Abstract
The current study aimed to investigate the relation of circulating tumor cell (CTC) with clinicopathological features. In addition, its longitudinal change during chemotherapy and its correlation with prognosis in advanced gallbladder carcinoma (GBC) patients were explored. Totally 45 unresectable, locally advanced or metastatic GBC patients who underwent chemotherapy were enrolled in this prospective study. The CTC in 7.5 ml blood was detected at pre-treatment and 3 months post-treatment. CTC was almost detectable in all advanced GBC patients before treatment, whose count was positively correlated with metastatic disease (vs. local advanced disease) (P=0.002), number of organs with metastases (P=0.006), and CA199 level (P=0.002). After treatment, CTC count declined from 4.0 (range: 0.0-83.0) at pre-treatment to 2.0 (range: 0.0-36.0) at post-treatment (P=0.003). Interestingly, pre-treatment CTC count (P=0.270) was of no difference, while post-treatment CTC count was lower (P=0.038) in objective-response patients compared to that in non-objective-response patients; meanwhile, both pre-treatment CTC count (P=0.017) and post-treatment CTC count (P<0.001) were lower in disease-control patients compared with those in non-disease-control patients. Importantly, pre-treatment CTC count ≥2 (versus <2) was only correlated with worse progression-free survival (PFS) (P=0.014) but not overall survival (OS) (P=0.057); while pre-treatment CTC count ≥5 (versus <5), post-treatment CTC count ≥2 (versus <2), post-treatment CTC count ≥5 (versus <5), CTC count up (versus equal/down) were all correlated with poor PFS and OS (all P<0.050). In conclusion, higher CTC count during chemotherapy correlates with worse treatment response, PFS and OS in advanced GBC patients, which implies that CTC measurement may optimize the prognostication and individualized treatment in these patients.
Keywords: Advanced gallbladder carcinoma, circulating tumor cell, clinicopathological features, chemotherapy, prognosis
Introduction
Gallbladder carcinoma (GBC) is one of the most fatal gastrointestinal malignances, which accounts for 80-95% of biliary tract carcinomas [1]. It is currently considered that screening of risk factors such as gallstones, chronic cholecystitis, chronic bacterial infection, primary sclerosing cholangitis, etc. would improve the early identification of GBC, and then promote the surgical accessibility to facilitate the better prognosis of GBC patients [2]. However, due to the unspecific clinical symptoms, GBCs are commonly diagnosed at advanced stages, which limits the optimal application of resection, and worsens the patients’ prognosis [3]. In addition, for the general GBC patients, their 5-year survival rate is already as low as 5%, not to mention that for those unresectable advanced-stage GBC patients, their survival prognosis is even much worse [1-3]. Therefore, it is of value to explore potential prognostic markers for unresectable, advanced stage GBC patients to improve their survival.
Circulating tumor cell (CTC) is a kind of cancer cells that are detached from primary tumor and then entered blood circulation, which is currently observed to be closely related to tumor metastasis [4]. Recently, CTC detection is proposed to be not only a diagnostic tool, but also a prognostic marker for several carcinomas [5,6], and it’s now recommended as a useful way to monitor progression-free survival (PFS) and overall survival (OS) in metastatic breast, prostate, and colorectal carcinomas by Food and Drug Administration of USA [4]. In terms of hepatobiliary carcinomas, several reports uncover that CTC correlates with advanced disease features and shows a potency for prognostication to facilitate precision medicine in hepatocellular carcinoma patients [7,8]. However, as for GBC, very limited data about CTC is revealed, and only a single recent study observes that CTC differentiates GBC patients from disease controls (cholecystitis patients) and normal controls, and correlates with more advanced TNM stage of GBC [9]. However, its prognostic role and longitudinal change during treatment in advanced GBC patients are unknown; meanwhile, based on the features of CTC and its previous application in other carcinomas, its investigation in advance GBC might optimize the prognostication of patients.
Therefore, the current study aimed to investigate the relation of CTC with clinicopathological features; furthermore, its longitudinal change during chemotherapy and its correlation with prognosis in advanced GBC patients were explored.
