Experimental cohort and genomic profiling of tissue
An overview of patient enrollment and study design is shown in Fig. S1. We initially enrolled 65 patients with locally advanced or metastatic breast cancer between May 2016 and March 2017. After further clinical assessment, 23 patients were excluded because they were ineligible for neoadjuvant chemotherapy. In addition, one patient was excluded due to insufficient tumor tissue for genomic analysis and seven patients were excluded due to drop-out during follow-up. Ultimately, 32 patients were included in the study, and 63 tissue and 206 blood samples were collected (Fig. S2). Baseline (pre-treatment) patient characteristics and clinical staging information are shown in Table 1. The median diagnostic age was 47 years (27–67). Twelve patients (37.5%) were menopausal. Most tumors (71.9%, 23/32) were moderately differentiated. Twenty-four (75.0%) patients were estrogen receptor (ER) positive and 21 (65.6%) were progesterone receptor (PR) positive. HER2 overexpression was identified in 11 patients (34.4%). Only four patients (12.5%) were identified with low Ki67 (< 14%). Except for five patients with metastatic disease (15.6%), 5 (15.6%) had stage IIb and 22 (68.8%) patients had stage Ⅲ disease.
Table 1
Clinicopathological characteristics and ctDNA detectability of analyzed patients
Characteristics
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Total = 32
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Age at diagnosis, years, median (range)
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47 (27 to67)
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Menstruation
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Menopausal, n (%)
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12 (37.5%)
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Premenopausal, n (%)
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20 (62.5%)
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Pathological examination of the biopsy
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|
Moderate differentiation, n (%)
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23 (71.9%)
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Poor differentiation, n (%)
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9 (28.1%)
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ER status
|
|
Positive, n (%)
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24 (75.0%)
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Negative, n (%)
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8 (25.0%)
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PR status
|
|
Positive, n (%)
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21 (65.6%)
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Negative, n (%)
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11 (34.4%)
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HER2 Overexpression
|
|
Positive, n (%)
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11 (34.4%)
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Negative, n (%)
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21 (65.6%)
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Ki67
|
|
< 14%, n (%)
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4 (12.5%)
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≥ 14%, n (%)
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28 (87.5%)
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Luminal typing
|
|
Luminal A, n (%)
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4 (12.5%)
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Luminal B, n (%)
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20 (62.5%)
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HER2 overexpression, n (%)
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3 (9.4%)
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TNBC, n (%)
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5 (15.6%)
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T stage
|
|
T1, n (%)
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4 (12.5%)
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T2, n (%)
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22 (68.8%)
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T3, n (%)
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6 (18.7%)
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N stage
|
|
N1, n (%)
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9 (28.1%)
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N2, n (%)
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17 (53.2%)
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N3, n (%)
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6 (18.7%)
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M stage
|
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M0, n (%)
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27 (84.4%)
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M1, n (%)
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5 (15.6%)
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Clinical stage
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|
IIb, n (%)
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5 (15.6%)
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III, n (%)
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22 (68.8%)
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IV, n (%)
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5 (15.6%)
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pCR
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|
Yes, n (%)
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4 (12.5%)
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No, n (%)
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28 (87.5%)
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Baseline ctDNA
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|
Positive, n (%)
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21 (65.6%)
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Negative, n (%)
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9 (28.1%)
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NA*, n (%)
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2 (6.3%)
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Chemo ctDNA
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Positive, n (%)
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3 (9.4%)
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Negative, n (%)
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29 (90.6%)
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Post-Chemo ctDNA
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|
Positive, n (%)
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9 (28.1%)
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Negative, n (%)
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22 (68.8%)
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NA*, n (%)
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1 (3.1%)
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Post-Op ctDNA
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|
Positive, n (%)
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5 (15.6%)
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Negative, n (%)
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27 (84.4%)
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Abbreviations: ER, estrogen receptor; PR, progesterone receptor; TNBC, triple-negative breast cancer; Chemo, chemotherapy; Op, operation.
*sample unattained.
The genomic status of tumor tissue was obviously changed under the pressure of neoadjuvant chemotherapy. Within 117 and 75 mutations identified in 31 pairs of tissue biopsy and surgically resected tissue samples, only 46 mutations were co-expressed in both samples, and the genomic status was stable in only two patients (P06, P25), indicating a longitudinal heterogeneity induced by chemotherapy (Fig. S3). To eliminate the temporal bias in the evaluation of plasma, we assessed the union set of tissue biopsy and resected tissue mutations in the same patient. As a result, at least one mutation was identified in each patient. In total, 151 mutations were detected with a median of 3 (range, 1–23). TP53 (n = 14, 43.8%) and PIK3CA (n = 13, 40.6%) were the most recurrent mutant genes as expected (Fig. 1A). We also evaluated the concordance between ERBB2 amplification at baseline and HER2 status determined by immunohistochemical staining and fluorescence in situ hybridization (Fig. 1B). Among 11 patients with HER2-positive tumors, 10 (90.9%) carried ERBB2 amplification (copy number > 3). All 21 patients with negative HER2 exhibited a normal copy number of ERBB2. The overall concordance was 96.9%. Even for the inconsistent one, the copy number of ERBB2 (2.6) was higher than that of patients with HER2-negative tumors. It did not reach the cut-off value possibly due to the insufficient proportion of tumor cells in tissue sample.
