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 results

Patient Pre-NAT Stage (TNM) Subtype No. of Mutations Targeted Baseline ctDNA (AF%, No. of Mutations) ctDNA after or during NAT (AF%, No. of Mutations) Residual Tumor (TNM) 
T3 N1 ER+ PR+ HER2- + (0.02%, 2) T2 N1 
T3 N0 TNBC 12 + (0.29%, 6) + (0.01%, 1) T1a N0 
T2 N1 TNBC 18 + (1.19%, 10) + (0.01%, 1) T1mi N0 
T3 N1 TNBC 10 + (0.02%, 3) + (0.05%, 3) T3 N1 
T2 N0 TNBC + (0.46%, 7) pathCR 
T1c N1 TNBC 14 pathCR 
Patient Pre-NAT Stage (TNM) Subtype No. of Mutations Targeted Baseline ctDNA (AF%, No. of Mutations) ctDNA after or during NAT (AF%, No. of Mutations) Residual Tumor (TNM) 
T3 N1 ER+ PR+ HER2- + (0.02%, 2) T2 N1 
T3 N0 TNBC 12 + (0.29%, 6) + (0.01%, 1) T1a N0 
T2 N1 TNBC 18 + (1.19%, 10) + (0.01%, 1) T1mi N0 
T3 N1 TNBC 10 + (0.02%, 3) + (0.05%, 3) T3 N1 
T2 N0 TNBC + (0.46%, 7) pathCR 
T1c N1 TNBC 14 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.