have not been investigated in DLBCL. Today, standard treatment for DLBCL patients is rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and methylation of MGMT has been shown to predict chemosensitivity to the alkylating agent cyclophosphamide in the era before rituximab. We developed a novel method for the analysis of allelic MGMT methylation patterns based on pyrosequencing of antisense methylation specific PCR (MSP) products including the rs16906252 SNP. Samples from 148 DLBCL patients, of which 75 received R-CHOP, were studied using this assay. Standard bisulfite pyrosequencing and bisulfite sequencing of single clones were used to confirm data obtained by allelic MSP pyrosequencing. The allelic MSP pyrosequencing assay could reliably analyze allelic methylation patterns in standards of known allelic methylation status even when diluted in unmethylated DNA down to 1.25% methylation. MGMT methylation was observed for 28 patients of which seven were heterozygous. Thus, an association between the T-allele and MGMT methylation was observed (p-value =0.04). Among the seven methylated and heterozygous patients, two were methylated at both alleles, four were methylated only at the T-allele, and one only at the C-allele. Finally, we found no statistical significant associations between MGMT methylation or SNP genotypes with clinical parameters among the R-CHOP treated patients. In conclusion, allelic MSP pyrosequencing is a fast and cost-efficient method for evaluation of allelic methylation patterns, which may have wide implications for locus specific methylation analysis of tumor suppressor genes in future cancer research. Citation Format: Lasse Sommer Kristensen, Marianne Bach Treppendahl, Mia Seremet Girkov, Fazila Asmar, Helene Myrtue Nielsen, Tina Kjeldsen, Elisabeth Ralfkiaer, Lise Lotte Hansen, Kirsten Gronbaek. Investigation of MGMT methylation patterns in diffuse large B-cell lymphoma using a novel allelic methylation-specific PCR pyrosequencing assay. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B28.