Odenheimer-Bergman, Ahuva

Abstract Background: Accurate circulating biomarkers for detecting residual disease can help guide therapy decisions, particularly in early-stage cancer patients. However, currently available methods lack the sensitivity required to confidently assess the presence of residual disease in patients with low tumor burden. To address this need, we have developed TARDIS (Targeted Digital Sequencing), a personalized, multiplexed amplicon sequencing method capable of tracking as many as 100 or more mutations simultaneously. Methods: We obtained tumor biopsies and longitudinal plasma samples from patients with early-stage breast cancer, glioblastoma, and pancreatic cancer. Each tumor biopsy was analyzed whole-exome sequencing. Founder mutations were selected, accounting for copy number alterations (analyzed using sequenza) and a consensus allele fraction approach that combined pyclone and custom in-house methods. Patient-specific TARDIS primers were designed to detect these mutations in plasma cfDNA. Error suppression in TARDIS was achieved using a combination of unique molecular identifiers and fragment sizes to group sequencing reads into read families. Results: In 33 patients with early-stage breast cancer treated with neoadjuvant therapy, we targeted between 3 and 116 (mean 30) mutations per patient and analyzed between 1 and 4 longitudinal plasma samples using TARDIS. Prior to treatment, we detected ctDNA in 100% patients with Stage I-III breast cancer (n=32, 95% CI= 89%-100%). We detected tumor-specific mutations in 100% of baseline breast cancer plasma samples. After completion of neoadjuvant therapy and before surgery, ctDNA levels were significantly lower in patients with pathologic complete response (pathCR, no evidence of disease at surgery) compared to patients with residual disease (median tumor fractions 0.003% and 0.017%, respectively, p=0.0058, AUC=0.83). Conclusions: TARDIS enables highly sensitive detection of ctDNA in patients with nonmetastatic cancers. Analysis of longitudinal plasma samples using TARDIS holds promise for personalizing the extent of treatment in patients with curable disease. Multiple clinical validation studies across cancer types are ongoing to define quantitative thresholds for changes in ctDNA levels that could improve clinical decision making. Citation Format: Bradon R. McDonald, Tania Contente-Cuomo, Stephen-John Sammut, Michelle D. Stephens, Ahuva Odenheimer-Bergman, Brenda Ernst, Nieves Perdigones, Suet-Feung Chin, Maria Farooq, Rosa Mejia, Patricia A. Cronin, Karen S. Anderson, Heidi E. Kosiorek, Donald W. Northfelt, Ann E. McCullough, Bhavika K. Patel, Jeffrey N. Weitzel, Thomas P. Slavin, Carlos Caldas, Barbara A. Pockaj, Muhammed Murtaza. Personalized monitoring of treatment response using Targeted Digital Sequencing of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A51.

A robust personalized ctDNA test, TARDIS, achieves high accuracy for residual disease after completion of neoadjuvant therapy.

Abstract Background: Circulating tumor DNA (ctDNA) analysis holds potential for minimal residual disease (MRD) detection in early stage breast cancer. However, sensitivity for MRD is limited due to low ctDNA levels in early stage patients and limited blood volumes. Loss of input DNA during library preparation, limited multiplexing or low sensitivity of current molecular methods further limit accuracy. To address this gap, we have developed TARgeted DIgital Sequencing (TARDIS), a novel method for simultaneous analysis of multiple patient-specific mutations in plasma DNA. Methods: Using tumor exome sequencing, we identify and prioritize somatic founder mutations, design nested primers and evaluate them for multiplex performance. Using 5-10 ng input plasma DNA, we perform 1) targeted linear pre-amplification to improve downstream molecular conversion, 2) single-stranded adapter ligation to incorporate unique molecular identifiers (UMIs) and 3) targeted PCR to prepare sequencing-ready libraries. The resulting sequencing reads have fixed target-specific ends and variable ligation ends. We utilize fragment size and UMIs to group sequencing reads into read families. To ensure specificity, we require targeted mutations are supported by 2 or more read families. Results: To assess analytical performance, we targeted 8 mutations in cell-free DNA reference samples with 0.25%-2% mutation allele fractions (AFs). Precision across 7-16 replicates at each AF level agreed with expectations of Poisson distribution, demonstrating effective analysis of ˜70% of input DNA. At 2%, 1%, 0.5% and 0.25% AFs, variant-level sensitivity was 96.4%, 96.4%, 91.1% and 65.8%, approaching the theoretical limit given input DNA. At 0.25% AF, 3-7 mutations were detected per sample, achieving 100% sample-level sensitivity. In 16 wild-type replicates, no targeted mutations were called (100% specificity). Averaging multiple mutations improved precision in sample-level AF estimates. Mean AFs from 8 mutations for the 2% sample were 2.34%-2.80% (5.8% CV). In 6 patients with breast cancer treated with neoadjuvant therapy (NAT), we analyzed 8-18 patient-specific mutations (mean 11.8). Before treatment, ctDNA was detected in 5/6 patients at mean AFs of 0.02%-1.19% (mean 0.40%), supported by 2-10 mutations (mean 5.6). Of these 5 patients, 4 had residual disease after NAT and ctDNA was detected pre-operatively or during NAT in 3/4 patients. 1 patient achieved pathological Complete Response and ctDNA was undetectable after NAT. Conclusions: Preliminary results suggest TARDIS enables accurate MRD detection after neoadjuvant therapy in patients with early stage breast cancer. On-going work is expanding this analysis to include additional patients and investigate the clinical validity of peri-operative ctDNA monitoring. Summary of clinical resultsPatientPre-NAT Stage (TNM)SubtypeNo. of Mutations TargetedBaseline ctDNA (AF%, No. of Mutations)ctDNA after or during NAT (AF%, No. of Mutations)Residual Tumor (TNM)1T3 N1ER+ PR+ HER2-8+ (0.02%, 2)-T2 N12T3 N0TNBC12+ (0.29%, 6)+ (0.01%, 1)T1a N03T2 N1TNBC18+ (1.19%, 10)+ (0.01%, 1)T1mi N04T3 N1TNBC10+ (0.02%, 3)+ (0.05%, 3)T3 N15T2 N0TNBC9+ (0.46%, 7)-pathCR6T1c N1TNBC14--pathCR Citation Format: McDonald BR, Contente-Cuomo T, Sammut S-J, Ernst B, Odenheimer-Bergman A, Perdigones N, Chin S-F, Farooq M, Cronin PA, Anderson KS, Kosiorek H, Northfelt D, McCullough A, Patel B, Caldas C, Pockaj B, Murtaza M. Multiplexed targeted digital sequencing of circulating tumor DNA to detect minimal residual disease in early and locally advanced breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-21.