Preoperative staging of suspicious axillary lymph nodes (ALNs) allows patients to be triaged to ALN dissection or to sentinel lymph node biopsy (SLNB). Ultrasound-guided fine needle aspiration (FNA) and cytology of ALN is moderately sensitive but its clinical utility relies heavily on the cytologist’s experience. We proposed that the 5-h automated GeneXpert system-based prototype breast cancer detection assay (BCDA) that quantitatively measures DNA methylation in ten tumor-specific gene markers could provide a facile, accurate test for detecting cancer in FNA of enlarged lymph nodes. We validated the assay in ALN-FNA samples from a prospective study of patients (N = 230) undergoing SLNB. In a blinded analysis of 218 evaluable LN-FNAs from 108 malignant and 110 benign LNs by histology, BCDA displayed a sensitivity of 90.7% and specificity of 99.1%, achieving an area under the ROC curve, AUC of 0.958 (95% CI: 0.928–0.989; P < 0.0001). Next, we conducted a study of archival FNAs of ipsilateral palpable LNs (malignant, N = 72, benign, N = 53 by cytology) collected in the outpatient setting prior to neoadjuvant chemotherapy (NAC). Using the ROC-threshold determined in the prospective study, compared to cytology, BCDA achieved a sensitivity of 94.4% and a specificity of 92.5% with a ROC-AUC = 0.977 (95% CI: 0.953–1.000; P < 0.0001). Our study shows that the automated assay detects cancer in suspicious lymph nodes with a high level of accuracy within 5 h. This cancer detection assay, scalable for analysis to scores of LN FNAs, could assist in determining eligibility of patients to different treatment regimens.

Current molecular liquid biopsy assays to detect recurrence or monitor response to treatment require sophisticated technology, highly trained personnel, and a turnaround time of weeks. We describe the development and technical validation of an automated Liquid Biopsy for Breast Cancer Methylation (LBx-BCM) prototype, a DNA methylation detection cartridge assay that is simple to perform and quantitatively detects nine methylated markers within 4.5 h. LBx-BCM demonstrated high interassay reproducibility when analyzing exogenous methylated DNA (75-300 DNA copies) spiked into plasma (Coefficient of Variation, CV = 7.1 - 10.9%) and serum (CV = 19.1 - 36.1%). It also demonstrated high interuser reproducibility (Spearman r = 0.887, P &lt; 0.0001) when samples of metastatic breast cancer (MBC, N = 11) and normal control (N = 4) were evaluated independently by two users. Analyses of interplatform reproducibility indicated very high concordance between LBx-BCM and the reference assay, cMethDNA, among 66 paired plasma samples (MBC N = 40, controls N = 26; Spearman r = 0.891; 95% CI = 0.825 - 0.933, P&lt; 0.0001). LBx-BCM achieved a ROC AUC = 0.909 (95% CI = 0.836 - 0.982), 83% sensitivity and 92% specificity; cMethDNA achieved a ROC AUC = 0.896 (95% CI = 0.817 - 0.974), 83% sensitivity and 92% specificity in test set samples. The automated LBx-BCM cartridge prototype is fast, with performance levels equivalent to the highly sensitive, manual cMethDNA method. Future prospective clinical studies will evaluate LBx-BCM detection sensitivity and its ability to monitor therapeutic response during treatment for advanced breast cancer.

BACKGROUND: Previously, we demonstrated the clinical validity of cMethDNA, a circulating methylated tumor DNA (ctDNA) assay, in serum samples from TBCRC 005 (J Clin Oncol, 2017; 35:751-758) to predict progression free- and overall-survival, and to monitor therapeutic response in patients with stage IV breast cancer. Here, Johns Hopkins (JH) and Cepheid partnered to develop an automated GeneXpert (GX) cartridge-based system to provide quantitative measures of DNA methylation within 5 hours.METHODS: With a goal of discriminating stage IV breast cancer from healthy and benign breast disease with high sensitivity and specificity, we evaluated breast cancer-specific DNA methylation markers (selected through comprehensive methylome analysis) in STRECK tube plasma of 46 patients with metastatic breast cancer enrolled in Individualized Molecular Analyses Guide Efforts in Breast Cancer (IMAGE II trial), 17 benign breast disease and 9 healthy normal controls (J0888 repository). Blood from IMAGE II participants was collected upon disease progression. A newly designed GX Breast Cancer Monitoring Assay for research use only (RUO*) first converted unmethylated CpG sites in ctDNA from 1 ml plasma with bisulfite. The sample was then split into two methylation detection cartridges, which quantitated DNA methylation of 9 markers along with an ACTB reference. Cumulative methylation (CM) of the 9-gene panel was calculated using a novel algorithm. Performance was assessed based on Receiver Operating Characteristic (ROC) curves and Mann-Whitney analyses.RESULTS: The GX Breast Cancer Monitoring Assay (RUO)* showed that the 9-gene panel was significantly more methylated in cancer compared to normal/benign plasma samples (median for cancer: 428.0 CM units versus for benign: 0.0 CM units; P&lt; 0.0001), and revealed a sensitivity of 85% and specificity of 92%, using a cumulative methylation threshold of 35.5 units based on ROC area under the curve (AUC) = 0.909 (95% CI 0.836 – 0.982, P&lt;0.0001). We will present comparisons of the GX results to cMethDNA, the gold standard assay, which reported 85-90% sensitivity at 90% specificity.CONCLUSIONS: We identified a panel of methylated DNA markers that discriminates stage IV breast from benign breast disease and healthy normal subjects using ctDNA. Our automated cartridge-based assay prototype demonstrates high sensitivity and specificity for detecting invasive breast cancer. Its ability to assess changes in DNA methylation will be tested next with clinical trial samples collected longitudinally during treatment. This assay has potential clinical utility in monitoring therapeutic response and predicting disease recurrence.* For Research Use Only. Not for use in diagnostic procedures. Not reviewed by any regulatory body. Citation Format: Mary Jo Fackler, Suzana Tulac, Neesha Venkatesan, Adam J. Aslam, Leslie M. Cope, Jennifer Lehman, Rita Denbow, Jeffrey Reynolds, Morgan Buckley, Bradley M. Downs, Kala Visvanathan, Christopher B. Umbricht, Antonio C. Wolff, Vered Stearns, Edwin W. Lai, Saraswati Sukumar. An automated DNA methylation assay for monitoring treatment response in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-03.

