Patient characteristics and availability of CTC, cfDNA and primary and metastatic tissue.
We performed DNA sequencing of CTC and cfDNA samples in a cohort of 38 women with metastatic breast cancer patients treated in our center. A valid sample was available in 34 of them. In 20 of them there was available tissue of metastasis, and 32 also had tissue from primary tumor. Median age of the patients was 58 (range, 37–85). As shown in Table 1, most patients (81.6%) had endocrine resistant disease, either primary (21.1%) or secondary (60.5%). The plasma samples were taken in different response settings corresponding to different moments of the clinical evolution: 2 at time of metastatic recurrence or after metastasis surgery (5.3%), 21 with stable disease (55.3%), 5 with partial or complete response (13.2%), and 9 of them at time of disease progression (23.7%). Treatment at time of sample obtention consisted in endocrine therapy in 60.5% of the patients and was chemotherapy for the remaining 39.5%. Median overall survival for the whole group has not been reached, and 2-years survival is 60% (95%CI, 45–75%).
Among the 38 patients, only 8 (21%) had detectable CTC. All CTC + patients showed endocrine resistance (37.5% primary and 62.5% secondary), although no significant association was found between endocrine resistance and CTC detection (Fisher’s exact test, p = 0.31). Detection of CTC was associated with progressive disease (45.5% vs. 10.3% of patients with SD/OR, p = 0.03) and showed a trend to worse overall survival (median: 13.2 months vs. not reached; p = 0.13).
Table 1
Patient characteristics (n = 38).
Characteristicsa | N (%) |
Age, median, range | | 58 (37–85) |
Metastasis location | Bone only | 23 (60.5%) |
| Visceral only | 6 (15.8%) |
| Visceral + Bone | 9 (23.7%) |
Metastatic disease | At diagnosis (M1) | 16 (42.1%) |
| Recurrence (M0) | 22 (57.9%) |
Type of endocrine resistance | No resistance | 7 (18.4%) |
Primary resistance PD < 6 months 1st line ET Recurrence < 2yrs adjuvant ET | 8 (21.1%) 5 (13.2%) 3 (7.9%) |
Secondary resistance PD > 6 months 1st line ET Recurrence > 2yrs adjuvant ET | 23 (60.5%) 15 (39.5%) 8 (21.1%) |
Drug related to endocrine resistance | Aromatase inhibitors | 28 (73.7%) |
Tamoxifen | 9 (23.7%) |
Fulvestrant | 1 (2.6%) |
Response setting | PD | 9 (23.7%) |
SD | 21 (55.3%) |
PC/CR | 5 (13.2%) |
Pre-treatment/NED | 3 (7.9%) |
Endocrine therapy | 1st line 2nd line 3rd line | 17 (44.7%) 4 (10.5%) 2 (5.3%) |
Chemotherapy | 1st or 2nd line CT 3rd line | 7 (18.4%) 8 (21.1%) |
CTC detection | CTC- | 30 (79%) |
CTC+ | 8 (21%) |
a CR: complete response; CT: chemotherapy; CTC: circulating tumor cell; ET: endocrine therapy; NE: non-evident disease; PD: progression of disease; PR: partial response; SD: stable disease; |
Sequencing of cfDNA, CTC, metastases and primary tumor
DNA sequencing was based on a targeted panel comprising 10 genes and 152 hotspots. The same panel was used for all samples. The rate of detection of genetic alterations was 87.5% (7/8) for ctcDNA, 61.5% for cfDNA, 63.2% for metastatic tissue and 67.7% for the primary tumor biopsy. The type and number of mutations for each type of samples are summarized on Fig. 1 and the complete list is shown in Supplementary Table S1.
The median number of genetic alterations detected by DNA sequencing did not significantly differ among the different types of samples, with a range of 0 to 4 for all of them, except for primary tumor biopsies (range: 0–8).
The specific type of genetic alterations also differed among the different types of samples (Table 2). In particular, sequencing of CTC showed a high frequency of TP53 mutations (75% of cases) and a comparable number of PIK3CA (25%), but no ESR1 mutations were found. In contrast with CTC results, cfDNA was enriched in ESR1 mutations (6/34; 17.6%) while keeping a comparable frequency of PIK3CA (23.5%) and AKT1 (14.7%) mutations and an intermediate number of TP53 mutations (50%).
