Clinical significance of circulating tumour cells and tumour marker detection in the chemotherapeutic evaluation of advanced colorectal cancer

Abstract Aim Systemic chemotherapy combining biological targeted therapies is the standard therapy for patients with metastatic colorectal cancer (mCRC), but effective markers are needed to identify clinical responders. Circulating tumour cells (CTCs) have been associated with prognosis in patients with mCRC. This study aimed to explore the relationship between CTC number and the clinical response of patients with advanced CRC. Method Epithelial cell adhesion molecule‐independent enrichment and CD45− fluorescence in situ hybridization immunofluorescence were used to detect peripheral blood CTCs in 79 patients with advanced CRC. Fisher's exact test and Spearman's rank correlation coefficient were used to analyse the correlation between CTC number and efficacy of chemotherapy. Kaplan–Meier and Cox regression analyses were used to evaluate progression‐free survival (PFS). Results Among the evaluable patients, CTCs were significantly correlated with clinical response (r =4.891, p = 0.031). High CTC numbers were associated with a poor treatment response (r = −0.250, p = 0.027). Dynamic decrease in CTC number was associated with clinical response (p = 0.046). High baseline CTC number and carcinoembryonic antigen levels were prognostic factors for unfavourable PFS in multivariable analysis [hazard ratio (HR) = 3.30, p = 0.011 and HR = 2.04, p = 0.044, respectively]. Compared with the CTC‐positive group, the CTC‐negative group showed superior PFS (median PFS 15.53 vs. 9.43 months, p = 0.041) among CRC patients receiving first‐line treatment. Conclusion CTC number is a feasible biomarker for predicting outcomes in mCRC patients receiving systemic chemotherapy.


INTRODUC TI ON
Colorectal cancer (CRC) is the third most common cancer and second most common cause of cancer-related death worldwide. More than 1.9 million new cases of CRC and 935,000 deaths occurred worldwide in 2020 [1]. The incidence and mortality of CRC in China are increasing, with 383,000 new cases and 187,000 deaths in 2015 [2]. The incidence and mortality rates in China in 2018 were 23.7 per 100,000 and 10.9 per 100,000, respectively [3]. Metastasis is the main cause of death from CRC, and is present in approximately 25% of patients with CRC at initial diagnosis [4].
Systemic chemotherapy combining biological targeting therapies is the standard therapy for patients with metastatic colorectal cancer (mCRC). Although the use of these combination therapies in mCRC has led to response rates of >50%, approximately 28%-44% of patients have no response to first-line treatment with double chemotherapy plus either cetuximab or bevacizuamb [5]. Assessment of tumour response by imaging and serum tumour markers has been associated with survival, but these measurements are not sufficient to predict the efficacy of systemic chemotherapy. Therefore, there is an urgent need to develop robust prognostic biomarkers for mCRC patients receiving chemotherapy to predict resistance and identify alternative strategies for overcoming chemotherapeutic resistance.
Unfortunately, no accepted biological or molecular marker of prognostic value is currently available for systemic chemotherapy.
Circulating tumour cells (CTCs) are cancer cells that detach from the primary tumour or its metastases and disseminate in the bloodstream; these cells can be isolated directly from peripheral blood, obviating the need for invasive tumour biopsies [6,7]. Recent refinement of an immunomagnetic separation technology to reliably and reproducibly isolate, enumerate and characterize CTCs in epithelial malignancies has enabled further study of CTCs as a prognostic and predictive marker [8]. In recent years, CTCs have been widely proposed to serve as biomarkers in various cancer types, including breast, prostate and colorectal cancer [9][10][11][12]. Studies have also shown that CTC number is an independent predictor of progression-free survival (PFS) and overall survival (OS) in mCRC [11,12]. Therefore, we hypothesized that CTC number might be a potential indicator for predicting the response to chemotherapy in patients with mCRC.
In this study, we aimed to investigate the impact of CTC number on the outcomes and prognoses of first-line treatment in mCRC patients.