Materials and methods
Patients
This prospective cohort study consecutively enrolled 45 newly-diagnosed advanced GBC patients who were treated in our hospital between March 2016 and April 2020. The enrollment criteria included: (1) confirmed diagnosis of GBC by pathological examination via aspiration biopsy; (2) unresectable, locally advanced or metastatic GBC confirmed by imagine examinations; (3) age ≥18 years; (4) Eastern Cooperative Oncology Group performance status (ECOG PS) score 0-1; (5) adequate liver, renal and bone marrow function to undergo chemotherapy; (6) had measurable lesions on computed tomography (CT) scan or magnetic resonance imaging (MRI). Patients with any of the following conditions were excluded: (1) contraindications to chemotherapy; (2) poor compliance to regular follow-up; (3) pregnant or lactating female patients. This study was approved by the Ethics Committee of Renji Hospital with approval number “Renji Hospital [2014] 51k”. All patients signed the informed consents.
Basic clinical data collection
After diagnostic workup and necessary examinations, the following clinical features of patients were recorded in case report form (CRF): age, gender, ECOG PS score, disease status (locally advanced or metastatic), organs with metastases, carbohydrate antigen 199 (CA199) level, and carcinoembryonic antigen (CEA) level.
Treatment regimens
Patients received one of the following gemcitabine-based or fluorouracil-based chemotherapy regimens as first-line therapy: (1) gemcitabine plus cisplatin: gemcitabine 1000 mg/m2 plus cisplatin 50 mg/m2 administered intravenously (IV) on days 1 and 8, every 3 weeks; (2) gemcitabine plus oxaliplatin: gemcitabine 1000 mg/m2 IV on day 1, followed by oxaliplatin 100 mg/m2 IV on day 2, every 2 weeks; (3) fluorouracil plus cisplatin/leucovorin: leucovorin 200 mg/m2, fluorouracil 400 mg/m2 on day 1 followed by fluorouracil 2400 mg/m2 continuous infusion over 46 h, plus cisplatin 50 mg/m2, every 2 weeks; (4) fluorouracil plus oxaliplatin/leucovorin: leucovorin 200 mg/m2 followed by a 400 mg/m2 bolus fluorouracil followed by a 22-hour infusion of fluorouracil 600 mg/m2 on two consecutive days, plus a 2-hour infusion of 85 mg/m2 oxaliplatin, on day 1, every 2 weeks. Chemotherapy was continued until the patient declined further doses or until limiting toxicity or disease progression occurred. The dosages of chemotherapy drugs or the chemotherapy cycles were adjusted by attending physicians when the intolerant toxicity or disease progression occurred.
CTC analysis
Peripheral blood samples (7.5 mL) of GBC patients were respectively collected before initiation of chemotherapy and after 3-month chemotherapy (4-6 cycles), using tubes containing acid citrate dextrose. All samples were processed within 1 hour after collection. The CTC was isolated and enriched by EasySepTM Direct Human CTC Enrichment Kit (Stem Cells Technologies, Inc., Vancouver, BC, Canada), and the CTC count was detected by flow cytometry. The specific procedures of CTC detection were performed as described in a previous study [9]. In the survival analysis, CTC count was classified using the cut-off value of 2 and 5, according to the previous study [8]. Change of CTC count was calculated by the pre-treatment CTC count minus the post-treatment CTC count.
Follow-up and assessment
After initiation of chemotherapy, imagine examinations, such as MRI or CT scanning, were performed to assess the disease status, every 6 weeks in the first six months of the year and then every 2-4 months. The tumor response was evaluated at 3 months after initiation of chemotherapy, which was classified in accordance with the Response Evaluation Criteria in Solid Tumors criteria [10], including complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Objective response rate (ORR) was calculated as CR+PR and disease control rate (DCR) was calculated as CR+PR+SD. Furthermore, survival status of patients was documented during follow-up, and the progression-free survival (PFS) and the overall survival (OS) were evaluated. PFS was defined as the time interval from the initiation of chemotherapy to disease progression or death, whichever occurred first. OS was defined as the time interval from the initiation of chemotherapy to death.
