Patient characteristics
A total of 13 women with ovarian cancer were enrolled in this study. Patient characteristics are presented in Table 1. The median age at diagnosis was 56 years (range 40–75). The histology of ovarian cancer was high-grade, serous carcinoma in 9 patients. The initial FIGO stage in 4, 7, and 2 patients was stage I-II, stage III, and stage IV, respectively. All patients underwent surgery and chemotherapy, and a complete response was observed in 11 patients. Six patients had a recurrence in lung, liver, or peritoneal seeding. Initial CA125 was high in all patients, and CTCs were detected in initial samples of 84.62% patients (11/13). All patients underwent debulking surgery including total abdominal hysterectomy, bilateral Salpingo-oophorectomy, bilateral pelvic lymph node dissection, para-aortic nodal dissection, appendectomy, and omentectomy. Thirteen patients received platinum doublet-based adjuvant chemotherapy such as paclitaxel plus carboplatin with or without bevacizumab for 4–6 cycles, and 4 patients received neoadjuvant chemotherapy before surgery because of the unresectable status. After the therapy, samples from 10 patients were collected and tested. In 90% patients, CA125 levels and CTC counts were lower than those in baseline samples. Only patient 6 had increased CTC counts after therapy. In addition, this atypical patient had a normal range of CA125 (< 30 U/ml) in the baseline sample.
Table 1. Clinical characteristics of patients with ovarian cancer
No.
|
Age at diagnosis (years)
|
Initial FIGO
Stage
|
Histology
|
Initial CA125 (U/mL)
|
Initial CTCs
(/3 mL)
|
Treatment
response
|
CA125
after therapy (U/mL)
|
CTCs
after therapy
(/3 mL)
|
Recurrence
|
No. of blood samples
|
1
|
65
|
IIIC
|
High-grade serous carcinoma
|
3186.5
|
76
|
CR
|
5.8
|
0
|
No
|
8
|
2
|
67
|
IIIC
|
High-grade serous carcinoma
|
76
|
26
|
CR
|
8.1
|
0
|
Yes
|
7
|
3
|
55
|
IIIC
|
High-grade serous carcinoma
|
4278.5
|
5
|
CR
|
11.3
|
0
|
Yes
|
6
|
4
|
53
|
IC
|
Mucinous carcinoma
|
18.2
|
64
|
CR
|
8.5
|
1
|
No
|
5
|
5
|
49
|
IIIA2
|
High-grade serous carcinoma
|
631.9
|
55
|
CR
|
9.3
|
2
|
No
|
4
|
6
|
56
|
IC
|
High-grade serous carcinoma
|
25.7
|
0
|
CR
|
7.7
|
3
|
No
|
4
|
7
|
75
|
IV
|
Adenocarcinoma
with serous carcinoma
|
10000
|
0
|
PR
|
244.5
|
0
|
Yes
|
4
|
8
|
40
|
IIB
|
Clear cell carcinoma
|
34.6
|
13
|
CR
|
41.6
|
3
|
Yes
|
3
|
9
|
59
|
IIB
|
High-grade serous carcinoma
|
696.4
|
1
|
CR
|
65.1
|
0
|
Yes
|
3
|
10
|
46
|
IIIC
|
Clear cell carcinoma
|
2432.8
|
6
|
CR
|
153.1
|
0
|
No
|
2
|
11
|
47
|
IV
|
High-grade serous carcinoma
|
553.3
|
2
|
CR
|
NA
|
NA
|
Yes
|
1
|
12
|
66
|
IIIC
|
High-grade serous carcinoma
|
3399.8
|
10
|
CR
|
NA
|
NA
|
No
|
1
|
13
|
65
|
IIIC
|
High-grade serous carcinoma
|
8767.4
|
4
|
PR
|
NA
|
NA
|
No
|
1
|
FIGO, International Federation of Gynecology and Obstetrics; CTCs, Circulating Tumor Cells; CR, complete response; PR, partial response.
From each patient sample, isolated cells were stained and discriminated based on the conventional criteria for CTC identification that is EpCAM/CK positive and DAPI positive cells without CD45 expression. Figure 2 shows the representative images of CTCs from patient samples.
