Published OnlineFirst October 7, 2014; DOI: 10.1158/1078-0432.CCR-14-0297 Clinical Cancer Personalized Medicine and Imaging Research DNA Methylation Status of Key Cell-Cycle Regulators Such as CDKNA2/p16 and CCNA1 Correlates with Treatment Response to Doxorubicin and 5-Fluorouracil in Locally Advanced Breast Tumors Jovana Klajic1,2,3, Florence Busato4, Hege Edvardsen3, Nizar Touleimat4, Thomas Fleischer2,3, Ida Bukholm5,6, Anne-Lise Børresen-Dale2,3, Per Eystein Lønning7,8,Jorg€ Tost4, and Vessela N. Kristensen1,2,3 Abstract Purpose: To explore alterations in gene promoter methylation as a potential cause of acquired drug resistance to doxorubicin or combined treatment with 5-fluorouracil and mitomycin C in human breast cancers. Experimental Design: Paired tumor samples from locally advanced breast cancer patients treated with doxorubicin and 5-fluorouracil-mitomycin C were used in the genome-wide DNA methylation analysis as discovery cohort. An enlarged cohort from the same two prospective studies as those in the discovery cohort was used as a validation set in pyrosequencing analysis. Results: A total of 469 genes were differentially methylated after treatment with doxorubicin and revealed a significant association with canonical pathways enriched for immune cell response and cell-cycle regulating genes including CDKN2A, CCND2, CCNA1, which were also associated to treatment response. Treatment with FUMI resulted in 343 differentially methylated genes representing canonical pathways such as retinoate biosynthesis, gai signaling, and LXR/RXR activation. Despite the clearly different genes and pathways involved in the metabolism and therapeutic effect of both drugs, 46 genes were differentially methylated before and after treatment with both doxorubicin and FUMI. DNA methylation profiles in genes such as BRCA1, FOXC1, and IGFBP3, and most notably repetitive elements like ALU and LINE1, were associated with TP53 mutations status. Conclusion: We identified and validated key cell-cycle regulators differentially methylated before and after neoadjuvant chemotherapy such as CDKN2A and CCNA1 and reported that methylation patterns of these genes may be potential predictive markers to anthracycline/mitomycine sensitivity. Clin Cancer Res; 1–10. Ó2014 AACR. Introduction 1Division of Medicine, Department of Clinical Molecular Biology and Initially implemented for locally advanced breast cancers, Laboratory Science (EpiGen), Akershus University Hospital, Lørenskog, primary (presurgery) systemic therapy is nowadays increas- 2 Norway. K.G. Jebsen Center for Breast Cancer Research, Institute for ingly used for operable breast carcinomas. Multiple studies Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway. 3Department of Genetics, Institute for Cancer Research, OUS Radiumhos- have correlated a pathologic complete response to primary pitalet Montebello, Oslo, Norway. 4Laboratory for Epigenetics and Envi- chemotherapy with improved prognosis (1). However, ronment, Centre National de Genotypage, CEA—Institut de Genomique, although factors like hormone receptor status, HER2 over- Evry, France. 5Department of Surgery, Akerhus University Hospital, Oslo, Norway. 6Institute for Clinical Medicine, Faculty of Medicine, University expression, and gene expression signatures do correlate with of Oslo, Norway. 7Section of Oncology, Institute of Clinical Science, pathologic complete response (2, 3), the predictive power University of Bergen, Bergen, Norway. 8Department of Oncology, Hauke- land University Hospital, Bergen, Norway. of each parameter is modest, and the cause of drug resis- tance remains poorly understood (4). TP53 mutation status Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). is another strong prognostic factor, being associated with worse prognosis after treatment with anthracycline as well Corresponding Author: Vessela N. Kristensen, Faculty of Medicine, Insti- tute for Clinical Medicine, University of Oslo, 0372, Oslo, Norway. Phone: as mitomycin-containing chemotherapy (5, 6). 