Abstract P6-03-07: An automated DNA methylation assay (QM-MSP) for rapid breast cancer diagnosis in underdeveloped countries
Authors: Fackler MJ , Downs BM , Mercado-Rodriguez C , Cimino-Mathews A , Chen C , Yuan J , Cope LM , Kohlway A , Kocmond K , Lai E , Weidler J , Visvanathan K , Umbricht CB , Harvey S , Wolff AC , Bates M , Sukumar S 
Published: February 2018

BACKGROUND: Underdeveloped countries reported 882,900 new cases of breast cancer and 324,000 deaths in 2012, likely to be a gross underestimation according to recent reports. Often, mammography screening is not available, primary care services are limited, and pathology and treatment services are available only in the regional hospitals. Because of the lack of access to diagnostic and treatment services, it is estimated that more than 90% of patients with breast cancer never present for medical treatment. To address this situation, an accurate, easy-to-perform diagnostic test appropriate for use in remote clinics is desperately needed. Johns Hopkins (JH) and Cepheid partnered to translate a robust Quantitative Multiplex Methylation-Specific PCR (QM-MSP) assay to an automated, cartridge-based system that provides quantitative measures of DNA methylation within hours of fine needle aspiration or core biopsy of image-detected suspicious lesions.

METHODS: With a goal of discriminating malignant from benign breast disease with high sensitivity and specificity, we evaluated 24 breast cancer-specific DNA methylation markers (selected through comprehensive methylome analysis) in 119 invasive ductal carcinomas and 186 benign breast tissues. QM-MSP was performed on sections of formalin-fixed paraffin-embedded (FFPE) tissues to quantify DNA methylation. The dynamic range and performance of quantitative methylation detection was tested using a subset of 9 genes in the cartridge-based system.

RESULTS: QM-MSP was performed in a Training set consisting of 93 tissues [n=43 IDC, n=50 benign lesions (25 usual ductal hyperplasia, UDH, and 25 papilloma)] from the US. We selected 9 DNA markers significantly (p&lt;0.05) more methylated in malignant compared to benign lesions, which had low or no methylation. An independent Test set consisted of benign (n=26) and malignant (n=10) tissues (mostly Caucasian; JH Test Set). As a panel, the 9 markers were significantly more methylated in malignant than benign tissue (p&lt;0.001), revealing a sensitivity of 90% and specificity of 92%, using a laboratory cutoff of 9.5 CMI units (900 unit scale) based on receiver operator characteristic statistics (ROC; p&lt;0.0001, AUC=0.977). To determine if the markers characterized in the JH Test Set could perform as well in samples from a different geography, the panel was tested on 176 tissues from Wuhan, China (China Test Set). In this cohort (66 IDC and 110 benign tissues - 49 fibroadenoma, 19 benign cyst, 12 UDH, 30 papilloma), sensitivity was 89% and specificity was 89% for detection of breast cancer with ROC AUC=0.945. An advanced version of the cartridge with up to 12 methylated DNA markers is under development, thus far showing robust signals in cancer and low background in benign tissues. Current work at JH is focused on optimizing the technical performance of the cartridge.

CONCLUSIONS: We identified a panel of methylated DNA markers that discriminate malignant from benign breast lesions and built a prototype automated cartridge-based assay with promising sensitivity and specificity for breast cancer. Such an assay has the potential to aid in specimen triage in the pathology lab and provide fast, low cost and accurate diagnosis of breast cancer in LMIC settings.

Citation Format: Fackler MJ, Downs BM, Mercado-Rodriguez C, Cimino-Mathews A, Chen C, Yuan J, Cope LM, Kohlway A, Kocmond K, Lai E, Weidler J, Visvanathan K, Umbricht CB, Harvey S, Wolff AC, Bates M, Sukumar S. An automated DNA methylation assay (QM-MSP) for rapid breast cancer diagnosis in underdeveloped countries [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-03-07.