Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer Francesca Riva, 1,2 Francois-Clement Bidard, 1,3* Alexandre Houy, 4 Adrien Saliou, 1 Jordan Madic, 1 Aurore Rampanou, 1,5 Caroline Hego, 1 Maud Milder, 1,5 Paul Cottu, 3 Marie-Paule Sablin, 3 Anne Vincent-Salomon, 6 Olivier Lantz, 5,6,7,8 Marc-Henri Stern, 4 Charlotte Proudhon, 1 and Jean-Yves Pierga 1,3,9 BACKGROUND: In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circu- lating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. METHODS: Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 (TP53) mu- tations previously characterized in tumor tissue by mas- sively parallel sequencing (MPS). RESULTS: Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS (r = 0.99), specificity (2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 pa- tients (75%). Its detection was associated with mitotic index (P = 0.003), tumor grade (P = 0.003), and stage (P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detect- able ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Patho- logical complete response (16/38 patients) was not corre- lated with ctDNA detection at any time point. ctDNA pos- itivity after 1 cycle of NCT was correlated with shorter disease-free (P  0.001) and overall (P = 0.006) survival. CONCLUSIONS: Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly asso- ciated with shorter survival. © 2016 American Association for Clinical Chemistry Triple-negative breast cancers (TNBCs) 10 represent 15%–20% of invasive breast cancers. This subgroup is defined by the absence of estrogen and progesterone re- ceptor expression, no amplification of the erb-b2 recep- tor tyrosine kinase 2 (ERBB2) gene, 11 and frequent tu- mor protein p53 (TP53) inactivating gene mutations (1, 2 ). At diagnosis, TNBCs tend to display larger tumor size and higher proliferation rate than other breast can- cers; in the absence of overt metastasis, TNBCs are often treated by neoadjuvant chemotherapy (NCT) followed by surgery. In addition to breast tumor shrinkage, NCT aims at eradicating any disseminated tumor cell (also known as micrometastasis) that may have spread throughout the body. The persistence of a minimal resid- ual disease at distant sites after the treatment of a localized breast cancer is a key parameter for posttreatment survival but cannot be reliably assessed by the current biological or radiological tools (3 ). In that context, the detection and quantification of circulating tumor DNA (ctDNA) is a very promising tool that can assess tumor burden, response to therapy, and minimal residual disease (4, 5 ). ctDNA corresponds to fragmented DNA released into the blood stream by tumor masses (6, 7 ). In meta- 1 Laboratory of Circulating Tumor Biomarkers, Institut Curie, PSL Research University, SiRIC, Paris, France; 2 Department of Medical Oncology, San Gerardo Hospital, Monza, Italy; 3 De- partment of Medical Oncology, Institut Curie, PSL Research University, Paris, France; 4 INSERM U830, Institut Curie, PSL Research University, Paris, France; 5 INSERM CIC-BT 1428, Institut Curie, PSL Research University, Paris, France; 6 Department of Biopathology, Institut Curie, PSL Research University, Paris, France; 7 Department of Tumor Biology, Institut Curie, PSL Research University, Paris, France; 8 INSERM U932, Institut Curie, PSL Research Univer- sity, Paris, France; 9 Universite ´ Paris Descartes, Sorbonne Paris Cite ´ , Paris, France. * Address correspondence to this author at: Institut Curie, PSL Research University, Depart- ment of Medical Oncology, 26 rue d’Ulm, 75005 Paris, France. Fax +33-153-104-041; e-mail francois-clement.bidard@curie.fr. Received July 2, 2016; accepted October 20, 2016. Previously published online at DOI: 10.1373/clinchem.2016.262337 © 2016 American Association for Clinical Chemistry 10 Nonstandard abbreviations: TNBCs, triple negative breast cancers; NCT, neoadjuvant chemotherapy; ctDNA, circulating tumor DNA; pCR, pathological complete response; RCB, residual cancer burden; ddcPCR, droplet digital PCR; MAF, mutant allele fre- quency; cfcDNA, cell-free circulating DNA. 11 Human genes: ERBB2, erb-b2 receptor tyrosine kinase 2; TP53, tumor protein p53; BRCA1, BRCA1, DNA repair associated; BRCA2, BRCA2, DNA repair associated. Clinical Chemistry 63:3 691–699 (2017) Cancer Diagnostics 691