Table 2
Frequency of genomic alteration according to type of sample.
| ctDNA (n = 34) | CTC (n = 8) | Metastasis (n = 20) | Primary tumor (n = 32) |
TP53 | 17 (50.0%) | 7 (87.50%) | 4 (20.0%) | 9 (28.0%) |
PIK3CA | 9 (26.5%) | 2 (25.0%) | 7 (35.0%) | 14 (43.8%) |
ESR1 | 6 (17.6%) | 0 (0%) | 2 (10.0%) | 1 (3.1%) |
SF3B1 | 4 (11.7%) | 1 (12.5%) | 2 (10.0%) | 3 (9.4%) |
AKT1 | 5 (14.7%) | 2 (25.0%) | 4 (20.0%) | 2 (6.3%) |
FBXW7 | 0 (0%) | 1 (12.50%) | 0 (0%) | 0 (0%) |
ERBB3 | 0 (0%) | 2 (25.0%) | 0 (0%) | 1 (3.1%) |
Others | 0 (0%) | 0 (0%) | KRAS:1 (5%); EGFR: 1 (5%) | ERBB2: 1 (3.1%); KRAS: 2 (6.2%) |
Sequencing of metastatic tissues revealed a similar pattern with predominance of PIK3CA (35%), AKT1 (20%) and ESR1 (10%) mutations. Finally, the distribution of primary tumor genetic alterations was different, with a very high rate of PIK3CA mutations (43.7%), a lower occurrence of TP53 mutations (28%) and only one case with an ESR1 mutation (3.1%). Interestingly, a few mutations were only found in some type of samples. In particular, FBXW7 and ERBB3 mutations were detectable only in CTC, while ERBB2 mutations were only present in the primary tumor sample of one patient.
The number of different mutations of the same gene for a particular patient was also higher for TP53 and PIK3CA, especially in the primary tumor and cfDNA, thus yielding a larger total number of genomic alterations (Fig. 2).
Matched asssesment and concordance of somatic mutations on liquid biopsies (CTC, ctDNA) and metastasis tissue
Globally, we found a low to intermediate concordance between CTC and cfDNA, for the detection of any genomic alterations (Table 3). Concordance was also low to moderate between primary and metastatic tissue. A higher overall concordance was found between CTC and primary tumor sequencing (k = 0.57; p = 0.12), which was significantly better between cfDNA and metastasis (k = 0.51, p = 0.03).
We performed a concordance analysis specifically focused on the three main groups of mutations related to endocrine resistance. Also, in order to determine the clinical utility of each type of sample to maximize the identification of targetable mutations, we calculated the percentage of positivity provided for each type of sample over the total number of cases with mutations identified by sequencing every pair of matched samples. As shown in Table 3, our findings are consistent with different degrees of overall concordance according to the type of mutation.
For PIK3CA, the concordance between CTC and cfDNA was low, and the same occurred when CTC results were compared with tissue biopsy of either the primary tumor or metastases, which suggest some degree of complementarity between CTC and other type of samples. Our results also showed a low concordance of cfDNA positive results with those provided by sequencing of tumor (25%) or metastasis (50%) biopsy, which might provide a higher sensitivity for detection of mutations (70-87.5% rate of positive results in comparison with 25–40% for cfDNA).
Regarding AKT1, complete concordance was found for detection of mutations between CTC and primary tumor or metastasis biopsy, while only 33% of mutations identified in cfDNA sequencing were found in the matched CTC samples. The concordance between cfDNA and primary or metastatic biopsies was also low, with the identification of only 16–25% of the mutations found in cfDNA.
ESR1 mutations were not found in any of the CTC samples, which has limited the concordance analysis with othder samples. However, the finding of ESR1 mutations in cfDNA again showed a low concordance (25%) with metastases biopsy detection, and null concordance with primary tumor, as expected. Finally, although not shown in Table 3, the concordance for TP53 mutations was very low in every pair of matched samples, with kappa values below 0.20 in all cases.
Association of genomic alterations in metastatic samples (CTC, cfDNA, metastatic tissue) with progressive disease and endocrine resistance
The average number of mutations found in cfDNA was significantly higher in those patients with disease progression (2.27 vs. 0.81; Mann-Whitney U, p = 0.002), but we did not find the same differences for CTC (p = 0.39) or metastatic tissue (p = 0.79) samples. Similarly, the number of patients with any mutation present in cfDNA showed a non-significant association with the response status (progression disease: 90.9%; stable disease or objective response: 60.9%) (Fisher’s exact test, p = 0.11). Endocrine resistant patients also showed a trend to a higher number of genetic alterations in cfDNA when compared with patients without resistance (1.42 vs. 0.43; p = 0.08), again without differences for the rest of samples (Fig. 1).
