Abstract Background: While mammography remains a standard screening tool for breast cancer, its high false positive rate and invasiveness of subsequent biopsies highlight the need for improved technologies. Liquid biopsy, leveraging circulating cell-free DNA (cfDNA), offers a noninvasive alternative, yet the low levels of cfDNA in blood demand highly sensitive detection techniques. In this study, we sought to improve our two-step lab assay, cMethDNA (1), by testing three commercial DNA isolation kits, and through innovative use of primer combinations to specifically enrich for methylated cfDNA in blood. Methods: Circulating cfDNA was isolated using three kits: QIAamp MinElute Virus Spin Kit (Virus kit), QIAamp MinElute ccfDNA Mini Kit (ccfDNA kit), and MAGicBead cfDNA Isolation Kit. A 9-gene panel consisting of AKR1B1, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1A, TM6SF1, TMEFF2, and ZNF671 (2) was amplified using bisulfite-converted DNA and optimized primer combinations in a cell free, multiplexed, methylation-specific PCR (cf-MMSP) assay. Technical validation of the assay was performed with normal plasma spiked with 12.5-50 copies of fully methylated human genomic DNA and 10 copies of STDHOXB4 plasmid. We quantified relative methylation levels for each gene, and cumulative methylation (CM) of all 9 markers in each sample. The cf-MMSP assay was validated in a pilot study of plasma samples from 21 stage IV breast cancer patients and 20 healthy controls or women with benign disease. Results: The Virus Spin kit outperformed the MAGicBead and ccfDNA kits in cfDNA recovery, showing a significant improvement in the detection of STDHOXB4 reference DNA (p < 0.0001 vs. MAGicBead). Using a combination of a methylation-agnostic (ExtF) and a methylation sequence-specific primer (IntMR) set in the first multiplex PCR step significantly reduced Ct values of target genes (median 17.9 vs. 19.8, p = 0.0001) and STDHOXB4 (median 16.8 vs. 18.8, p < 0.0001), enhancing detection sensitivity by at least 4-fold compared to cMethDNA. In clinical samples, the cf-MMSP assay distinguished stage IV breast cancer patients from controls with 86% sensitivity (95% CI = 65.4-95.0) and 90% specificity (95% CI = 69.9-98.2), achieving an area under the curve (AUC) of 0.8929 (p < 0.0001). Conclusions: The cf-MMSP assay, which included the use of the Virus kit for cfDNA isolation, choice of primers that selectively amplified methylated products, and a single STDHOXB4 standard, significantly reduced assay cost and time and enhanced its sensitivity for detecting methylated cfDNA in plasma from breast cancer patients. Reference: 1) Fackler et al, PMID: 24737128. 2) Fackler et al, PMID: 36046124. Citation Format: Liqun Zhang, Wenfei Xia, Mary Jo Fackler, Gang Yu, Madison Pleas, Leslie Cope, Saraswati V. Sukumar. Development of the cf-MMSP assay for enhanced detection of methylated cfDNA in breast cancer by liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2115.

BACKGROUND:The use of liquid biopsy to analyze cell-free DNA (cfDNA) provides a noninvasive approach for breast cancer detection, but its low abundance necessitates highly sensitive assays. METHODS:We developed cell-free multiplex methylation-specific PCR (cf-MMSP) through optimized cfDNA extraction and primer design. We compared the extraction efficiency of three commercial kits using normal plasma spiked with 12.5 to 50 copies of fully methylated genomic DNA and 50 copies of STD_HOXB4 plasmid. A nine-gene methylation panel (AKR1B1, COL6A2, HIST1H3C, HOXB4, RASGRF2, RASSF1A, TM6SF1, TMEFF2, and ZNF671) was amplified from bisulfite-converted DNA using a nested PCR that pairs a methylation-independent external forward primer with a reverse methylation (RM)-specific primer in the first multiplex reaction. RESULTS:The QIAamp MinElute Virus Spin Kit yielded superior cfDNA recovery of the STD_HOXB4 control (P < 0.0001 vs. both MAGicBead cfDNA Isolation Kit and QIAamp MinElute ccfDNA Mini Kit). The new primer combination significantly lowered cycle threshold values for genes (median, 17.9 vs. 19.8; P = 0.0001) and STD_HOXB4 (median, 16.8 vs. 18.8; P < 0.0001), translating to a fourfold improvement in analytical detection sensitivity. In an evaluation of 21 stage IV breast cancer plasma samples and 20 benign controls, cf-MMSP distinguished cases from controls with 81% sensitivity [95% confidence interval (CI), 58.1%-94.6%], 85% specificity (95% CI, 62.1%-96.8%), and area under the receiver operating characteristic curve = 0.89 (P < 0.0001). CONCLUSIONS:Optimization of DNA extraction and primer design in cf-MMSP improved low-input analytical sensitivity while maintaining performance comparable with cMethDNA in stage IV breast cancer. IMPACT:cf-MMSP improves low-input methylated cfDNA detection and warrants further validation in earlier-stage breast cancer.