Statistical analysis
R 4.0.3 and GraphPad Prism 7.02 (GraphPad Software Inc., San Diego, California, USA) were used for data analysis and plotting. Data were shown as mean ± standard deviation (SD), median with range, or number with percentage. Correlation of pre-treatment CTC count with clinical features was determined by Wilcoxon rank sum test or Kruskal-Wallis H rank sum test. The change of CTC count before and after treatment was determined by Wilcoxon signed-rank test. Comparison of CTC count between two groups was determined by Wilcoxon rank sum test. Survival curve distribution was displayed by Kaplan-Meier method, and the comparison of survival curve between two groups was determined by Log-rank test. Independent related factors of DFS and OS were determined by multivariate Cox’s analysis. P value less than 0.050 was considered as statistically significant.
Results
Patients characteristics
Forty-five advanced GBC patients with a mean age of 61.3±10.0 years were enrolled, among which 33 (73.3%) were females and 12 (26.7%) were males (Table 1). ECOG PS 0 and 1 appeared in 28 (62.2%) cases and 17 (37.8%) cases, respectively. Besides, 13 (28.9%) cases presented with locally advanced disease, while the other 32 (71.1%) cases presented with metastatic disease. In addition, the information about other detailed clinical features and chemotherapy regimens were shown in Table 1.
Table 1.
Characteristics of GBC patients
| Items | GBC patients (n=45) |
|---|---|
| Demographics | |
| Age (years), mean ± SD | 61.3±10.0 |
| ≤60 years, No. (%) | 19 (42.2) |
| >60 years, No. (%) | 26 (57.8) |
| Gender, No. (%) | |
| Male | 12 (26.7) |
| Female | 33 (73.3) |
| Disease-related features | |
| ECOG PS score, No. (%) | |
| 0 | 28 (62.2) |
| 1 | 17 (37.8) |
| Disease status, No. (%) | |
| Locally advanced | 13 (28.9) |
| Metastatic | 32 (71.1) |
| Number of organs with metastases, No. (%) | |
| 0 | 13 (28.9) |
| 1 | 19 (42.2) |
| 2 | 11 (24.5) |
| 3 | 2 (4.4) |
| Metastatic organs, No. (%) | |
| Liver | 25 (55.6) |
| Peritoneum | 13 (28.9) |
| Bone | 5 (11.1) |
| Lung | 4 (8.9) |
| CA199 (kU/L), median (range) | 81.6 (1.4-15238.6) |
| Normal (≤40 kU/L), No. (%) | 14 (31.1) |
| Abnormal (>40 kU/L), No. (%) | 31 (68.9) |
| CEA (ng/mL), median (range) | 6.6 (0.9-613.3) |
| Normal (≤5 ng/mL), No. (%) | 21 (46.7) |
| Abnormal (>5 ng/mL), No. (%) | 24 (53.3) |
| Treatment regimens | |
| Gemcitabine plus cisplatin, No. (%) | 30 (66.7) |
| Gemcitabine plus oxaliplatin, No. (%) | 8 (17.8) |
| Fluorouracil plus cisplatin/leucovorin, No. (%) | 4 (8.8) |
| Fluorouracil plus oxaliplatin/leucovorin, No. (%) | 3 (6.7) |
GBC, gallbladder carcinoma; SD, standard deviation; ECOG, Eastern Cooperative Oncology Group; PS, performance status; CA199, carbohydrate antigen 199; CEA, carcinoembryonic antigen.
Pre-treatment CTC and its relation to clinical features
Pre-treatment CTC showed a mean count of 8.6±15.1 and median count of 4.0 (IQR: 1.0-8.0) (Range: 0.0-83.0) in advanced GBC patients. Its distribution in these patients was illuminated in Figure 1. Interestingly, pre-treatment CTC count was positively correlated with metastatic disease (vs. locally advanced disease) (P=0.002), number of organs with metastases (P=0.006), liver metastases (P=0.004) and abnormal CA199 level (P=0.002), but not correlated with other clinical features in the advanced GBC patients (Table 2).
Figure 1.
Distribution of pre-treatment CTC count.
Table 2.
