Alla Slynko

Influence of human papillomavirus and p16INK4a on treatment outcome of patients with anal cancer

BACKGROUND The purpose of this study was to evaluate HPV-DNA and p16(INK4a) (p16) expression as prognostic markers for outcome in patients with anal cancer. METHODS From January 2000 to December 2011 a cohort of 105 anal cancer patients was treated with definitive chemoradiation at our institution. Tumor biopsies from 90 patients were analyzed for HPV-DNA by polymerase chain reaction and for p16 expression by immunohistochemistry. RESULTS Median follow-up was 48.6months (range 2.8-169.1months). HPV-DNA or p16-expression was found in 75 anal cancers each (83.3%), concordance was detectable in 70 tumors (77.8%). Significantly improved overall survival (OS) [77.1% vs. 51.4%, p=0.005], progression-free survival (PFS) [64.0% vs. 35.0%, p<0.001] and improved local control [81.0% vs. 55.9%, p=0.023] was found for concomitant HPV- and p16-positive anal carcinomas (cHPPAC) in univariate analysis. Multivariate analysis showed better OS [p=0.015] and PFS [p=0.002] for cHPPAC. CONCLUSION The combination of HPV-DNA and p16 can be used as an independent prognostic parameter in anal cancer patients.

Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis

Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy.

Dissecting and modeling the emergent murine TEC compartment during ontogeny

The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage‐specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co‐expression of EpCAM, CD80 and MHC class II (MHCII) on non‐hematopoietic (CD45−) thymic stromal cells in wild‐type BL6 mice. Using a combination of ex vivo analysis, Re‐aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modeling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII−CD80− → MHCIIloCD80− → MHCIIhiCD80− → MHCIIhiCD80hi TECs, whereby MHCIIhiCD80− and MHCIIhiCD80hi TECs bear features of cTECs and mTECs respectively. These emergent MHCIIhiCD80− cTECs directly generate mature MHCIIhiCD80hi mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment.

DNA methylation array analysis identifies breast cancer associated RPTOR , MGRN1 and RAPSN hypomethylation in peripheral blood DNA

DNA methylation changes in peripheral blood DNA have been shown to be associated with solid tumors. We sought to identify methylation alterations in whole blood DNA that are associated with breast cancer (BC). Epigenome-wide DNA methylation profiling on blood DNA from BC cases and healthy controls was performed by applying Infinium HumanMethylation450K BeadChips. Promising CpG sites were selected and validated in three independent larger sample cohorts via MassARRAY EpiTyper assays. CpG sites located in three genes (cg06418238 in RPTOR, cg00736299 in MGRN1 and cg27466532 in RAPSN), which showed significant hypomethylation in BC patients compared to healthy controls in the discovery cohort (p < 1.00 × 10−6) were selected and successfully validated in three independent cohorts (validation I, n =211; validation II, n=378; validation III, n=520). The observed methylation differences are likely not cell-type specific, as the differences were only seen in whole blood, but not in specific sub cell-types of leucocytes. Moreover, we observed in quartile analysis that women in the lower methylation quartiles of these three loci had higher ORs than women in the higher quartiles. The combined AUC of three loci was 0.79 (95%CI 0.73-0.85) in validation cohort I, and was 0.60 (95%CI 0.54-0.66) and 0.62 (95%CI 0.57-0.67) in validation cohort II and III, respectively. Our study suggests that hypomethylation of CpG sites in RPTOR, MGRN1 and RAPSN in blood is associated with BC and might serve as blood-based marker supplements for BC if these could be verified in prospective studies.

Abstract 3445: Diacylglycerol kinase alpha as a novel epigenetically regulated risk marker for radiotherapy-induced fibrosis

Ionizing radiation is a common treatment option for cancer but its use is limited by the unpredictable and highly heterogeneous onset of late side effects, especially radiation-induced fibrosis. Clinically applicable biomarkers and effective treatments for radiation fibrosis are currently unavailable. In order to identify novel markers we ran a genome-wide DNA methylation screen in primary dermal fibroblasts obtained from breast cancer patients before intraoperative radiotherapy. Cells from patients developing fibrosis within a three-year follow up were compared to those without fibrosis (12 individuals per group). Illumina Infinium HumanMethylation450 BeadChip analysis revealed differentially methylated sites which are associated with fibrosis. Notably, we identified a differentially methylated region (DMR) at the diacylglycerol kinase alpha (DGKA) locus as a potential fibrosis marker. This DGKA DMR was confirmed using quantitative MassARRAY technology in 75 patient fibroblast samples. We first investigated whether high or low DNA methylation at this DGKA DMR affects cellular radiation response. Functional in vitro analysis showed that the methylation status of the DGKA DMR inversely correlated with its radiation-induced mRNA and protein expression as well as with its enzymatic activity. We next examined the DMR for its role as a regulatory site. The intragenically located DMR was identified as a potential enhancer sequence using chromatin immunoprecipitation (ChIP) for H3K4me1 and H3K27ac as well as luciferase reporter assays. Chromatin conformation capture (3C) analysis revealed interaction of this enhancer with the DGKA promoter in fibroblasts with low DNA methylation, and further ChIP experiments showed a DNA methylation-dependent recruitment of the profibrotic transcription factor Early Growth Response 1 (EGR1) to this site. We finally asked how epigenetically altered DGKA expression could impact on cellular processes relevant to fibrosis such as fibroblast transactivation or stress response. Results in primary fibroblasts showed that, in response to ionizing radiation and other stress factors, DGKA affects global levels of its substrate diacylglycerol, as well as expression of the fibroblast activation markers Alpha Smooth Muscle Actin (ACTA2) and collagen 1 (COL1A1). Upon overexpression of DGKA in HEK293T cells, a luciferase-based screening of 15 stress-responsive signaling reporters revealed functional consequences on several response pathways. In summary, DGKA has emerged as a novel, epigenetically regulated signaling protein that has a role in radiation fibrosis and may serve as a new biomarker and therapeutic target. Citation Format: Christoph Weigel, Marlon R. Veldwijk, Christopher C. Oakes, Petra Seibold, Alla Slynko, David B. Liesenfeld, Carsten Herskind, Elena Sperk, Axel Benner, Christoph Plass, Frederik Wenz, Jenny Chang-Claude, Peter Schmezer, Odilia Popanda. Diacylglycerol kinase alpha as a novel epigenetically regulated risk marker for radiotherapy-induced fibrosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3445. doi:10.1158/1538-7445.AM2015-3445