Collection, enrichment and identification of circulating tumour cells
Peripheral blood samples were collected by venepuncture. For CTC tests, 3.2 ml of blood was used after discarding the first 1.8 ml to avoid contamination with epithelial cells. An additional 5 ml of blood was collected for the analysis of serum tumour markers. The samples for analysis were collected in a tube containing an anticoagulant. The strategy for CTC enrichment was essentially similar to that in the literature [13,14]. Briefly, red blood cell lysis was performed within 12 h of collection of the blood sample. The samples were then resuspended in phosphate-buffered saline and incubated with magnetic beads coated with anti-CD45 monoclonal antibody for 30 min, followed by separation of magnetic beads using a magnetic stand. The supernatants were subjected to identification.

Data collection
The clinicopathological data, including patient age and sex, sur- Survival data were obtained from the medical charts. PFS was defined as the time from the start of chemotherapy to documented disease progression or death, whichever occurred first. OS was defined as the time between the date of diagnosis of mCRC and the date of disease-related death or last follow-up visit.

Assessment of tumour response and follow-up
Assessment of tumour response was performed every three cycles of treatment by computed tomography or magnetic resonance imaging. The efficacy assessment included complete response (CR), partial response (PR), progressive disease (PD) and stable disease (SD) according to the Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST 1.1) [15]. The best clinical response during chemotherapy for each patient was recorded as the tumour response.
Follow-up was obtained every 3 months after chemotherapy ended.
The PFS data, defined as the time from the start of chemotherapy to documented disease progression or death, were obtained from the medical records and telephone follow-up.

Statistical analysis
The patient characteristics are expressed as medians (25th-75th percentiles) and categorical data are expressed as numbers (percentages

Patient characteristics
A total of 79 patients were enrolled in the study (Appendix S1).
As shown in Table 1

Correlation between dynamically decreasing CTCs and the efficacy of systemic chemotherapy
Continuously detected CTCs of 59 patients were available during chemotherapy. The correlation between the dynamic decrease in CTC number and the clinical response was explored. Dynamically decreasing CTC number was significantly correlated with clinical response (r = 6.16, p = 0.046) ( Table 3).  Table 4). The median PFS values were 15.53 and 15.57 months in the CTC-negative and CEA-negative groups, respectively, which were significantly higher than those in the positive groups (Figure 2). Thus far, the median OS of these patients has not been reached.

DISCUSS ION
In this single-institution retrospective study, we enrolled 78 evalu-     78.57% and 85.71%. [26]. The authors also successfully found that DNA mutational analysis of CTCs may enable noninvasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients [27].  for patients with small cell lung cancer who received first-line chemotherapy. The reduction of CTC number after two cycles of chemotherapy was a potential predictor of chemotherapeutic response in small cell lung cancer [24].
High baseline CEA levels were prognostic factors for PFS in the univariate and multivariate analyses. However, no associations were found between the baseline tumour markers and clinical response in

CON CLUS ION
Positivity for CTCs at baseline without a reduction in CTC number during chemotherapy in mCRC patients was positively correlated with PD.
Higher CTC numbers are related to a poor prognosis. Because blood collection is simple, convenient and minimally invasive, CTC number may be used as a new biomarker for predicting disease progression and survival. The relationship between CTC number and efficacy of chemotherapy in patients with mCRC should be investigated further.

E TH I C S S TATEM ENT
The study was approved by the Ethics Board of Shanghai General Hospital.

PATI ENT CO N S ENT S TATEM ENT
Informed consent to participate in the study was obtained from all the patients.

PER M I SS I O N TO R EPRO D U CE M ATER I A L FRO M OTH ER SO U RCE S
No material has been reproduced from other sources.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Li Qi conceived of the presented idea. All authors collected the data.
Shen Feifei and Zhu Yiwen analysed, interpreted the data and wrote the manuscript. Li Qi and Liu Chuan supervised and supported the study and critically revised the manuscript. All authors approved the final version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.