Clinical Characteristics And Baseline Ctdna
Based on the aforementioned tissue analysis, we tracked so-called tumor-derived mutations in blood ctDNA, including SNVs, Indels and CNV of ERBB2. As a result, at least one tumor-derived mutation could be detected at baseline in 21/30 patients (70%), during chemotherapy in 3/32 patients (9.4%), post-chemotherapy in 9/31 patients (29.0%), and post-operatively in 5/32 patients (15.6%) (Table 1). All patients with negative ctDNA at baseline demonstrated no mutation in blood ctDNA at any other time point, indicating the importance of determining the baseline status of ctDNA (Table 1).
At baseline, premenopausal patients had significantly more positive ctDNA than menopausal patients (p = 0.013, Fig. 2A). We also evaluated the variant allele frequency (VAF) for each plasma sample to reflect the circulating tumor burden. For plasma samples with more than one mutation, the VAF was identified as the maximal one. Similarly, the premenopausal cohort showed a higher VAF than the menopausal cohort (p = 0.007, Fig. 2A). In addition, patients with more lymph involvement had a higher proportion of positive ctDNA than those with less lymph involvement (p = 0.011, Fig. 2B). Although not reaching statistical significance, a higher VAF was observed in patients with more lymph involvement (p = 0.073, Fig. 2B). Higher fractions of positive ctDNA (p = 0.007) and VAF (p = 0.043) were identified in patients with advanced stage than in those with early stage tumors (Fig. 2C). Furthermore, the fraction of patients with positive ctDNA associated with high recurrence risk factors, including poor differentiation, hormone receptor negative, HER2 positive, high Ki67, and metastasis (Fig. S4). The VAF was also relatively high for patients with positive progesterone receptor and metastasis (Fig. S4). Taken together, these results indicate that the baseline ctDNA status is associated with multiple clinical factors; however, they should be further validated in large-scale cohorts.
The peri-neoadjuvant ctDNA associated with the therapeutic efficacy
The cfDNA concentration and ctDNA VAF varied at different sampling time points. At baseline, the cfDNA concentration was the lowest (p < 0.001, Fig. 2D), whereas the VAF was the highest (p < 0.001, Fig. 2D). For the dynamic change of ctDNA, four types of patients were present: (1) ctDNA was negative at baseline, during chemotherapy, and after chemotherapy (n = 9); (2) ctDNA was positive at baseline, but negative during and after chemotherapy (n = 11, Fig. S5A); (3) ctDNA was positive at baseline and during chemotherapy (n = 3, Fig. S5B); and (4) ctDNA was positive at baseline and after chemotherapy, but was negative during chemotherapy (n = 7, Fig. S5C). For type 3 and 4, the ctDNA residue during and after chemotherapy may be a clue about the refractory disease and be associated with therapeutic efficacy.
Whereafter, we analyzed the correlation between ctDNA status and therapeutic efficacy. Miller-Payne classification was used to assess the pathological response of carcinoma in situ. For N stage (ypN) after NCT, one patient was excluded because the post-chemotherapy blood sample was unavailable. Overall, 21 patients (67.7%) exhibited negative ctDNA during and after chemotherapy, including four pCR patients (Fig. 3). The median decrease in primary tumor volume was 54.6% (range, 17.1–98.0%) and 90.2% (range, 7.7–100%) for patients with positive and negative ctDNA, respectively (Fig. 3). With respect to Miller-Payne classification, grade 4–5 was found in only 10% (1/10) of patients with positive ctDNA but over half (52.38%, 11/21) of patients with negative ctDNA (p = 0.046; Fig. 3, Table S2). Furthermore, we found the median number of involved lymph nodes was 3.5 (range, 0–6) for patients with positive ctDNA and 1 (range, 0–9) for patients with negative ctDNA. Over 10 regional lymph nodes were involved (ypN3) in four patients with positive ctDNA. Nevertheless, no negative patients demonstrated stage ypN3 (p = 0.0067; Fig. 3, Table S2). To determine whether baseline ctDNA status is related to mutational detection of serial ctDNA based on the aforementioned results, we next performed the same analysis in 22 patients with positive baseline ctDNA and drew similar conclusions as expected (for Miller-Payne grade, p = 0.0244; for stage ypN, p = 0.0260; Table S2). These results indicated that peri-neoadjuvant ctDNA status before surgery could predict the pathological decrease in carcinoma in situ, as well as the involvement of regional lymph nodes evaluated post-operatively.