Detection of CTCs and correlation with CA125
At baseline, 84.62% of patients had a positive CTC count with 1 or more CTCs in 3 ml of blood (range 1–76). The mean and median CTC count of all patients were 20.2 and 6.0 at baseline. The median follow-up duration was 22.7 months (range 5.2–28.7). Most of the patients presented a decreased CTC count after surgery and chemotherapy, except for 2 patients who had no CTC at baseline. At the time of complete response, CTC count evaluated in 8 patients was < 3.
For the correlation analysis between CTC count and CA125 according to clinical course, patients were selected according to the following criteria: 1) patients who gave blood samples > 3 times, 2) patients who had higher than normal (30 U/ml) initial CA125 level, and 3) patients diagnosed with complete response based on the imaging result after surgery. Based on these criteria, 6 patients were selected, and the patients were divided into two groups: no recurrence group and recurrence group.
For the 3 patients in the no recurrence group, the CTC count and CA125 had similar patterns and were correlated with the clinical course. Patient 1 was diagnosed with ovarian cancer, high-grade serous carcinoma, and initial FIGO stage IIIC. The pre-treatment CTC count and CA125 were 76 and 3186.5, respectively. After perioperative chemotherapy and surgery, both parameters decreased to baseline levels, and a complete response was observed in the computed tomography (CT) scan at the end of chemotherapy (Fig. 3A). Patient 5 was diagnosed with ovarian cancer, high-grade serous carcinoma, and initial FIGO stage IIIA2. The pre-treatment CTC count and CA125 were 55 and 631.9, respectively. After surgery and chemotherapy, a durable complete response was observed in the CT scan, and CTC count and CA125 were 2 and 9.3, respectively, which further decreased to 0 and 10.0, respectively after 14 months (Fig. 3D). Patient 8 was diagnosed with ovarian cancer, clear cell carcinoma, and initial FIGO stage IIB. The pre-treatment CTC count and CA125 were 13 and 67.8, respectively. Both parameters were similar during treatment, and a complete response was observed after 2 months (Fig. 3E).
For the 3 patients in the recurrence group, the CTC count and CA125 showed similar patterns. However, CTC count was elevated before or after recurrence while CA125 remained in the normal range. For example, patient 2 was diagnosed with ovarian cancer, high-grade serous carcinoma, and initial FIGO stage IIIC. The pre-treatment CTC count and CA125 were 26 and 76.0, respectively. After chemotherapy and surgery, CTC count and CA125 were 0 and 8.1, respectively, and a complete response was observed in the CT scan. Recurrence occurred 5 months later with the appearance of new lung nodules. The increase in CTC count was followed by a recurrence, although CA125 remained within the normal range (Fig. 3B). Patient 3 was diagnosed with ovarian cancer, high-grade serous carcinoma, and initial FIGO stage IIIC. The pre-treatment CTC count and CA125 were 5 and 4278.5, respectively. After surgery and chemotherapy, CTC count and CA125 decreased to 0 and 11.3, respectively, and a complete response was observed in the CT scan. Five months later, an increase in CTC count with CA125 in the normal range was observed followed by a recurrence of peritoneal seeding (Fig. 3C). Patient 9 was diagnosed with ovarian cancer, high-grade serous carcinoma, and initial FIGO stage IIB. After 20 months from diagnosis, CTC count was slightly increased with a normal range of CA125, and recurrence was detected in the peritoneum (Fig. 3F).
All the changes in CTCs and CA125 were analyzed to check concordance and association with the corresponding clinical assessments. First, to confirm the concordance between CA125 and CTC count, each point of change in the 6 cases was classified into 4 categories: both increased, only CTC increased, only CA125 increased, and no change in both (no change or decrease). Concordance in increasing change was calculated in cases where both increased, and it was 71.4%. On the other hand, concordance in no change was calculated in cases where there was no change in both, and it was 75.0%. According to the directional concordance analysis, CTC count had a high concordance with a change in CA125 (Fig. 4A). To analyze the association with the corresponding clinical status, each point of change in the 6 cases was classified according to recurrence. Both CA125 and CTCs had the same specificity of 77.8%. However, CTCs had higher values in other aspects including sensitivity (CTCs 100.0% vs. CA125 60.0%), positive predictive value (PPV, CTCs 55.6% vs. CA125 42.9%), and negative predictive value (NPV, CTCs, 100.0% vs. CA125 87.5%) than CA125 (Fig. 4B).