Elisabeth Müller-Holzner

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.

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.

Interferon-gamma in the first-line therapy of ovarian cancer: a randomized phase III trial

Intraperitoneal treatment with interferon-γ (IFN-γ) has been shown to achieve surgically documented responses in the second-line therapy of ovarian cancer. To assess its efficacy in the first-line therapy, we conducted a randomized controlled trial with 148 patients who had undergone primary surgery for FIGO stage Ic–IIIc ovarian cancer. In the control arm women received 100 mg m–2cisplatin and 600 mg m–2cyclophosphamide, the experimental arm included the above regimen with IFN-γ 0.1 mg subcutaneously on days 1, 3, 5, 15, 17 and 19 of each 28-day cycle. Progression-free survival at 3 years was improved from 38% in controls to 51% in the treatment group corresponding to median times to progression of 17 and 48 months (P = 0.031, relative risk of progression 0.48, confidence interval 0.28–0.82). Three-year overall survival was 58% and 74% accordingly (n.s., median not yet reached). Complete clinical responses were observed in 68% with IFN-γ versus 56% in controls (n.s.). Toxicity was comparable in both groups except for a mild flu-like syndrome, experienced by most patients after administration of IFN-γ. Thus, with acceptable toxicity, the inclusion of IFN-γ in the first-line chemotherapy of ovarian cancer yielded a benefit in prolonging progression-free survival.

Breast cancer DNA methylation profiles in cancer cells and tumor stroma: association with HER-2/neu status in primary breast cancer.

The HER-2/neu gene is amplified and overexpressed in 20% to 30% of invasive breast carcinomas and is associated with increased metastatic potential and less tamoxifen sensitivity. We generated the DNA methylation profiles of 143 human breast tumors and found significant differences in HER-2/neu expression and DNA methylation of five genes. For three of these five genes [PGR (coding for the progesterone receptor), HSD17B4 (coding for type 4 17-beta-hydroxysteroid dehydrogenase, an enzyme that mainly degrades active 17-beta-estradiol into inactive metabolites), and CDH13 (coding for H-cadherin)] a higher prevalence of DNA methylation in HER-2/neu-positive cancers was confirmed in an independent set of microdissected primary breast cancers. DNA methylation was not only present in cancer cells but also in the tumor stroma fraction. Of the isolated fractions in HER-2/neu-positive versus -negative cancers, 27.1% versus 10.5%, respectively, showed DNA methylation of the five genes (P = 0.011, Fishers exact test). In Her-2++/+++ breast cancers, HSD17B4 mRNA expression was inversely associated with HSD17B4 methylation (P = 0.04). These data support the view that in addition to HER-2/neu-associated signaling, epigenetic changes in cancer as well as in tumor stroma cells might attribute to the specific biological features of HER-2/neu-positive cancers.

Mammaglobin gene expression : A superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin-19

Various molecular markers have been used for the detection of circulating breast cancer cells in blood by reverse transcriptase-polymerase chain reaction (RT-PCR). Using nested RT-PCR, we compared the specificity and sensitivity of human mammaglobin (hMAM), epidermal-growth-factor receptor (EGF-R), and cytokeratin 19 (CK-19) expression as markers for circulating carcinoma cells in the peripheral blood of patients with breast cancer. Blood samples from 12 patients with ductal carcinoma in situ, 133 patients with invasive breast cancer, 20 patients with hematological malignancies, 31 healthy volunteers, and tumor tissues from 40 patients with invasive breast cancer were screened for mRNA encoding hMAM, EGF-R, or CK-19 by nested RT-PCR. In all breast cancer tissues, mRNA for hMAM, EGF-R, and CK-19 was detectable. In blood samples from patients with invasive breast cancer, 11 (8%), 13 (10%), and 64 (48%) were positive for mRNA encoding hMAM, EGF-R, or CK-19, respectively. Blood samples from none of the healthy volunteers and patients with hematological disorders were positive for hMAM, while CK-19 mRNA was found in the blood of 12 (39%) healthy volunteers and transcripts for EGF-R and CK-19 were detectable in 5 (25%) and 2 (10%), respectively, of the patients with hematological malignancies. Only hMAM mRNA expression in blood correlated with clinical parameters such as nodal status, metastasis, and CA 15–3 serum levels. In summary, hMAM transcripts detectable in blood by RT-PCR represent the most specific molecular marker for hematogenous spread of breast cancer cells. With the nested RT-PCR method, aberrant EGF-R mRNA expression might occasionally be found in hematological malignancies, whereas CK-19 mRNA expression proved to be rather nonspecific. The prognostic value of hMAM RT-PCR–based tumor cell detection in peripheral blood should be further tested and validated in prospective studies.

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.

DNA hypomethylation is prevalent even in low-grade breast cancers

Hypomethylation of some portions of the genome and hypermethylation of others are very frequent attributes of human cancer. We previously showed that cancer-associated DNA hypomethylation often involves satellite 2 (Sat2), the main DNA component of the large juxtacentromeric (centromere-adjacent) heterochromatin of chromosome 1. In this study, we compared methylation of Sat2 and centromeric satellite DNA (Sat?) as well as overall genomic methylation in 41 breast adenocarcinomas of known tumor grade and stage, 16 non-neoplastic breast tissues (mostly fibroadenomas), and a variety of normal somatic tissue controls. The cancers were significantly hypomethylated at Sat2 relative to the fibroadenomas or normal somatic tissues and at Sat? relative to the normal somatic tissues. However, unlike Sat2, Sat? did not display significant differences in methylation between the cancers and the non-neoplastic breast tissues. Therefore, hypomethylation at Sat2 is a much better marker of breast cancer than is Sat? hypomethylation. There was a significant association of Sat2 hypomethylation with global DNA hypomethylation in the cancers but not with tumor grade, stage, axillary lymph node involvement, or hormone receptor status. Extensive cancer-associated hypomethylation of juxtacentromeric satellite DNA and global DNA hypomethylation were common even in grade-1 or stage-1 carcinomas, which suggests that demethylation of the genome is an early event in breast carcinogenesis.