Correlation of pre-treatment CTC count with clinical features of GBC patients
| Clinical features | Pre-treatment CTC count, median (range) | P value |
|---|---|---|
| Age | 0.781 | |
| ≤60 years | 3.0 (1.0-83.0) | |
| >60 years | 5.0 (0.0-18.0) | |
| Gender | 0.990 | |
| Male | 4.0 (1.0-15.0) | |
| Female | 3.0 (0.0-83.0) | |
| ECOG PS score | 0.111 | |
| 0 | 3.0 (0.0-83.0) | |
| 1 | 5.0 (1.0-27.0) | |
| Disease status | 0.002 | |
| Locally advanced | 1.0 (0.0-12.0) | |
| Metastatic | 5.0 (1.0-83.0) | |
| Number of organs with metastases | 0.006 | |
| 0 | 1.0 (0.0-12.0) | |
| 1 | 5.0 (1.0-31.0) | |
| 2 | 6.0 (1.0-83.0) | |
| 3 | 16.5 (15.0-18.0) | |
| Liver metastases | 0.004 | |
| No | 2.0 (0.0-12.0) | |
| Yes | 5.0 (1.0-83.0) | |
| Peritoneum metastases | 0.067 | |
| No | 3.0 (0.0-56.0) | |
| Yes | 6.0 (1.0-83.0) | |
| Bone metastases | 0.148 | |
| No | 3.5 (0.0-83.0) | |
| Yes | 10.0 (2.0-56.0) | |
| Lung metastases | 0.631 | |
| No | 4.0 (0.0-83.0) | |
| Yes | 7.0 (1.0-15.0) | |
| CA199 | 0.002 | |
| Normal (≤40 kU/L) | 2.0 (0.0-7.0) | |
| Abnormal (>40 kU/L) | 6.0 (1.0-83.0) | |
| CEA | 0.590 | |
| Normal (≤5 ng/mL) | 4.0 (0.0-83.0) | |
| Abnormal (>5 ng/mL) | 3.5 (1.0-56.0) |
GBC, gallbladder carcinoma; CTC, circulating tumor cell; ECOG, Eastern Cooperative Oncology Group; PS, performance status; CA199, carbohydrate antigen 199; CEA, carcinoembryonic antigen.
Treatment response, PFS and OS
After chemotherapy, 0 (0.0%), 15 (33.3%), 16 (35.6%) and 14 (31.1) cases had CR, PR, SD and PD, respectively, with ORR of 33.3% and DCR of 68.9% in advanced GBC patients (Figure 2A). Furthermore, median PFS was 5.0 (95% CI: 3.7-6.3) months (Figure 2B) and median OS was 10.0 (95% CI: 8.7-11.3) months (Figure 2C) in these patients, respectively.
Figure 2.
Treatment response and survival data. Treatment response at 3 months (A), PFS (B) and OS (C) in chemotherapy treated advanced GBC patients.
Longitudinal change of CTC count and treatment response
After treatment, CTC count declined from 4.0 (range: 0.0-83.0) at pre-treatment to 2.0 (range: 0.0-36.0) at post-treatment in advanced GBC patients (P=0.003) (Figure 3). Importantly, post-treatment CTC count was lower in ORR patients compared to that in non-ORR patients (P=0.038), while pre-treatment CTC count (P=0.270) and change of CTC count (P=0.091) were of no difference between them (Figure 4A-C). Furthermore, both pre-treatment CTC count (P=0.017) and post-treatment CTC count (P<0.001) were lower, while change of CTC count (P=0.003) was higher in DCR patients compared with those in non-DCR patients (Figure 4D-F).
Figure 3.
Change of CTC count.
Figure 4.
Correlation of pre-treatment/post-treatment/change of CTC count with treatment response. Comparison of pre-treatment CTC count (A), post-treatment CTC count (B) and change of CTC count (C) between ORR patients and non-ORR patients. Comparison of pre-treatment CTC count (D), post-treatment CTC count (E) and change of CTC count (F) between DCR patients and non-DCR patients.
Correlation of CTC count with PFS and OS
Pre-treatment and post-treatment CTC count was cut off by 2 or 5, meanwhile, change of CTC count was categorized as up or equal/down, so as to assess their correlations with PFS and OS. Pre-treatment CTC count ≥2 (vs. <2, P=0.014, hazard ratio (HR) =2.199) (Figure 5A), pre-treatment CTC count ≥5 (vs. <5, P=0.002, HR=2.381) (Figure 5B), post-treatment CTC count ≥2 (vs. <2, P<0.001, HR=4.550) (Figure 5C), post-treatment CTC count ≥5 (vs. <5, P<0.001, HR=3.151) (Figure 5D), and CTC count up (vs. equal/down, P<0.001, HR=3.519) (Figure 5E) were all correlated with worse PFS. By comparison of HR, post-treatment CTC count ≥2 (vs. <2) showed best value for predicting PFS.
