Circulating Tumor DNA Using Tagged Targeted Deep Sequencing to Assess Minimal Residual Disease in Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy

Abstract

In breast cancer patients undergoing neoadjuvant chemotherapy before surgery, there is an unmet need for noninvasive predictive biomarkers of response. The analysis of circulating tumor DNA (ctDNA) in particular has been the object of several reports, but few of them have studied the applicability of tagged targeted deep sequencing (tTDS) to clinical practice and its performance compared with droplet digital PCR (ddPCR). Here, we present the first results from an ongoing study involving a prospectively accrued, monocentric cohort of patients affected by invasive breast cancer, undergoing neoadjuvant chemotherapy followed by surgery with curative intent as per clinical practice. A pretreatment tumor biopsy and plasma samples were collected before and during treatment, after surgery, and every six months henceforth or until relapse, whichever came first. Pretreatment biopsies were sequenced with a 409-gene massive parallel sequencing (MPS) panel, allowing the identification of target mutations and their research in plasma by tTDS and ddPCR as a complementary approach. Using tTDS, we demonstrated the presence of at least one deleterious mutation in all the relapsed cases we studied (n = 4), with an average lead time of six months before clinical relapse. The association with ddPCR was suboptimal, and only one relapsed patient could be identified with such method. tTDS shows potential as an early noninvasive method for the detection of MRD in BC patients.

Figure 1

Diagram workflow of the planned sample collection and analyses: at diagnosis, tumor biopsies of patients presenting with invasive breast cancer and indication for NACT were analysed by MPS to identify somatic tumor-specific mutations and then confirmed by ddPCR and tTDS. Mutations in plasma were analysed by ddPCR at baseline, half treatment completed, before surgery, at 12 weeks, 24 weeks, 1 year after surgery, and every 24 weeks until 3 years of follow-up or upon relapse. tTDS was assessed at baseline on biopsies and plasma, as well as at relapse or last follow-up time point.

Figure 2

ddPCR anticipates clinical relapse in one patient with a TP53 mutation. (a) Assessment of target mutation by ddPCR during the monitoring on ctDNA in a patient (UPN1) relapsed at 12 months. TP53 p. Y205C c.614A>G was present at 6 months (T4) after surgery, anticipating the patient's clinical relapse by six months. (b) ddPCR scatterplot of target mutation (y-axis) vs. wild type (x-axis) on FFPE biopsy at diagnosis (upper plot) and on ctDNA at T4 (lower plot).

Figure 3

Scatterplot highlighting the correlation between cfDNA input amount (x-axis) and limit of detection (LOD, y-axis).

Figure 4

Comparison case in point of MPS, MPS, and tTDS for mutation tracking: PIK3CA mutation H1047R could be detected in plasma and in the FFPE pretreatment biopsy by MPS, ddPCR, and tTDS. However, ddPCR allowed the detection of such mutation only at baseline, whereas by tTDS we could track the presence of H1047R six months before relapse.