Hannes M. Müller

Association of Breast Cancer DNA Methylation Profiles with Hormone Receptor Status and Response to Tamoxifen

We have generated DNA methylation profiles of 148 human breast tumors and found significant differences in hormone receptor (HR) status between clusters of DNA methylation profiles. Of 35 DNA methylation markers analyzed, the ESR1 gene, encoding estrogen receptor α, proved to be the best predictor of progesterone receptor status, whereas methylation of the PGR gene, encoding progesterone receptor, was the best predictor of estrogen receptor status. ESR1 methylation outperformed HR status as a predictor of clinical response in patients treated with the antiestrogen tamoxifen, whereas promoter methylation of the CYP1B1 gene, encoding a tamoxifen- and estradiol-metabolizing cytochrome P450, predicted response differentially in tamoxifen-treated and nontamoxifen-treated patients. High levels of promoter methylation of the ARHI gene, encoding a RAS-related small G-protein, were strongly predictive of good survival in patients who had not received tamoxifen therapy. Our results reveal an as yet unrecognized degree of interaction between DNA methylation and HR biology in breast cancer cells and suggest potentially clinically useful novel DNA methylation predictors of response to hormonal and non-hormonal breast cancer therapy.

Methylation changes in faecal DNA: a marker for colorectal cancer screening?

DNA methylation is a common molecular alteration in colorectal cancer cells. We report an assessment of faecal DNA from patients with colorectal cancer and controls to determine the feasibility, sensitivity, and specificity of this approach. By use of MethyLight analysis of faecal DNA from three independent sets of patients, we identified SFRP2 methylation as a sensitive single DNA-based marker for identification of colorectal cancer in stool samples (sensitivity 90% [CI 56-100] and specificity 77% [46-95] in the training set [n=23]; sensitivity 77% [46-95] and specificity 77% [46-95] in an independent test set [n=26]). Whether a combination of genetic and epigenetic markers will identify colorectal cancer at an early stage remains to be shown.

DNA Hypomethylation and Ovarian Cancer Biology

Hypomethylation of some portions of the genome and hypermethylation of others are very frequent in human cancer. The hypomethylation often involves satellite 2 (Sat2) DNA in the juxtacentromeric (centromere-adjacent) region of chromosome 1. In this study, we analyzed methylation in centromeric and juxtacentromeric satellite DNA in 115 ovarian cancers, 26 non-neoplastic ovarian specimens, and various normal somatic tissue standards. We found that hypomethylation of both types of satellite DNA in ovarian samples increased significantly from non-neoplastic toward cancer tissue. Furthermore, strong hypomethylation was significantly more prevalent in tumors of advanced stage or high grade. Importantly, extensive hypomethylation of Sat2 DNA in chromosome 1 was a highly significant marker of poor prognosis (relative risk for relapse, 4.1, and death, 9.4) and more informative than tumor grade or stage. Also, comparing methylation of satellite DNA and 15 5′ gene regions, which are often hypermethylated in cancer or implicated in ovarian carcinogenesis, we generally found no positive or negative association between methylation changes in satellite DNA and in the gene regions. However, hypermethylation at two loci, CDH13 (at 16q24) and RNR1 (at 13p12), was correlated strongly with lower levels of Sat2 hypomethylation. The CDH13/Sat2 epigenetic correlation was seen also in breast cancers. We conclude that satellite DNA hypomethylation is an important issue in ovarian carcinogenesis as demonstrated by: (a) an increase from non-neoplastic tissue toward ovarian cancer; (b) an increase within the ovarian cancer group toward advanced grade and stage; and (c) the finding that strong hypomethylation was an independent marker of poor prognosis.

DNA Methylation in Serum and Tumors of Cervical Cancer Patients

Purpose: Promoter hypermethylation has been recognized to play an important role in carcinogenesis. Numerous studies have demonstrated tumor-specific alterations, such as aberrant promoter hypermethylation, in DNA recovered from plasma or serum of patients with various malignancies. The aim of this study was to investigate the methylation status of various genes in cervical cancer patients and their association with clinicopathological characteristics and outcome of the disease. Experimental Design: The methylation status of CALCA, hTERT, MYOD1, PGR (progesterone receptor), and TIMP3 was investigated in serum samples from 93 cervical cancer patients and 19 corresponding tissue samples using the MethyLight technique. Results: Aberrant promoter hypermethylation was detected in any of these genes in 87% (81 of 93) of the serum samples studied. Methylation of MYOD1 was detected more frequently in advanced stage. All of the genes found to be methylated in serum samples were also methylated in the corresponding tissue sample, except in one patient. Patients with unmethylated MYOD1 serum DNA had significantly better disease-free (P = 0.04) and overall survival (P = 0.02) in comparison with patients with methylated MYOD1. Conclusions: To the best of our knowledge, this is, thus far, the largest study investigating aberrant promoter hypermethylation in serum samples from cancer patients and the first study investigating methylation patterns in sera of cervical cancer patients. Our results suggest that serological detection of MYOD1 promoter hypermethylation may be of potential use as a prognostic marker for discriminating cervical cancer patients at high risk for lymph node metastasis or relapse. Additional studies, including a panel of additional genes, are necessary to elucidate the role of aberrant methylation in serum as a tool for surveillance of cervical cancer.