Figure 5.
Correlation of pre-treatment/post-treatment/change of CTC count with PFS. Correlation of pre-treatment CTC count ≥2 (vs. <2) (A), pre-treatment CTC count ≥5 (vs. <5) (B), post-treatment CTC count ≥2 (vs. <2) (C), post-treatment CTC count ≥5 (vs. <5) (D), and CTC count change up (vs. equal/down) (E) with PFS.
In addition, pre-treatment CTC count ≥2 (vs. <2, P=0.057, HR=1.942) (Figure 6A) did not associate with OS; while pre-treatment CTC count ≥5 (vs. <5, P=0.027, HR=1.961) (Figure 6B), post-treatment CTC count ≥2 (vs. <2, P<0.001, HR=4.294) (Figure 6C), post-treatment CTC count ≥5 (vs. <5, P<0.001, HR=2.979) (Figure 6D), and CTC count up (vs. equal/down, P=0.002, HR=3.222) (Figure 6E) were all correlated with worse OS. By comparison of HR, post-treatment CTC count ≥2 (vs. <2) showed best potency for predicting OS.
Figure 6.
Correlation of pre-treatment/post-treatment/change of CTC count with OS. Correlation of pre-treatment CTC count ≥2 (vs. <2) (A), pre-treatment CTC count ≥5 (vs. <5) (B), post-treatment CTC count ≥2 (vs. <2) (C), post-treatment CTC count ≥5 (vs. <5) (D), and CTC count change up (vs. equal/down) (E) with OS.
Independent factor of PFS and OS
Post-treatment CTC ≥2 is independently correlated with both worse PFS (P=0.004, HR=2.913) and OS (P<0.001, HR=5.791) (Table 3). Furthermore, number of organs with metastases, peritoneum metastases, abnormal CA199 and abnormal CEA are also independently related to PFS or OS (all P<0.05).
Table 3.
Cox’s proportional hazard regression analysis for PFS and OS
| Factors | HR (95% CI) | P value |
|---|---|---|
| Multivariate Cox’s regression for PFS | ||
| Post-treatment CTC ≥2 | 2.913 (1.416-5.993) | 0.004 |
| Number of organs with metastases | 2.027 (1.276-3.220) | 0.003 |
| CA199 abnormal (>40 kU/L) | 2.208 (1.081-4.508) | 0.030 |
| Multivariate Cox’s regression for OS | ||
| Post-treatment CTC ≥2 | 5.791 (2.631-12.745) | <0.001 |
| Peritoneum metastases | 2.099 (1.055-4.175) | 0.035 |
| CEA abnormal (>5 ng/mL) | 2.272 (1.157-4.465) | 0.017 |
PFS, progression-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; CTC, circulating tumor cell; CA199, carbohydrate antigen 199; CEA, carcinoembryonic antigen.
Discussion
Several interesting findings were discovered in our present study, which padded the data of CTC detection in GBC: (1) CTC was almost detectable in all advanced GBC patients before treatment, whose count was positively correlated with metastatic disease (vs. local advanced disease), number of organs with metastases, and CA199; (2) After treatment, CTC count was obviously declined in advanced GBC patients, especially in DCR patients; (3) Pre-treatment CTC count, post-treatment CTC count and its change all presented potential for predicting prognosis in advanced GBC patients.
