Circulating Tumor DNA as a Predictive Marker of Recurrence for Patients With Stage II-III Breast Cancer Treated With Neoadjuvant Therapy

Background Patients with stage II to III breast cancer have a high recurrence rate. The early detection of recurrent breast cancer remains a major unmet need. Circulating tumor DNA (ctDNA) has been proven to be a marker of disease progression in metastatic breast cancer. We aimed to evaluate the prognostic value of ctDNA in the setting of neoadjuvant therapy (NAT). Methods Plasma was sampled at the initial diagnosis (defined as before NAT) and after breast surgery and neoadjuvant therapy(defined as after NAT). We extracted ctDNA from the plasma and performed deep sequencing of a target gene panel. ctDNA positivity was marked by the detection of alterations, such as mutations and copy number variations. Results A total of 95 patients were enrolled in this study; 60 patients exhibited ctDNA positivity before NAT, and 31 patients exhibited ctDNA positivity after NAT. A pathologic complete response (pCR) was observed in 13 patients, including one ER(+)Her2(-) patient, six Her2(+) patients and six triple-negative breast cancer (TNBC) patients. Among the entire cohort, multivariate analysis showed that N3 classification and ctDNA positivity after NAT were independent risk factors that predicted recurrence (N3, hazard ratio (HR) 3.34, 95% confidence interval (CI) 1.26 – 8.87, p = 0.016; ctDNA, HR 4.29, 95% CI 2.06 – 8.92, p < 0.0001). The presence of ctDNA before NAT did not affect the rate of recurrence-free survival. For patients with Her2(+) or TNBC, patients who did not achieve pCR were associated with a trend of higher recurrence (p = 0.105). Advanced nodal status and ctDNA positivity after NAT were significant risk factors for recurrence (N2 – 3, HR 3.753, 95% CI 1.146 – 12.297, p = 0.029; ctDNA, HR 3.123, 95% CI 1.139 – 8.564, p = 0.027). Two patients who achieved pCR had ctDNA positivity after NAT; one TNBC patient had hepatic metastases six months after surgery, and one Her2(+) breast cancer patient had brain metastasis 13 months after surgery. Conclusions This study suggested that the presence of ctDNA after NAT is a robust marker for predicting relapse in stage II to III breast cancer patients.

used to test whether the experimental method could detect these mutants.
First, we needed to distinguish the true existence of low-abundance mutants from background errors due to polymerase chain reaction (PCR) or deep sequencing. We calculated the mean and standard error (SD) of the background errors of each DNA region from raw FASTQ data with a quality score of 30. The mean background error per base was 9.2 × 10 -4 , and the SD was 5.09 × 10 -5 . The upper limit of the 99% confidence interval for background errors was 2.23 × 10 -3 (Supplementary Figure S1A).
The measured level of the 0.1% mutant was 3.17× 10 -3 ,which is significantly higher than the range of background errors. This result suggests that our NGS testing procedures can accurately detect 0.1% mutants. Second, we observed that the mutation level could be measured in a linear manner. In reference samples, we achieved a mutation-level sensitivity of 99.50%, 11.76%, 1.35% and 0.32% for 100%, 10%, 1%, and 0.1% mutants, respectively, consistent with the decreasing number of mutant molecules (Supplementary Figure S1B).    Each bar indicates the frequency of correct and erroneous nucleotides from sequencing within the region of the reference variant. The mean background error per base was 9.2 × 10 -4 , and the upper limit of the 99% confidence interval of the background error was 2.23 × 10 -3 (dotted plane). A variant was considered correct when the frequency of variants was more than 2.23 × 10 -3 . The measured frequency of the 0.1% reference variant was 3.17 × 10 -3 , which was higher than upper limit of background errors, showing that the variant was detected. (B) High degree of linearity between the added and measured ratio of variants.

c-Myc
Illustration of the genetic alterations in DNA of breast tumor, ctDNA before NAT and ctDNA after NAT. We selected 5 patients who had TP53 variants in their ctDNA and their biopsy tumors were available for DNA extraction. We performed NGS on the DNA of the biopsy tumor to check whether the TP53 variants that were detected in ctDNA existed in the DNA of breast tumors. If the TP53 variant could be parallel identified in the DNA of the breast tumor and ctDNA, we could confirm that the source of the genetic variants detected in the ctDNA originated from breast tumors.
The TP53 gene variants of patients #8 and #91 were detected in DNA of breast tumors, ctDNA before and after NAT. These results suggest that the TP53 variants in the ctDNA came from breast cancer cells. The presence of ctDNA after NAT was related to a high risk of recurrence; accordingly, both patients experienced recurrence.
For patient #45, the source of the TP53 variant (TP53:NM_000546: c.818G>A) was detected both in DNA of breast tumors and the ctDNA before NAT, suggesting the variants of ctDNA originating from breast tumor. The variant was not identified in the ctDNA after NAT. She did not experience recurrence.
Patients #27 and #83 had more than one genetic alteration in their tumors; these mutations have the potential to be biomarkers for the presence of ctDNA. Patient #27 had Her2 and c-MYC amplification in the breast tumor, ctDNA before and after NAT. The tumor DNA also contained the mutation TP53:NM_000546: c.701A>G, and this variant also observed in ctDNA before and after NAT. Patient #27 experienced recurrence. Patient #83 had variants in the TP53 and PIK3CA genes in tumor cells. Both variants were identified in ctDNA before NAT but not after NAT. Patient #83 did not experience recurrence. The data show good concordance between TP53 variants and other genetic alterations in ctDNA.
Supplementary Figure S2 (continued) p < 0.001 Supplementary Figure S3 ctDNA positivity after NAT was significantly associated with RFS among 72 patients with detectable genetic variants before and after NAT, p < 0.001.

Recurrence-free
survival Years A Before NAT

B
Supplementary Figure S4 case #50 After NAT case #5 For case #50, staging computed tomography (CT) showed no hepatic metastasis before NAT; however, follow-up CT revealed multiple hepatic metastases 6 months after NAT. Case #5 did not have neurologic symptoms before NAT. However, unsteady gait occurred 13 months after NAT, and magnetic resonance imaging found cerebellar metastasis.