Purpose: To examine circulating tumor DNA (ctDNA) and its association with residual cancer burden (RCB) using an ultrasensitive assay in patients with triple-negative breast cancer (TNBC) receiving neoadjuvant chemotherapy (NAT). Patients and Methods: We identified responders (RCB-0/1) and matched non-responders (RCB-2/3) from the phase II TBCRC 030 prospective study of neoadjuvant paclitaxel vs. cisplatin in TNBC. We collected plasma samples at baseline, three weeks, and twelve weeks (end of therapy). We created personalized ctDNA assays utilizing MAESTRO mutation enrichment sequencing. We explored associations between ctDNA and RCB status and disease recurrence. Results: Of 139 patients, 68 had complete samples and no additional NAT. Twenty-two were responders and 19 of those had sufficient tissue for whole-genome sequencing. We identified an additional 19 non-responders for a matched case-control analysis of 38 patients using a MAESTRO ctDNA assay tracking 319-1000 variants (median 1000) to 114 plasma samples from 3 timepoints. Overall, ctDNA positivity was 100% at baseline, 79% at week 3, and 55% at week 12. Median tumor fraction (TFx) was 3.7 x 10-4 (range: 7.9 x 10-7 to 4.9 x 10-1). TFx decreased 285-fold from baseline to week 3 in responders and 24-fold in non-responders. Week 12 ctDNA clearance correlated with RCB: clearance was observed in 10/11 patients with RCB-0, 3/8 with RCB-1, 4/15 with RCB-2, and 0/4 with RCB-3. Among 6 patients with known recurrence five had persistent ctDNA at week 12. Conclusion: NAT for TNBC reduced ctDNA TFx by 285-fold in responders and 24-fold in non-responders. In 58% (22/38) of patients, ctDNA TFx dropped below the detection level of a commercially available test, emphasizing the need for sensitive tests. Additional studies will determine if ctDNA-guided approaches can improve outcomes.

HAP reports research funding from Puma Biotechnology (paid to institution) and advisory roles at Illumina and Caris. JC reports royalties from ArcherDx and ownership interest (stocks, stock options, patent or other intellectual property or other ownership interest excluding diversified mutual funds) in SeekIn (思勤医疗). AB reports consulting fees (e.g., advisory boards) from Abbvie, Agendia, Bayer, BioTheranostics, Coherus Biosciences, Daiichi Sankyo/Lilly, Eisai, Genentech/Roche, General Electric, Gilead Sciences, Immunomedics, Merck, Michael J. Hennessy Associates, Myriad Pharmaceuticals, Novartis, Onc Live, Pfizer, Puma Biotechnology, Seattle Genetics, Tyme; research support from AstraZeneca/Daiichi Sankyo, Daiichi Sankyo/Lilly, Genentech/Roche, Gilead Sciences, Merck, Novartis, Puma Biotechnology; expert testimony fees from Pfizer; and third-party writing assistance for this abstract, furnished by Eleanor Porteous, MSc, of Health Interactions, was provided by F. Hoffmann-La Roche. JF reports consulting fees (e.g., advisory boards) from Exact Sciences, G1 Therapeutics, OncoSec, Pfizer, PRIME; and ownership interest (stocks, stock options, patent or other intellectual property or other ownership interest excluding diversified mutual funds) in Exact Sciences, Rain Therapeutics, Veracyte. TAT reports research support from Astellas Pharma, AstraZeneca, Ayala Pharmaceuticals, Daiichi Sankyo, Genentech/Roche; consulting fees (e.g., advisory boards) from AstraZeneca, Daiichi Sankyo, Gilead, Novartis, Pfizer; and participation on a data safety monitoring board for Genentech. LAC reports uncompensated relationships with AstraZeneca/Daiichi Sankyo, Eisai, Exact Sciences, G1 Therapeutics, Genentech/Roche, GSK, Lilly, Novartis, Sanofi, Seagen; receipt of intellectual property rights / patent holder from Falcon Therapeutics; and research support from NanoStringTechnologies, Novartis, Seattle Genetics, Syndax, Veracyte. MFR reports research support from F. Hoffmann-La Roche Ltd and Pfizer; fees for non-CME services received directly from commercial interest or their agents (e.g., speakers' bureaus) from Daiichi Sankyo, Genentech, Macrogenics, Seattle Genetics; and third-party writing assistance for this abstract, furnished by Sunaina Indermun, BPharm, PhD, of Health Interactions, was provided by F. Hoffmann-La Roche. VS reports research support from Abbvie, Biocept, Novartis, Pfizer, Puma Biotechnology, QUE Oncology; and consulting fees (e.g., advisory boards) from AstraZeneca and Novartis. JS reports research support from Abbvie, Inc, Cascadian Therapeutics, Celcuity, Inc., Genentech, Merck, Minerva Biotechnologies, Myriad Pharmaceuticals, Novartis, Pfizer, Seagen, Inc, Seattle Genetics, Xencor; consulting fees (e.g., advisory boards) from Daiichi Sankyo, GE Healthcare, GlaxoSmithKline, Sensei Biotherapeutics, Volastra; and honoraria for travel, accommodations, expenses from GE Healthcare, GlaxoSmithKline, Nektar. EPW reports honoraria from Genentech, GSK, Seattle Genetics. IEK reports consulting fees (e.g., advisory boards) from AstraZeneca, Bristol Meyers Squibb, Daiichi Sankyo, Genentech/Roche, Macrogenics, Seattle Genetics, Taiho Oncology; fees from non-CME services received directly from commercial interest or their agents (e.g., speakers bureaus) from AstraZeneca; manuscript funding paid to institution from study sponsor AstraZeneca; medical writing support provided Articulate Science, LLC and funded by Daiichi Sankyo (study sponsor); research support (paid to institution) from Genentech/Roche, Macrogenics, Pfizer; participation on a data safety monitoring board or advisory board at Merck and Novartis; leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid and ownership interest (stocks, stock options, patent or other intellectual property or other ownership interest excluding diversified mutual funds) in PureTech. GMM reports being a co-inventor on the patent for the MAESTRO mutation enrichment sequencing methods used in this study: patent application (PCT/US2021/013520). TRG reports consulting fees (e.g., advisory boards) from Anjli Pharmaceuticals and Dewpoint Therapeutics; research funding from Bayer HealthCare, Calico Life Sciences, Novo Holdings; ownership interest (stocks, stock options, patent or other intellectual property or other ownership interest excluding diversified mutual funds) in Dewpoint Therapeutics; and founder and ownership interest in Sherlock Biosciences. ELM reports consulting fees (e.g., advisory boards) from AstraZeneca, Gilead, Lilly, Novartis; third-party writing assistance for this abstract, furnished by Eleanor Porteous, MSc, of Health Interactions, was provided by F. Hoffmann-La Roche. VAA reports being a co-inventor on the patent for the MAESTRO mutation enrichment sequencing methods used in this study: patent application (PCT/US2021/013520). All other authors report no disclosures.

NCT01982448

The work was supported by NCI Mentored Clinical Scientist Research Career Development Award (1K08CA252639, H.A.P.); Gerstner Family Foundation; Forget-Me-Not Fund.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study was approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board. This study was conducted in compliance with the principles of the Declaration of Helsinki.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

The data that support the findings of this study are available from the corresponding authors [HAP, ELM, and VAA], upon reasonable request.