Circulating nucleic acids in plasma or serum (CNAPS) as prognostic and predictive markers in patients with solid neoplasias

It is now widely accepted that there is a need for the development of molecular markers of cancer that can be used for clinical prognostication and monitoring. Approximately a decade ago tumor-derived circulating nucleic acids in the plasma or serum (CNAPS) of cancer patients were introduced as a noninvasive tool for cancer detection. This review focuses on the various types of CNAPS of patients with solid neoplasias (genetic alterations in circulating DNA, microsatellites, methylated DNA, viral DNA, nucleosomes, mitochondrial DNA and cell-free mRNA) and their putative potential as prognostic or predictive parameter or even as a tool for therapy monitoring during follow-up. Additionally, this review aims to point out the difference between a prognostic and a predictive factor in patient bloodstream. However, with rapid technical improvement and well-designed studies we conclude that the next years will see CNAPS analysis integrated in the prognostication and monitoring of cancer patients, thus producing more specific treatment regimens for patients with various stages of neoplastic disease and ultimately longer survival and better quality of life.

Analysis of Aberrant DNA Methylation and Human Papillomavirus DNA in Cervicovaginal Specimens to Detect Invasive Cervical Cancer and Its Precursors

Purpose: Cancer of the uterine cervix is an important cause of death in women worldwide. Pap smears as a tool for screening decreased the incidence and mortality of cervical cancer dramatically. This proof of principle study aimed to develop a potential tool for cervical screening using a test that can be applied by patients without visiting a physician and to increase the coverage rate, especially of the high-risk population with low socioeconomic status. Experimental Design: Human papillomavirus (HPV) DNA testing and methylation analysis of DNA obtained from cervicovaginal specimens of 13, 31, and 11 patients with no dysplasia/low-grade squamous intraepithelial lesion (SIL), high-grade SIL, and invasive cervical cancer, respectively, collected on a tampon, was performed using PCR-based methods to detect invasive cervical cancer and study whether these changes are already present in the precursor lesions. Results: High-risk HPV DNA was present in 68 and 82% of patients with high-grade SIL and invasive cervical cancer. DNA methylation of the 11 genes tested increased with severity of the cervical lesion. Unsupervised hierarchical cluster analysis using solely information on DNA methylation of the 11 genes was able to predict the presence of invasive cervical cancers: one of the two clusters formed contained 9 of 11 invasive cervical cancers, as well as two high-grade SILs. Conclusions: HPV DNA and DNA methylation analyzed in cervicovaginal specimens are able to predict invasive cervical cancers. To detect all high-grade SILs when applying this test, genes that become methylated earlier throughout cervical carcinogenesis have to be defined.

Prognostic DNA Methylation Marker in Serum of Cancer Patients

Abstract: Changes in the status of DNA methylation are among the most common molecular alterations in human neoplasia. Recent demonstrations of tumor‐derived methylated DNA in the blood stream of cancer patients allow the use of these epigenetic markers for risk assessment in cancer patients. We were interested in evaluating the prognostic value of several methylated genes in the serum of cancer patients. Using MethyLight, a high‐throughput DNA methylation assay, we analyzed 215 serum samples from patients with cervical (n= 93) or breast cancer (n= 122) for DNA methylation changes. In cervical cancer, hypermethylation of three genes (MYOD1, CDH1, and CDH13) in pretreatment sera was statistically significantly associated with a poorer disease outcome. Additionally, for the first time we used a so‐called gene evaluation set to identify the most important DNA methylation changes in the serum of breast cancer patients from a long list of candidate genes. In the gene evaluation set, we detected five genes (ESR1, APC, HSD17B4, HIC1, and RASSF1A) using our criteria for further analysis. Finally, two of the evaluated genes (APC and RASSF1A) proved to be independent prognostic parameters in breast cancer patients. In summary, we detected several prognostic DNA methylation markers in the serum of cervical and breast cancer patients. This finding indicates great potential for the use of these epigenetic markers in clinical, routine risk assessment in patients with various malignancies.

