Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival

Abstract

Background: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence.

Patients and methods: Cell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years.

Results: At T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P = 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5).

Conclusions: Lack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Keywords: breast cancer; circulating tumor DNA; neoadjuvant chemotherapy; pathologic complete response.

Fig 1.. Study schema, methods for ctDNA analysis, patients and samples

A) Diagram showing the study schema of the I-SPY 2 TRIAL. Prior to study entry, tumor biopsy from each patient is analyzed to assess hormone-receptor (HR) and human epidermal growth factor receptor 2 (HER2) status and MammaPrint scores. Blood samples are collected at the following time points: T0- baseline/pretreatment, T1– 3 weeks after initiation of therapy, T2- between two treatment regimens (paclitaxel +/− MK-2206 and anthracycline (AC)), T3- after neoadjuvant chemotherapy (NAC) prior to surgery. B) Flow chart showing patients and samples evaluated in the study and sample performance at different quality control (QC) points. C) Schema of the methods for ctDNA analysis. PCR-polymerase chain reaction.

Fig 2.. Association between ctDNA and clinicopathologic characteristics

A) Overview of patient and tumor characteristics according to ctDNA status at baseline (T0). HR-hormone receptor, TNBC-triple negative breast cancer, pCR- pathological complete response. B) Proportion of ctDNA-positive and negative patients at baseline (T0) according to clinical characteristics. P values were calculated using Fisher’s exact test. C) Mean tumor molecules per mL of plasma according to clinical characteristics. Distributions were compared using Wilcoxon rank sum (binary variable) or Kruskal Wallis (ternary variable) tests.

Fig 3.. ctDNA dynamics over the course of neoadjuvant chemotherapy

A) Proportion of patients according to ctDNA positivity based on number of samples available per time point. B) Mean tumor molecules per mL of plasma across time points. C) Patients with complete ctDNA data for four time points (n=58) grouped according to observed patterns of ctDNA clearance or non-clearance. D) Survival in patients grouped according to ctDNA clearance. Of the 58 patients, 54 had survival data. Patients who cleared ctDNA at T1, T2 or T3 were combined into one group and their survival was compared with those of patients who did not clear ctDNA at T3 and those who were ctDNA-negative at T0 (reference group).

Fig 4.. Association of ctDNA with response to neoadjuvant chemotherapy and its positive predictive value

A) Sankey plot showing ctDNA dynamics (clearance or non-clearance) early during treatment vs. response (pathologic complete response, pCR, or no pCR). Analysis was focused on patients who were ctDNA-positive at baseline (T0) and had corresponding ctDNA testing results at T1, 3 weeks after initiation of therapy. B) Positive predictive value (PPV) of ctDNA-positivity in predicting failure to achieve pCR. PPV is the proportion of patients with a positive ctDNA test (at a specific time point) with residual cancer after NAC.

Fig 5.. ctDNA and clinical outcomes

A) Overview of the ctDNA detection across different time points [T0: baseline/pretreatment, T1: 3 weeks after initiation of therapy, T2: between two treatment regimens (paclitaxel and AC), T3: after NAC prior to surgery]. The right panel shows a swimmer plot depicting the length of follow-up and events in 75 patients with survival data. The primary endpoint of the study was distant recurrence-free survival. B) Proportion of subtypes according to groups based on pCR and ctDNA status at T3. C) Patient survival stratified based on ctDNA status after NAC (T3) and response to treatment (pathological complete response, pCR). Inset table shows the numbers and percentages of patients according to subtype and response/ctDNA status.