Since the introduction of CTC detection in clinical settings, it has been recognized as a potential biomarker for carcinomas diagnosis and progression monitor [11-13]. For instance, CTC capture presents with a certain value for diagnosis of early-stage lung cancer with 52.94% sensitivity and 90.00% specificity [14], and CTC count correlates with lymph node and distant metastases in newly diagnosed lung cancer patients [14]. CTC is also identified in 90.76% non-metastatic breast cancer patients, whose count positively relates to tumor size and hormone receptor negative status [14]. In aspect of hepatobiliary carcinomas, a recent meta-analysis exhibits that CTC detection achieves 0.60 (95% CI: 0.57-0.63) specificity and 0.95 (95% CI: 0.93-0.96) sensitivity for the diagnosis of hepatocellular carcinoma [15]. Then, another cohort study finds that preoperative CTC count associates with Edmondson stage and postoperative CTC count correlates with TP53 positive status in hepatocellular carcinoma patients [16]. However, possibly due to the low incidence of GBC, its study about CTC detection is very limited. Only a current study reveals that CTC appears 92.6% sensitivity and 91.7% specificity for GBC diagnosis, and its count could discriminate GBC with different TNM stages from each other with cut-off threshold of 3 or 4 [9]. However, the detection of CTC and its clinical relation with disease features in advanced GBC is never investigated. In our current study, we observe that CTC was almost detectable in all advanced GBC patients before treatment, whose count is positively correlated with metastatic disease (vs. local advanced disease), number of organs with metastases, and CA199. The possible explanations were as follows: (1) Higher CTC count increased the possibility of tumor cell planting in other organs apart from the GBC [1-3], therefore it is positively correlated with metastatic disease (vs. local advanced disease) and metastatic organ number. (2) CA199 reflected the worse disease conditions, which is reported to be highly expressed in metastatic GBC. Meanwhile, higher CTCs is related to metastatic GBC. Taken together, CTCs showed a positive correlation with CA199 indirectly.
Most studies about CTC in carcinomas focus on its diagnostic and prognostic potency, while its longitudinal change during treatment is seldom explored [14-16]. In our study, we found that CTC count was decreased after chemotherapy in advanced GBC patients, which might be on account of that after the chemotherapy drug flooded into blood circulation, the CTC was diminished to some extent. Furthermore, we observed that post-treatment CTC count correlated with decreased ORR, and then pre-treatment CTC count, post-treatment CTC count and its change all presented relation to declined DCR in advanced GBC patients, indicating the close association of CTC count with treatment response in these patients. These might result from: (1) for the pre-treatment CTC count, its high level reflected higher severity of GBC (such as more metastatic organs, elevated CA199 level), and therefore, it correlated with chemotherapy response indirectly; (2) for the post-treatment CTC count and change of CTC count, the patients realizing better treatment response reflected the inhibition of disease progression, and therefore, the CTC count was repressed in the meanwhile.
Apart from the diagnostic, disease monitoring, and treatment response predicting role of CTC in carcinomas, its prognostic role is especially encouraging. For instance, pre-treatment CTC count above or equal to 4 tightly relates to worse PFS, and then the decline of CTC count after treatment predicts favorable PFS and OS in metastatic breast cancer patients [17]; CTC-positive status at each sample time all correlates with poor PFS and OS in gastric cancer patients revealed by a meta-analysis [18]. With regard to hepatobiliary carcinomas, a previous meta-analysis enrolling 23 studies uncovers that CTC positive status predicts poor relapse free survival and OS in hepatocellular carcinoma [19]. However, there has been no study revealing the prognostic role of CTC in GBC patients until now. In our present study, we observed that pre-treatment CTC count, post-treatment CTC count and its change all presented potential for predicting prognosis (reflected by PFS and/or OS) in advanced GBC patients. The following reasons might explain these results: (1) CTC count correlated with advanced disease conditions such as more metastatic organs and elevated CA199 level, which resulted in the worse PFS and OS indirectly in advanced GBC patients; (2) CTC count was related to worse treatment response of chemotherapy, which directly led to poor PFS and OS in advanced GBC patients.
Some limitations still existed in this present study: (1) Due to the low incidence of GBC, the sample size in the study was relatively small, so the findings needed to be further validated by large sample-sized study; (2) Only unresectable and advanced GBC patients were investigated in this study, so the findings were limited to these patients but not resectable GBC patients, which needed further exploration as well; (3) In order to reduce the bias and compounders, other therapies apart from chemotherapy treated advance GBC patients were not included (such as targeted therapy, immune check-point therapy, etc.), which also needed further assessment.
In conclusion, higher CTC count during chemotherapy correlates with worse treatment response, PFS and OS in advanced GBC patients, which implies that CTC measurement may optimize the prognostication and individualized treatment in these patients.
Acknowledgements
This study was registered on Chinese Clinical Trial Registry website (www.chictr.org.cn) with the approval number “ChiCTR2100052139”.
Disclosure of conflict of interest
None.
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