Background: Axillary lymph node metastasis is associated with a high risk of breast cancer recurrence and is the single most powerful predictor of patient prognosis. Various imaging tests alone or in combination with preoperative fine needle aspiration (FNA) cytology are used to evaluate suspicious lymph nodes (LNs). However, these methods have low sensitivity (70%) and their utility varies depending on the local surgical practice and experience of the cytologists. In previous work, we have demonstrated higher than 90% sensitivity/specificity of a panel of ten hypermethylated biomarkers in FNA to distinguish between cancer and benign breast lesions. Analysis of methylated genes in FNA of suspicious LNs may improve sensitivity of cancer detection and assist in staging disease more accurately. Methods: We used Quantitative Multiplex Methylation-Specific PCR (QM-MSP) to determine the utility of the 10-gene panel for detecting tumor cells in LN FNAs. Two studies were performed to test the accuracy of a newly developed automated, cartridge-based GeneXpert® Breast Cancer Detection Assay (RUO*). First, we conducted a prospective case-control study in China in breast cancer patients with palpable LNs undergoing sentinel lymph node biopsy. FNA specimens were collected intraoperatively from a single palpable LN and assayed. Histopathology of the same LN (88 metastatic and 104 benign), was used as the gold standard for comparison. Next, using the automated assay, a pilot validation study was conducted on archival ultrasound-guided FNAs (69 metastatic and 53 benign by cytopathology) collected preoperatively from enlarged axillary LNs in patients with and without breast cancer in the outpatient centers in China and U.S.A. We calculated sensitivity, specificity and area under the receiver operating characteristic curve (AUC) for the marker panel tested by the GeneXpert® assay compared to histopathology for the case control study, and cytopathology for the pilot validation study. We also compared its performance to the highly sensitive laboratory assay, QM-MSP. Results: In the case-control study in China, compared to histopathology, cytopathology of the palpable enlarged LNs achieved cancer detection sensitivity of 85% and specificity of 95% (ROC; AUC=0.898, 95% CI: 0.843-0.953). Using the GeneXpert® assay on the same samples, compared to histopathology, the methylated biomarker panel showed a sensitivity of 92.0% and a specificity of 98.1% based on receiver operating characteristic statistics (ROC; AUC = 0.960, 95% CI: 0.928-0.993). QM-MSP yielded a similar sensitivity of 91.8% and a specificity of 97.7% (ROC; AUC = 0.966, 95% CI: 0.934-0.998). In the pilot validation set, compared to cytopathology, the automated assay achieved a sensitivity of 94.2% and a specificity of 92.5% (ROC; AUC=0.976 (95%CI: 0.950-1.001). Conclusions: We have developed and piloted a 5-hour, automated GeneXpert® Breast Cancer Detection Assay (RUO*) on FNA to detect cancer in suspicious LNs. This assay performs with an accuracy equivalent to that of the highly sensitive but labor-intensive assay, QM-MSP. This test has the potential to determine axillary involvement with higher sensitivity than cytopathology within a very short time in the outpatient setting. A positive test preoperatively could provide guidance for decision-making regarding eligibility of patients for neoadjuvant treatment, post-mastectomy radiotherapy and breast reconstruction. Further evaluation of this automated assay in larger, blinded, prospective studies is warranted. Citation Format: Juanjuan Li, Bradley Downs, Leslie Cope, Xiuyun Zhang, Chuan-gui Song, Kejing Zhang, Yong Han, Mary Jo Fackler, Edwin Lai, Suzana Tulac, Neesha Venkatesan, Timothy Guzman, Chuang Chen, Jingping Yuan, Saraswati Sukumar. Automated molecular diagnosis of suspicious axillary lymph nodes in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-02-06.