47-22-78-13-75/47-920-68-432; Fax: 47-22-78-13-95; E-mail: Beside genetic abnormalities, epigenetic alterations [email protected] may contribute to breast carcinogenesis and tumor doi: 10.1158/1078-0432.CCR-14-0297 growth. A large number of genes have been shown to be Ó2014 American Association for Cancer Research. inactivated in breast cancer through gene silencing by www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst October 7, 2014; DOI: 10.1158/1078-0432.CCR-14-0297 Klajic et al. Discovery cohort (genome-wide screening) Translational Relevance Paired tumor samples from locally advanced breast can- We investigated treatment-associated changes in cer were taken before and after neoadjuvant treatment. The the DNA methylation profiles of locally advanced study comprised 19 tumors treated with doxorubicin and breast cancer patients treated with doxorubicin and 14 tumors treated with FUMI. These patients were admitted 0 5 fluorouracil-mitomycin C, prior and after neoadju- to the Haukeland University Hospital in Bergen, Norway vant chemotherapy and validates these profiles in a between 1991 and 2001 (5, 6). DNA from nine normal larger cohort. We identify and validate methylation breast tissues were collected at the Akershus University patterns of key cell-cycle regulators such as CDKN2A Hospital (Lørenskog, Norway) and included as a control and CCNA1 as potential predictive markers to anthra- of DNA methylation background to which tumor-specific cycline/mitomycine sensitivity. We report on the asso- methylation events are compared with. ciation of differentially methylated genes before and after treatment with canonical pathways enriched for Validation cohort cell-cycle regulating genes and immune cell response. An enlarged cohort comprising 85 paired tumors treated WedemonstrateanassociationbetweenTP53 muta- with doxorubicin and 39 paired tumors treated with FUMI tion status and DNA methylation levels in repetitive from the same two prospective studies as those in the elements like ALU and LINE1 and suggest that meth- discovery cohort was used as a validation set. Samples that ylation levels of these genes together with TP53 status were used for 27K arrays were also among those in valida- could be of value in predicting response to chemo- tion cohort. Clinical and molecular parameters were avail- therapy treatment. We also show an association of able for all samples. DNA from fresh-frozen tumor tissues CDKN2A to molecular subtypes and suggest that the was used in both cohorts. Study design is shown in Fig. 1. Luminal B subtype could be used in defining a group, which will benefit from chemotherapy treatment. Classification of Treatment Response The protocols enrolled patients between 1991 and 2001. Response to therapy was classified by the UICC system DNA methylation. Such findings include genes involved commonly applied at that time (11) and not the more in cell-cycle regulation (CDKN2A, CCND2), DNA repair recent RECIST system (12). Responses were classified as CR (MGMT, BRCA1, MLH1, GSTP1), regulation of cell tran- (complete response-complete disappearance of all tumor scription (HOXA5), hormone and receptor-mediated cell lesions), PR (partial response reduction 50% in the sum of signaling (ER and THRb), apoptosis, and tissue invasion all tumor lesions), PD (progressive disease-increase in the and metastasis (RASSF1A, RARb,TWIST,HIN1,CDH1; diameter of any individual tumor lesion by 25%), and ref. 7). Epigenetic silencing of these genes by DNA meth- StbD (stable disease; the condition between PR and PD). ylation is frequent and, in contrast with genetic mutation, Similar to what has been done for previous reports on these reversible (8, 9), making them potential candidates for materials (5, 6), we compared PD tumors as nonresponders pharmacologic manipulation, for example, through tar- with the combined group of tumors classified as StbD/PR as geted inhibition of DNA methyltransferases. Experimen- responders. tal studies have indicated drug-induced alterations in gene promoter methylation as a potential cause of TP53 analysis acquired drug resistance (10). Mutations in the TP53 gene were analyzed with use of The aim of this study was to explore alterations in gene genomic DNA and the temporal temperature gradient gel promoter methylation as a potential cause of acquired drug electrophoresis (TTGE) strategy (5, 6). DNA fragments resistance to doxorubicin or combined treatment with 5- covering exons 2–11 were amplified, all with a GC clamp fluorouracil (5-FU) and mitomycin C (FUMI) in human on one of the primers, and submitted to analysis by TTGE. breast cancers. To do so, we took advantage of our tissue Mutations of the TP53 gene were correlated with response to bank providing unique access to paired tumor samples chemotherapy with use of the c2 method, including Yates collected from the same patients before and after primary correction for a limited number of observations. In addi- treatment with doxorubicin or FUMI. We used the Illumina tion, the differences in the distribution of TP53 mutation Infinium 27K Human DNA Methylation BeadChip to assess among patients revealing a PD and the remaining groups to which degree the genome-wide methylation status is were analyzed with the use of Fisher exact test. associated with treatment response. Methylation