Abstract P2-23-07: DNA Methylation Analysis of Triple Negative Breast Cancers
Authors: Lin Lawrence H. , Tran Ivy , Yang Yiying , Cotzia Paolo , Roses Daniel , Schnabel Freya , Darvishian Farbod , Snuderl Matija 
Published: March 2023

Abstract
Background: Triple negative breast cancers (TNBC) are characterized by the absence of estrogen receptors, progesterone receptors, and HER2 receptors on immunohistochemical analysis (IHC). This subtype of breast cancer has been traditionally associated with unfavorable prognosis. TNBC includes morphologically diverse mammary carcinomas including apocrine, metaplastic, medullary and other less common variants with heterogeneous clinical behavior and outcome. Despite previous efforts to further characterize TNBC with genomic and transcriptomic techniques, there is no specific targeted therapy for TNBC. Cytotoxic chemotherapy remains the mainstay of systemic treatment for these patients. DNA methylation has proved to be a promising tool in classifying a variety of cancers. In this study, we aim to analyze TNBC using comprehensive DNA methylation profiling. Method: DNA methylation profiling was performed in TNBC cases from a single academic institution using Illumina Infinium MethylationEPIC Kit. The tumors were obtained from formalin-fixed paraffin-embedded tissue from surgical specimens. Unsupervised clustering analysis based on the methylation profile was performed. Clinical, pathological, and genetic features were analyzed between the clusters. Results: We analyzed 44 cases (all female; median age 61 years) with treatment naïve TNBC diagnosed from March 2011 to April 2018. Median follow-up time was 58 months. Thirty-four (77%) patients were identified as white with the remaining 10 (23%) as non-white. Thirty-four tumors (77%) were classified as invasive ductal carcinoma of no special type, six (14%) as apocrine carcinoma, and four (9%) as metaplastic carcinoma. Six tumors (14%) were grade 2 and 38 (86%) were grade 3. Lymphovascular invasion was noted in 7 patients (16%). Tumor size ranged from 4 to 45 mm (median: 20 mm). Lymph node involvement was identified in 8 (18%) patients. Eleven (25%) patients harbored germline BRCA1/2 mutations. During the follow up period, 7 patients developed recurrent disease: 3 had local recurrences, 4 patients were found to have metastatic disease, and 1 patient had both local and distant recurrence. At last follow-up, 34 patients (77%) showed no evidence of disease, while 4 (9%) were alive with disease and 5 (11%) had died of disease. In this cohort, we identified three distinct DNA methylation clusters. In Cluster 1 (n=9), the patients were significantly older (mean age: 72 years; p=0.008) and tumors were more likely to be of apocrine morphology (56%, p= 0.001), of lower grade (55% were grade 3; p=0.009), and showed lower proliferation index (mean ki-67: 32%; p= 0.002). Cluster 3 (n=28) included younger patients (mean: 55 years) and tumors with higher grade (92% were grade 3) and proliferation index (mean ki-67: 75%). Cluster 2 (n=7) represented cases with intermediate features between Clusters 1 and 3. All patients in Cluster 1 were white, while Clusters 2 and 3 included non-white women. Cluster 1 included a significantly higher percentage of HER2-low tumors (HER2 1+ or 2+ by IHC and negative fluorescence in situ hybridization) (p=0.03; cluster 1: 89%, cluster 2: 28%, cluster 3: 46%). The vast majority of patients with germline BRCA1/2 mutation were found in Cluster 3 (67% of patients with genetic testing in Cluster 3 had germline BRCA1/2 mutation). There was no difference between the clusters in relation to stage, recurrence, and outcome. Conclusion: DNA methylation profiling is a promising tool to classify TNBC patients into clinicopathologically relevant groups, and we are continuing to expand this cohort. Classification by DNA methylation profile may result in better risk stratification for TNBC patients, which can inform their system therapy. In addition, specific methylation profiles have the potential to lead to the development of specific targeted therapies to improve the prognosis and survival for these patients.
Citation Format: Lawrence H. Lin, Ivy Tran, Yiying Yang, Paolo Cotzia, Daniel Roses, Freya Schnabel, Farbod Darvishian, Matija Snuderl. DNA Methylation Analysis of Triple Negative Breast Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-07.