Spontaneous regression of CIN and delayed-type hypersensitivity to HPV-16 oncoprotein E7

We investigated delayed-type hypersensitivity to human papillomavirus (HPV) in women with cervical dysplasia or cancer. Women were challenged by skin tests with synthetic HPV-16 E7 oncoprotein peptides. 11 women were regressors (cleared disease without treatment) and 37 were progressors (required surgery). Antibodies to early antigens (markers for progression) were detectable in a higher proportion of cancer patients than all other patients, particularly progressors with cervical intraepithelial neoplasia (CIN). By contrast, cellular immunity to HPV-16 E7, measured by skin test, was significantly (p=0.0001) associated with clinical and cytological resolution of HPV-induced CIN, indicating that E7-specific T-helper cells have a role in control of HPV.

The PAM50 Risk-of-Recurrence Score Predicts Risk for Late Distant Recurrence after Endocrine Therapy in Postmenopausal Women with Endocrine-Responsive Early Breast Cancer

Purpose: To assess the prognostic value of the PAM50 risk-of-recurrence (ROR) score on late distant recurrence (beyond 5 years after diagnosis and treatment) in a large cohort of postmenopausal, endocrine-responsive breast cancer patients. Experimental Design: The PAM50 assay was performed on formalin-fixed paraffin-embedded whole-tumor sections of patients who had been enrolled in the Austrian Breast and Colorectal Cancer Study Group Trial 8 (ABCSG-8). RNA expression levels of the PAM50 genes were determined centrally using the nCounter Dx Analysis System. Late distant recurrence-free survival (DRFS) was analyzed using Cox models adjusted for clinical and pathologic parameters. Results: PAM50 analysis was successfully performed in 1,246 ABCSG-8 patients. PAM50 ROR score and ROR-based risk groups provided significant additional prognostic information with respect to late DRFS compared with a combined score of clinical factors alone (ROR score: ΔLRχ2 15.32, P < 0.001; ROR-based risk groups: ΔLRχ2 14.83, P < 0.001). Between years 5 and 15, we observed an absolute risk of distant recurrence of 2.4% in the low ROR-based risk group, as compared with 17.5% in the high ROR-based risk group. The DRFS differences according to the PAM50 ROR score were observed for both node-positive and node-negative disease. Conclusion: PAM50 ROR score and ROR-based risk groups can differentiate patients with breast cancer with respect to their risk for late distant recurrence beyond what can be achieved with established clinicopathologic risk factors. Clin Cancer Res; 20(5); 1298–305. ©2014 AACR.

Clinical Relevance of E2F Family Members in Ovarian Cancer—An Evaluation in a Training Set of 77 Patients

Purpose: The major obstacle in treating ovarian cancer is the rapid development of platinum resistance during therapy. Deregulation of members of the E2F family of transcription factors is crucially involved in carcinogenesis and probably in mechanisms underlying platinum resistance. We therefore investigated the relevance of the whole set of E2F family members in predicting clinical outcome and their significance in predicting platinum resistance. Experimental Design: Real-time PCR of all E2F family members was done from 77 ovarian carcinomas, defined as our training set, and 8 healthy control samples. The correlation with clinicopathologic characteristics, platinum resistance, and survival was investigated. Furthermore, the cross-talk of E2F family members was assessed for its value in predicting survival and platinum resistance. Results: The proliferation-promoting E2F1 and E2F2 were associated with grade 3 tumors and residual disease >2 cm in diameter after initial surgery. Survival analyses showed low expression of E2F1 or E2F2 to be significantly associated with favorable disease-free and overall survival (E2F1, P = 0.039 and 0.047, respectively; E2F2, P = 0.009 and 0.006, respectively). In contrast, high expression of inhibiting E2F4 or E2F7 predicted favorable disease-free and overall survival (E2F4, P = 0.047 and 0.042, respectively; E2F7, P = 0.048 and 0.042, respectively). A high E2F2 to E2F4 ratio was the most valuable prognostic variable for disease-free survival in multivariate analysis (hazard ratio, 6.494; P = 0.002). Tumors considered platinum resistant were associated with lower E2F4 and E2F7 expression (P = 0.012 and 0.009, respectively) compared with platinum-sensitive tumors. Again, ratios of E2F1 or E2F2 to E2F7 were the most favorable variables in predicting platinum resistance. Conclusions: We here show that deregulation of both proliferation-promoting and proliferation-inhibiting E2F transcription factors and their cross-talk is crucially involved in the tumor biology of ovarian cancer and influences clinical outcome. Furthermore, down-regulation of E2F7 may contribute to mechanisms underlying platinum resistance, and calculation of ratios of proliferation-promoting E2F1 to E2F7 could serve as a putative predictor of platinum resistance.