Abstract Purpose: Cervical smear cytology exhibits high variability and limited sensitivity in detecting cervical lesions. While the HPV test, recommended by WHO, offers high sensitivity, it has limited specificity. Our goal was to determine whether DNA methylation markers could improve both sensitivity and specificity, thereby enabling more effective triage to colposcopy and biopsy than cytology and HPV testing. Experimental Design: We previously published a 5-gene (TBXT, MOS, FMN, EDNRB, and ZNF671) DNA methylation assay with high sensitivity and specificity for detecting cervical cancer in cytology and histology samples [1]. In the current study, we tested cervical brush samples (n = 222) with known cytology but no available histology that were preserved in CytoLyt, selected and deidentified at the National Health Service screening clinic in Johannesburg, South Africa. Samples were tested for both high-risk HPV variants (Atila Biosystems) and for cumulative 5-gene methylation utilizing Quantitative Multiplex Methylation-Specific PCR (QM-MSP). Statistical tests included the Youden Index for gene-specific thresholds, odds ratio scores for individual genes, Mann-Whitney test, and Receiver Operating Characteristic Area Under the Curve (ROC AUC); a P value ≤ 0.05 was considered significant. Results: Among the 222 cervical brush samples, HPV was prevalent in similar numbers of HSIL (87/95, 92%) and LSIL (47/57, 82%), and in about half of the NIEL (39/70, 56%). Thus, HPV was highly sensitive for the detection of HSIL, but very poorly specific for low-risk LSIL or NIEL. In contrast, among HPV+ samples, to distinguish between high risk HSIL and low risk LSIL/NEIL, the QM-MSP assay showed a high level of sensitivity: 78.16% [CI 68.39 - 85.55] and, in addition, a high level of specificity: 95.35% [CI 88.64 - 98.18], and ROC AUC = 0.890 [CI 83.71 - 94.37]. A highly significant (P<0.0001) difference in methylation levels was observed between HSIL and LSIL/NIELs. In the small set of HPV-negative samples, hypermethylation of the 5-gene panel was observed in 5/8 (63%) HSIL, 0/10 (0%) LSIL, and 1/31 (3%) NIEL, suggesting that QM-MSP may be independent of the HPV status. Conclusions: These findings suggest that the 5-gene panel detected a large proportion of HSIL, while the majority of LSIL and NIEL remained negative. Thus, this assay could serve as an effective triage tool for HPV+ cases, expediting care for women with HSIL while potentially reducing referrals for colposcopy in LSIL and NIEL by up to 95%. This could reduce costs and risks associated with repeated biopsy procedures for low-risk lesions. Reference: [1] DOI: 10.1186/s13148-024-01669-z Citation Format: Roshni Saravanan, Mary Jo Fackler, Madison Pleas, Wenfei Xia, Tomisin Adebari, Gang Yu, Liqun Zhang, Suzette Jordaan, Eunice Van Den Berg, Pamela Michelow, Reubina Wadee, Maureen Joffe, Wenlong Carl Chen, Youxiang Wang, Leslie Cope, Saraswati Sukumar. The potential of methylated DNA markers for accurate detection of high-risk, HPV-positive cervical lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1959.

