Supplementary Materials and Methods, Figures 1 -9, Tables 1 - 4 from Novel Methylated Biomarkers and a Robust Assay to Detect Circulating Tumor DNA in Metastatic Breast Cancer

Fackler, Mary Jo; Lopez Bujanda, Zoila; Umbricht, Christopher; Teo, Wei Wen; Cho, Soonweng; Zhang, Zhe; Visvanathan, Kala; Jeter, Stacie; Argani, Pedram; Wang, Chenguang; Lyman, Jaclyn P.; de Brot, Marina; Ingle, James N.; Boughey, Judy; McGuire, Kandace; King, Tari A.; Carey, Lisa A.; Cope, Leslie; Wolff, Antonio C.; Sukumar, Saraswati

doi:10.1158/0008-5472.22402176.v1

00085472can133392-sup-can-13-3392matsmetstabs1-4figs1-9.pdf (1.49 MB)

Supplementary Materials and Methods, Figures 1 -9, Tables 1 - 4 from Novel Methylated Biomarkers and a Robust Assay to Detect Circulating Tumor DNA in Metastatic Breast Cancer

journal contribution

posted on 2023-03-30, 22:31 authored by Mary Jo Fackler, Zoila Lopez Bujanda, Christopher Umbricht, Wei Wen Teo, Soonweng Cho, Zhe Zhang, Kala Visvanathan, Stacie Jeter, Pedram Argani, Chenguang Wang, Jaclyn P. Lyman, Marina de Brot, James N. Ingle, Judy Boughey, Kandace McGuire, Tari A. King, Lisa A. Carey, Leslie Cope, Antonio C. Wolff, Saraswati Sukumar

PDF file - 1559KB, This file contains detailed methods, tables of study samples and source, primer and probe sequences,and genes belonging to the panel. Supplementary Table 1. Patient sample sets used in this study. Supplementary Table 2. 10-gene marker panel and its performance. Supplementary Table 3A. cMethDNA and QM-MSP primer and probe sequences. Supplementary Table 3B. cMethDNA and QM-MSP primer and probe sequences. Supplementary Table 3C. cMethDNA and QM-MSP primer and probe sequences. Supplementary Table 4. Comparison and reproducibility of the cMethDFNA assay using three DNA purification methods. Supplementary Figure 1. Infinium Human Methylation27 tissue DNA methylation array profile of the 10-gene marker panel. Supplementary Figure 2. Illumina Infinium Human Methylation27 serum DNA array profile of the 10-gene marker panel. Supplementary Figure 3. cMethDNA assay using a 6-gene marker panel. Supplementary Figure 4. Scatter plot and data analysis of cumulative methylation of the 10-gene panel in normal serum DNA. Supplementary Figure 5. Monitoring of treatment response by the cMethDNA assay. Supplementary Figure 6. Methylation Profiles in serum, primary, and metastatic tumor DNA. Supplementary Figure 7. Methylation profiles of serum, primary tumor and/or metastasis in patients with Stage 4 breast cancer. Supplementary Figure 8A. Performance of the 10-gene panel in arrays of a variety of tumor types. Supplementary Figure 8B. Performance of the 10-gene panel in arrays of a variety of tumor types. Supplementary Figure 8C. Performance of the 10-gene panel in arrays of a variety of tumor types. Supplementary Figure 8D. Performance of the 10-gene panel in arrays of a variety of tumor types. Supplementary Figure 8E. Performance of the 10-gene panel in arrays of a variety of tumor types. Supplementary Figure 8F. Performance of the 10-gene panel in arrays of a variety of tumor types. Supplementary Figure 9. Representative examples of amplification plots of cMethDNA real-time PCR.

History

ARTICLE ABSTRACT

The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. We developed cMethDNA, a quantitative multiplexed methylation-specific PCR assay for a panel of ten genes, consisting of novel and known breast cancer hypermethylated markers identified by mining our previously reported study of DNA methylation patterns in breast tissue (103 cancer, 21 normal on the Illumina HumanMethylation27 Beadchip) and then validating the 10-gene panel in The Cancer Genome Atlas project breast cancer methylome database. For cMethDNA, a fixed physiologic level (50 copies) of artificially constructed, standard nonhuman reference DNA specific for each gene is introduced in a constant volume of serum (300 μL) before purification of the DNA, facilitating a sensitive, specific, robust, and quantitative assay of tumor DNA, with broad dynamic range. Cancer-specific methylated DNA was detected in training (28 normal, 24 cancer) and test (27 normal, 33 cancer) sets of recurrent stage IV patient sera with a sensitivity of 91% and a specificity of 96% in the test set. In a pilot study, cMethDNA assay faithfully reflected patient response to chemotherapy (N = 29). A core methylation signature present in the primary breast cancer was retained in serum and metastatic tissues collected at autopsy two to 11 years after diagnosis of the disease. Together, our data suggest that the cMethDNA assay can detect advanced breast cancer, and monitor tumor burden and treatment response in women with metastatic breast cancer. Cancer Res; 74(8); 2160–70. ©2014 AACR.