Post-operative Ctdna Status Related To Recurrence And Survival
The median follow-up was 23.2 months (range, 16–26.3 months). All patients underwent post-operative adjuvant agents, including radiotherapy (n = 32), aromatase inhibitor (n = 21), and trastuzumab (n = 9). During follow-up, seven patients (21.9%) experienced distant recurrence, including five LABC and two MBC. We evaluated the prognostic discrepancy between patients grouped by different pathological factors, CEA/CA15-3 level, and serial ctDNA status. As a result, the recurrence rate and DFS performed similarly between patients with different Luminal isoforms, Miller-Payne grade, ypN stage, pathologic response, CEA/CA15-3 level, as well as cycle 3, and post-chemotherapy ctDNA status (Fig. S6). Although not demonstrating statistical significance, patients with clinical stage Ⅳ or positive baseline ctDNA tended to present poorer DFS compared with their counterparts (Fig. S6). Dramatically, patients with positive ctDNA after surgery had a higher risk of recurrence and worse DFS than those with negative ctDNA (Fig. 4A). All five patients with positive ctDNA post-operatively experienced distant metastasis during follow-up, while only 7.41% (2/27) of those with negative ctDNA relapsed. The median DFS was 9.8 months for the positive ctDNA cohort but not reached for the negative ctDNA cohort (HR 23.53; 95% CI, 1.904–290.9; p < 0.0001). Considering the potential prognostic impact of MBC and baseline ctDNA status, we further performed subgroup analysis only focusing on patients with clinical stage Ⅱ/Ⅲ or positive baseline ctDNA. Similar to the total cohort, those with positive ctDNA after surgery also displayed poorer prognosis compared with their counterparts (for patients with clinical stage Ⅱ/Ⅲ, recurrence rate, 100% vs. 0%, median DFS, 9.8 months vs. not reached, HR and 95% CI were unevaluable, p < 0.0001, Fig. 4B; for patients with positive baseline ctDNA, recurrence rate, 100% vs. 11.8%, median DFS, 9.8 months vs. not reached, HR, 15.59, 95% CI 1.822–133.4, p < 0.0001, Fig. 4C).
Sequential Ctdna Analysis In Tumor Surveillance
For seven patients with distant recurrence, blood samples were collected 3–5 times post-operatively and during follow-up. We used the VAF to depict the dynamic change of tumor burden for each patient. The CEA/CA15-3 levels at some time points were also taken into consideration. As shown in Fig. 5, the quantitative VAF of ctDNA fluctuated with the clinical performance of each patient. All patients experienced a dramatic increase in ctDNA VAF at relapse, and multiple ctDNA samples were positive between surgery and relapse, except for those of P25. P03 and P08 underwent adjuvant radiotherapy and a sequential aromatase inhibitor regimen, and ctDNA samples collected post-operatively and at recurrence were both positive. The VAF increased despite therapeutic agents and persisted in subsequent time points. Particularly for P03, a blood sample was collected at the end of chemotherapy after recurrence. The therapeutic evaluation was partial response, and a significant decrease in the VAF was observed. For P19, the VAF was stable during adjuvant radiotherapy but increased dramatically at recurrence. The therapeutic evaluation of second-line radiotherapy was PR, and the tumor progressed 112 days later, both of which were reflected in the dynamic change in ctDNA VAF. For P24 and P39, two blood samples were collected during adjuvant radiotherapy and sequential trastuzumab intake, the latter of which was detected with tumor-derived mutations. One hundred and sixty days later, both patients experienced bone or chest metastasis, and ctDNA VAF also increased to a relatively high level. For P24, the ctDNA burden then decreased significantly due to subsequent chemotherapy and lapatinib intake. For P25, only blood collected at recurrence was detectable with tumor-derived mutations. P41 underwent adjuvant radiotherapy and a sequential aromatase inhibitor regimen with trastuzumab. CtDNA VAF was initially stable and then increased when chest, lymph node, and bone metastasis arose. In consideration of the hypothesis that positive ctDNA represents the post-operative minimal residual disease before recurrence, tumor relapse could be predicted in advance with a median interval of 6.9 months (range, 0–12.1 months).
However, the sensitivity of conventional CEA/CA15-3 in tumor surveillance was unsatisfactory. Only one (P25) patients exhibited elevated CEA/CA15-3 post-operatively; two (P08, P25) had elevated CEA/CA15-3 at recurrence (Fig. 5). Furthermore, P24 and P25 showed elevated CEA/CA15-3 levels at 6.9 and 7.3 months before recurrence, respectively, indicating an advantage in relapse prediction over ctDNA (3.3 and 0 months) (Fig. 5). Altogether, the combination of CEA/CA15-3 and ctDNA may improve treatment monitoring in patients with locally advanced or metastatic breast cancer.