Abstract Purpose: We sought to develop a highly sensitive, reproducible cell-free methylated DNA assay for accurately detecting metastatic prostate cancer (mPCa) in plasma. Experimental Design: We identified a new 10-marker panel for accurate detection of PCa in the TCGA prostate adenocarcinoma (PRAD) 450K DNA methylation data. Marker sensitivity and specificity were verified in two independent public prostate cancer datasets (GSE112047, GSE76938). A quantitative multiplex methylation-specific PCR (QM-MSP) assay incorporating these markers was validated in archival benign and prostate cancer tissues from the United States (US, 61 cancer, 26 benign) and South Africa (SA, 80 cancer, 24 benign). We used a newly developed cell-free multiplex methylation-specific PCR (cf-MMSP) assay for the 10-marker panel. Intra-assay and intra-operator reproducibility assays tested its robustness. cf-MMSP performance was assessed in a Training set (40 benign, 40 cancer), and an independent Test set (19 benign, 20 cancer) of plasma. Shapley value regression was used to rank performance of the 10 genes in the cf-MMSP assay. We selected a 5-gene panel based on their high performance. Statistical methods used included the Youden index threshold for ROC analysis and Kruskal-Wallis tests. Results: In tissues, the 10-gene panel detected all stages of prostate cancer: In tissue from the US (n=87), we achieved ROC AUC = 1.00 (CI. 0.99 - 1.00), sensitivity of 98.36% (CI. 91.28 - 99.91) and specificity of 100.00% (CI. 87.13 - 100.00); in SA samples (n=104), ROC AUC was 0.99 (CI. 0.99 - 1.00), sensitivity of 98.28% (CI. 90.86 - 99.91), and specificity of 100.00% (CI. 86.20 - 100.00). cf-MMSP intra-assay test showed low CVs (1.70-5.70% across copy levels) and strong linearity (R2 = 0.93). The intra-operator test showed excellent reproducibility, with an ICC of 0.99. In plasma, in the training set, the 10-gene panel showed ROC AUC of 0.89 (CI. 0.81-0.97), sensitivity of 75.00% (CI. 59.81 - 85.81) and specificity of 93.88% (CI: 83.48 - 97.90) in distinguishing mPCa from benign. In the test set, the assay performed with ROC AUC of 0.91 (CI. 0.80 -1.00), sensitivity of 85.00% (CI. 63.96 - 94.76), and specificity of 93.88% (CI. 83.48 - 97.90). Using a standardized Shapley value cutoff of 0.07, we identified the five best-performing genes- HAAO, DLEC1, FBN1, TMEM155 and RTDR1. In the training set of plasma, the refined 5-gene panel subset showed ROC AUC of 0.92 (CI. 0.84 - 0.99), sensitivity of 90.00% (CI. 76.95 - 96.04) and specificity of 90.00% (CI. 76.95 - 96.04). In the test set, this 5-gene panel achieved ROC AUC of 0.92 (CI. 0.84 - 0.99), with a sensitivity of 90.00% (CI. 80.00 - 100.00) and a specificity of 84.20% (CI. 53.00 - 100.00). Conclusions: The 5-gene cf-MMSP panel showed strong performance indices in detecting mPCa in plasma. The assay shows potential for broadly accessible early detection and disease monitoring for molecular management of mPCa. Citation Format: Gang Yu, Mary Jo Fackler, Liqun Zhang, Wenfei Xia, Roshni Saravanan, Ezra Baraban, Eunice Van Den Berg, Adam Botha, Pamela Michelow, Reubina Wadee, Maureen Joffe, Wenlong Carl Chen, Thomas Pisanic, Michelle Petri, Jun Luo, Channing Paller, Samuel Denmeade, Leslie Cope, Saraswati Sukumar. Development of a methylated gene panel for detection of cell-free, metastatic prostate cancer DNA in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2587.

Association between week 4 CM and disease status at first restaging after adjusting for CTC

Overall and within batch Coefficient of Variation for CM and each Gene.

Association between week 4 CM and disease status at first restaging after adjusting for CEA.

Supplementary Figure 2 displays the relationship between the week 8 CM level and the change in CM level (Week 8 – baseline). Figure S4B and C use box plots to display the relationship between disease status at first restaging and week 8 CM level or change in CM level (Week 8 – baseline).