We also analyzed the association of finding specific genetic alterations in any of the metastatic samples (CTC, cfDNA, metastasis biopsy) with endocrine resistance. The association differed according to the specific gene mutation: the frequency of PIK3CA mutations trended to be higher (p = 0.22) in patients with endocrine resistance (primary: 50%, secondary: 34.7%) than in patients without resistance (14.3%). The same non-significant tendency was found for AKT1 mutations, which only occurred in endocrine resistant cases (primary: 37.5%; secondary: 17.4%). Similarly, ESR1 mutations were, only present in cases with endocrine resistance (22.5%), most of them (85.7%) corresponding to secondary resistance to aromatase inhibitors. Finally, only ESR1 mutations were associated with disease progression (45.5% mutated vs. 7.4% of patients with SD or OR; p = 0.01). This association was observed neither for PIK3CA (p = 0.46) nor for AKT1 mutations (p = 0.39).
Table 3
Concordance of matched samples for endocrine resistance-related genomic alterations.
| CTC-cfDNA | CTC-MTS | CTC-Tumor | cfDNA-MTS | cfDNA-Tumor | MTS-Tumor |
| N = 8 | N = 4 | N = 6 | N = 20 | N = 32 | N = 16 |
Any genomic alteration Overall concordance Mutation detection rate Positivity concordance Statistics | 62.5% CTC 87.5% cfDNA 75% 62.5% K = 0.20, p = 0.54 | 50% CTC 100% MTS 50% 50% --- | 83.3% CTC 83.3% Tumor 66.7% 66.6% K = 0.57, p = 0.12 | 77.7% cfDNA 72.2% MTS 61.1% 71.4% K = 0.51, p = 0.03 | 60.7% cfDNA 67.9% Tumor 71.4% 56.0% K = 0.07, p = 0.70 | 60% MTS 60% Tumor 73.3% 53.8% K = 0.12, p = 0.63 |
PIK3CA Overall concordance Mutation detection rate Positivity concordance Statistics | 62.5% Low CTC 50% (2/4) ctDNA 75% (3/4) 25% (1/4) κ = 0.14, p = 0.67 | 75% Moderate CTC 100% (2/2) Mts 50% (1/2) 50% (1/2) κ = 0.50, p = 0.25 | 50% Low CTC 33.3% (1/3) Tumor 66.7% (2/3) 0% (0/3) κ = 0.28, p = 0.45 | 80% Moderate ctDNA 62.5% (5/8) MTS 87.5% (7/8) 50% (4/8) κ = 0.53, p = 0.01 | 62.5% Low ctDNA 37.5% (6/16) Tumor 87.5% (14/16) 25% (4/16) κ = 0.19, p = 0.21 | 62.5% Low MTS 70% (7/10) Tumor 70% (7/10) 40% (4/10) κ = 0.24, p = 0.34 |
AKT1 Overall concordance Mutation detection rate Positivity concordance Statistics | 75% Low CTC 33% (1/3) ctDNA 100% (3/3) 33% (1/3) κ = 0.38, p = 0.17 | 100% Very high CTC 100% (1/1) MTS 100% (1/1) 100% (1/1) κ = 1.0, p = 0.04 | 100% Very high CTC 100% (1/1) Tumor 100% (1/1) 100% (1/1) κ = 1.0, p = 0.01 | 75% Low ctDNA 50% (3/6) MTS 66.7% (4/6) 16.7% (1/6) κ = 0.14, p = 0.53 | 90.6% Low ctDNA 75% (3/4) Tumor 50% (2/4) 25% (1/4) κ = 0.35, p = 0.04 | 93.8% High MTS 100% (3/3) Tumor 66.7% (2/3) 66.7% (2/3) κ = 0.76, p = 0.002 |
ESR1 Overall concordance Mutation rate Positivity concordance Statistics | -- CTC 0% (0/8) ctDNA 100% (1/1) 0% -- | -- CTC 0% (0/4) MTS 0% -- | -- CTC 0% (0/6) Tumor 0% (0/6) 0% -- | 85% Low ctDNA 75% (3/4) MTS 50% (2/4) 25% (1/4) κ = 0.32, p = 0.14 | 84.4% Very low ctDNA 80% (4/5) Tumor 20% (1/5) 0% (0/5) κ=-0.05, p = 0.70 | -- MTS 100% (2/2) Tumor 0% (0/2) 0% (0/2) -- |