Next-generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy

Abstract

Next-generation sequencing to detect circulating tumor DNA is a minimally invasive method for tumor genotyping and monitoring therapeutic response. The majority of studies have focused on detecting circulating tumor DNA from patients with metastatic disease. Herein, we tested whether circulating tumor DNA could be used as a biomarker to predict relapse in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy. In this study, we analyzed samples from 38 early-stage triple-negative breast cancer patients with matched tumor, blood, and plasma. Extracted DNA underwent library preparation and amplification using the Oncomine Research Panel consisting of 134 cancer genes, followed by high-coverage sequencing and bioinformatics. We detected high-quality somatic mutations from primary tumors in 33 of 38 patients. TP53 mutations were the most prevalent (82%) followed by PIK3CA (16%). Of the 33 patients who had a mutation identified in their primary tumor, we were able to detect circulating tumor DNA mutations in the plasma of four patients (three TP53 mutations, one AKT1 mutation, one CDKN2A mutation). All four patients had recurrence of their disease (100% specificity), but sensitivity was limited to detecting only 4 of 13 patients who clinically relapsed (31% sensitivity). Notably, all four patients had a rapid recurrence (0.3, 4.0, 5.3, and 8.9 months). Patients with detectable circulating tumor DNA had an inferior disease free survival (p < 0.0001; median disease-free survival: 4.6 mos. vs. not reached; hazard ratio = 12.6, 95% confidence interval: 3.06-52.2). Our study shows that next-generation circulating tumor DNA sequencing of triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy can predict recurrence with high specificity, but moderate sensitivity. For those patients where circulating tumor DNA is detected, recurrence is rapid.

Fig. 1

Trial schema for BRE09-146.BRE09-146 was a Phase II clinical trial to evaluate 2-year disease-free survival (DFS) in TNBC patients, treated with either Cisplatin (Arm A) or Cisplatin in combination with PARP inhibitor Rucaparib (Arm B) after neoadjuvant chemotherapy. Tumor tissue, whole blood, and plasma from four time points after surgery were collected as indicated. In this trial, plasma samples were collected only in Arm B of (the area enclosed by the red rectangle). Plasma samples were collected at four timepoints: Cycles 1 and 2 of the combination phase, and during weeks 1 and 5 of the maintenance phase

Fig. 2

CONSORT diagram. There were 135 patients enrolled in BRE09-146. In this study, we focused on 70 patients from Arm B. In Arm B, 27 patients did not have matched tumor tissue, whole blood, and at least one plasma collection and were excluded from this study leaving an N = 43. A further five patients were removed due to the inability to successfully create a plasma DNA library. In total, 38 patients reached the criteria for analysis

Fig. 3

Somatic mutations identified from sequencing of tumor tissues. Among the 38 patients in our study, 33 of them had at least one somatic mutation identified (87%); 21 of them had two or more somatic mutations (55%). TP53 mutations were the most prevalent in this study, followed by PIK3CA pathway mutations. Notably, there were 14 different mutations exclusively present in individual patients, representing the genomic heterogeneity of TNBC patients

Fig. 4

Longitudinal allele frequency tracking of ctDNA mutations. Somatic mutations were first identified in the primary tumor. These mutations were then searched for in matched plasma samples. ctDNA mutations were identified in four patients at varying allele frequencies. From patient 146-0005(a) and patient 146-0013(b), the increasing allele frequency of ctDNA was observed before clinically recurrence was diagnosed. Patient 146-0102(c) and patient 146-0112(d) had only one timepoint plasma sample available, and we were able to detect the ctDNA before clinical recurrence as well. The lead-time range was 0.07 to 8.87 months

Fig. 5

Kaplan–Meier plot: disease-free survival stratified by presence of tumor mutation in plasma. Four patients from this study who had mutation identified from plasma samples relapsed rapidly (0.3, 4.0, 5.3, and 8.9 months). The yellow line represents patients with detectable ctDNA in plasma. The blue line represents patients with no detectable ctDNA in plasma.The difference in median DFS between patients with detectable ctDNA vs. those without was statistically significant (p < 0.0001, median DFS: 4.6 mos. vs. NR; HR = 12.6, 95% CI: 3.06-52.2)