Supplementary figure 4 displays time updates ROC curves for week 4 CM level using two additional methods (A) binary thresholding and (B) random forest.

Association between week8 CM and change in CM and disease status at first restaging.

Association between week 4 CM and PFS and OS

Supplementary figure 5 displays time updated ROC curves for (A) CM level at week 8, (B) Change in CM level (Week8 – Week4) and (C) the Combination of week 8 CM level and Change in CM level (Week 8-Week 4) using linear discriminant analysis.

Supplementary Figure 3 displays baseline CM level by disease status at first restaging.

Association between week 4 CM and disease status at first restaging after adjusting for CA27-29.

Abstract Background Cervical cancer remains a leading cause of death, particularly in developing countries. WHO screening guidelines recommend human papilloma virus (HPV) detection as a means to identify women at risk of developing cervical cancer. While HPV testing identifies those at risk, it does not specifically distinguish individuals with neoplasia. We investigated whether a quantitative molecular test that measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy. Results Marker discovery was performed in TCGA-CESC Infinium Methylation 450 K Array database and verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation-Specific PCR in tissue sections (N = 252) and cervical smears (N = 244) from the USA, South Africa, and Vietnam. The gene panel consisted of FMN2, EDNRB, ZNF671, TBXT, and MOS. Cervical tissue samples from all three countries showed highly significant differential methylation in squamous cell carcinoma (SCC) with a sensitivity of 100% [95% CI 74.12–100.00], and specificity of 91% [95% CI 62.26–99.53] to 96% [95% CI 79.01–99.78], and receiver operating characteristic area under the curve (ROC AUC) = 1.000 [95% CI 1.00–1.00] compared to benign cervical tissue, and cervical intraepithelial neoplasia 2/3 with sensitivity of 55% [95% CI 37.77–70.84] to 89% [95% CI 67.20–98.03], specificity of 93% [95% CI 84.07–97.38] to 96% [95% CI 79.01–99.78], and a ROC AUC ranging from 0.793 [95% CI 0.68–0.89] to 0.99 [95% CI 0.97–1.00] compared to CIN1. In cervical smears, the marker panel detected SCC with a sensitivity of 87% [95% CI 77.45–92.69], specificity 95% [95% CI 88.64–98.18], and ROC AUC = 0.925 [95% CI 0.878–0.974] compared to normal, and high-grade squamous intraepithelial lesion (HSIL) at a sensitivity of 70% (95% CI 58.11–80.44), specificity of 94% (95% CI 88.30–97.40), and ROC AUC = 0.884 (95% CI 0.822–0.945) compared to low-grade intraepithelial lesion (LSIL)/normal in an analysis of pooled data from the three countries. Similar to HPV-positive, HPV-negative cervical carcinomas were frequently hypermethylated for these markers. Conclusions This 5-marker panel detected SCC and HSIL in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk HSIL will lead to timely treatment for those in need and prevent unnecessary procedures in women with low-risk lesions throughout the world. Validation of these markers in prospectively collected cervical smear cells followed by the development of a hypermethylated marker-based cervical cancer detection test is warranted.

Table contains sequences of primers and probes used to amplify the nine markers and ACTB in the LBx-BCM assay

Supplementary Figure 2 from Epigenetic Inactivation of the Potential Tumor Suppressor Gene FOXF1 in Breast Cancer

Figure S1: Performance of a minimal 5-gene marker panel.

Figure shows changes in LBx-BCM methylation in response to chemotherapy in 7 additional patient longitudinal serial samples. LBx-BCM was performed and cumulative methylation (CM) (Y-axis) is plotted from serum samples drawn at baseline (0 days) and immediately before each treatment cycle (X-axis). For each patient, treatment cycles are shown as a shaded area. PD, progressive disease; SD, stable disease (SD).

Figure shows ACTB reference gene DNA levels plotted for each of the 132 study samples. Stage IV breast cancer patient sera had significantly higher total ACTB DNA (lower Ct) compared to normal (Mann Whitney P < 0.0001). Descriptive statistics are shown for normal vs. cancer and for methylation Cartridge A vs Cartridge B