CDH1 and CDH13 methylation in serum is an independent prognostic marker in cervical cancer patients.

Cervical cancer is the principal cause of death due to cancer in women. Five‐year survival rate ranges from 15–80%, depending on the extent of the disease. New predictive markers for relapse may increase survival rates by improving treatment of patients at high risk for relapse. The gene products of CDH1 and CDH13, namely E‐cadherin and H‐cadherin, play a key role in cell–cell adhesion. Inactivation of the cadherin‐mediated cell adhesion system, caused by aberrant methylation, is a common finding in human cancers. To test the hypothesis that CDH1/CDH13 methylation is a prognostic marker in cervical cancer we determined the methylation status of CDH1/CDH13 in serum samples from 93 cervical cancer patients. Methylation analysis was carried out using MethyLight. Aberrant methylation of the 5′‐region of CDH1 or CDH13 was observed in 43% (40 of 93) of the patients. Cervical cancer patients with unmethylated CDH1/CDH13 in serum samples showed significantly better disease‐free survival in univariate and multivariate analysis. Median disease‐free survival for CDH1/CDH13 methylation negative and positive patients was 4.3 years and 1.2 years, respectively. Our results suggest that detection of aberrant methylation of CDH1/CDH13 may be of potential use as a marker for selecting cervical cancer patients at high risk for relapse who could benefit from additional systemic therapy.

Epigenetic downregulation of the retinoic acid receptor-beta2 gene in breast cancer.

A growing body of evidence supports the hypothesis that the retinoic acid receptor β2 (RAR-β2) gene is a tumor suppressor gene which induces apoptosis and that the chemopreventive and therapeutic effects of retinoids are due to induction of RAR-β2. During breast cancer progression, RAR-β2 is reduced or even lost. It is known from studies of other tumor-suppressor genes that methylation of the 5′-region is the cause of loss of expression. Several groups demonstrated that this is also true for the RAR-β2 in breast cancer by treating breast cancer cell lines with a demethylating agent and examining expression of the RAR-β2 gene in response to a challenge with retinoic acid. Studies using sodium bisulfite genomic sequencing as well as methylation specific PCR showed that a number of breast cancer cell lines as well as breast cancer tissue showed signs of methylation. The RAR-β2 gene was unmethylated in non-neoplastic breast tissue as well as in other normal tissues. A combination of retinoic acid with demethylating agents as well as with histone deacetylase inhibitors acts synergistically to inhibit growth. This review presents data that suggest that treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality. Both the time of commencement of chemoprevention and the choice of substances that are able either to prevent de novo methylation or to reverse methylation-caused gene silencing may be important considerations.

SFRP2 methylation in fecal DNA—a marker for colorectal polyps

IntroductionDNA methylation of secreted frizzled-related proteins (SFRPs) can be detected in colorectal cancer (CRC) tissue, in tissue of adenomas, and in aberrant crypt foci, whereas in normal colorectal mucosa tissue, SFRP genes are unmethylated. Recently, our study group was able to demonstrate SFRP2 methylation as the most sensitive single DNA-based marker in stool for identification of CRC. The purpose of this study was to clarify whether SFRP2 methylation in fecal DNA can be found in stool of individuals with hyperplastic and adenomatous colorectal polyps.Materials and methodsPatients who were diagnosed with colorectal polyps or showed negative colonoscopy were included in this study. DNA from stool samples was isolated. SFRP2 methylation was assessed by means of MethyLight.ResultsStool samples from 68 individuals were checked for DNA content; 23% of the samples (6 of 26) from healthy controls, 46% of the samples (6 of 13) from patients with hyperplastic polyps, and 45% of the samples (13 of 29) from patients with adenomas were positive for human DNA. SFRP2 methylation in stool samples was found in none of the healthy controls, in 33% (2 of 6) patients with hyperplastic polyps, and in 46% (6 of 13) patients with adenomas. Statistical analysis revealed that the frequency of SFRP2 methylation increased significantly (P = 0.028) from healthy controls to patients with hyperplastic polyps and to patients with adenomas.ConclusionsIn the current study, we report for the first time that SFRP2 methylation in fecal DNA increases significantly from healthy controls to patients with hyperplastic polyps and to patients with adenomas. SFRP2 methylation may serve as a marker for molecular stool-based adenoma and CRC screening.