Abstract A016: Identification of methylated regions in ductal carcinoma in situ and association with disease progression
Authors: Shah Vandna , Roman-Escorza Maria , Clements Karen Clements , Mulder Lennart , Lips Esther H. , Wesseling Jelle , Pinder Sarah , Thompson Alastair M. , Sawyer Elinor J. 
Published: December 2022

Abstract
Background: The advent of breast screening has led to a 4-fold increase in the diagnosis of ductal carcinoma in situ (DCIS). Studies following the clinical outcomes of patients show variable progression free survival rates, with only up to 35% of patients progressing to invasive disease without treatment. This highlights the need to find a biomarker to accurately predict which DCIS lesions will recur as invasive tumors. Epigenetics changes are events which occur early in tumorigenesis, this makes DNA methylation a potential biomarker of DCIS progression. However, a lack of DCIS methylation profiling with long term follow-up data exists. This study investigates genome-wide methylation profiles in women with primary DCIS and associates the data with their overall recurrence free survival. Methods: DCIS was macrodissected from 89 formalin-fixed paraffin embedded (FFPE) to extract tumor enriched DNA from patients with DCIS and long term follow up. 39 women had developed an ipsilateral invasive recurrence (classified as cases) and 50 had no evidence of recurrent disease (classified as controls). Genome wide methylation was assessed using the human methylation EPIC BeadChip which, interrogates over 850,000 methylation sites. Data was assessed for quality both at the sample and probe level by the wateRmelon. Further analysis was performed in ChAMP to identify differentially methylated regions (DMR) and by DMRcate packages in R to identify variably methylated regions (VMR). Genes annotated in the most significant VMRs were analysed through Metascape, a web-based tool to perform functional gene set enrichment analysis (GSEA). A cox proportional hazards model was used to calculate the association between the methylation of the VMRs and recurrence free survival. This model was adjusted for DCIS receptor status and grade. Results: 59 samples passed data quality assessment (35 controls and 24 cases). 10 differentially methylated regions were identified. The most significant of which, was a hypomethylated region on chromosome 4, containing CDKL2 (p = 0.001), known to promote the epithelial-mesenchymal transition in breast cancer progression. 5813 VMRs were identified across the genome, (P-value range between 0 and 10-321). 82% of the VMRs aligned to the body of the gene or the surrounding regulatory features. GSEA revealed that VMRs were predominantly involved in pathways involved in cell adhesion (GO:0007156) Assessment of the significant VMRs by COX proportional hazards model showed that a VMR on chromosome 6p was associated with the development of invasive disease after adjusting for oestrogen receptor, human epidermal growth factor 2 status and grade (p=0.001). Conclusions: This preliminary study shows altered sites of methylation could be observed across the genome, in DCIS. The function of the VMRs is currently being investigated to understand how methylation in this region predisposes to invasive recurrence of DCIS. Correlation of methylation status and RNA expression data will be used to understand the biological relevance.
Citation Format: Vandna Shah, Maria Roman-Escorza, Karen Clements Clements, Lennart Mulder, Esther H. Lips, Jelle Wesseling, Sarah Pinder, Alastair M. Thompson, Elinor J. Sawyer. Identification of methylated regions in ductal carcinoma in situ and association with disease progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr A016.