Patients.

The ctDNA study was conducted in the context of the I-SPY2 trial. Patients had MammaPrint high tumors and therefore are at high risk of metastatic recurrence within five years after diagnosis. ⁴⁴

This study involved patients with HER2-negative tumors. Breast cancer is generally classified into one of the 4 receptor subtypes based on hormone receptor (HR; estrogen and progesterone receptor) and HER2 expression. The HER2-negative receptor subtypes—HR-positive/HER2-negative and HR-negative/HER2-negative (also known as triple-negative breast cancer or TNBC)—represent about 85% of all breast cancers [73% HR-positive/HER2-negative and 12% TNBC]. ²⁹ Receptor subtypes have distinct biological characteristics that are reflected in differences in clinical outcomes ⁴⁵ and recommended treatment modalities in the early-stage setting; ³ for example, HR-positive/HER2-negative tumors are treated with endocrine therapy with or without chemotherapy, while HER2-positive tumors are treated with HER2-targeted drugs in combination with chemotherapy. Early-stage TNBC (stage II and higher) is most often treated with the checkpoint inhibitor, pembrolizumab, in the neoadjuvant setting in combination with chemotherapy, followed by pembrolizumab post-surgery. ⁴⁶

In the I-SPY2 trial, HER2-negative patients in the control arm of I-SPY2 received paclitaxel followed by AC. Investigational regimens are given in combination with paclitaxel or as a replacement for paclitaxel. The trial limits eligibility to women >18 years with stage II or III breast cancer >2.5cm and high MammaPrint score; ⁴⁷ these are patients at high risk of metastatic recurrence within 5 years after diagnosis. The I-SPY2 protocol was approved by Institutional Review Boards at all participating institutions and all patients signed written informed consent. Patients included in the current study were HER2-negative, with pretreatment tumor biopsy specimens available and whose plasma samples were analyzed for ctDNA. These patients were enrolled in I-SPY2 between March 2010 and July 2018. All patients provided written informed consent for subsequent use of their specimens for research purposes.

Of the 283 evaluable patients, 26.5% (75) were stage T3/T4; 41.7% (118) were node-positive; 55.1% (156) had grade 3 disease; 57.6% (163) were MammaPrint (ultra) high-risk 2; and 48.1% (136) received standard NAC, while the rest received NAC combined with an investigational drug (Table 1). Clinicopathologic characteristics and treatment assignment were balanced between subtypes except for a significantly higher proportion of patients with grade 3 (68.1% vs. 42.8%) and MammaPrint (ultra) high-risk 2 tumors (88.4% vs. 28.3%), and RCB-0 (24.6% vs. 15.2%) in the TNBC group compared with the HR-positive/HER2-negative group (all Fisher’s exact test p<0.05).