Circulating tumour cells and outcome in non-metastatic colorectal cancer: a prospective study

ABSTRACT BACKGROUND: Circulating tumour cells (CTC) in the blood have been accepted as a prognostic marker in patients with metastatic colorectal cancer (CRC). Only limited data exist on the

prognostic impact of CTC in patients with early stage CRC using standardised detection assays. The aim of this study was to elucidate the role of CTC in patients with non-metastatic CRC.

METHODS: A total of 287 patients with potentially curable CRC were enrolled, including 239 patients with UICC stage I–III. CTC were measured in the blood using the CellSearch system

preoperatively and on postoperative days 3 and 7. The complete patient group (UICC I–IV) and the non-metastatic cohort (UICC I–III) were analysed independently. Patients were followed for 28

(0–53) months. Prognostic factors for overall and progression-free survival were analysed using univariate and multivariate analyses. RESULTS: CTC were detected more frequently in patients

with metastatic disease. No clinicopathological variables were associated with CTC detection in non-metastatic patients. CTC detection (⩾1 CTC per 7.5 ml blood) in the blood was

significantly associated with worse overall survival (49.8 _vs_ 38.4 months; _P_<0.001) in the non-metastatic group (UICC I–III), as well as in the complete cohort (48.4 _vs_ 33.6 months;

_P_<0.001). On multivariate analysis CTC were the strongest prognostic factor in non-metastatic patients (hazard ratio (HR) 5.5; 95% confidence interval (CI) 2.3–13.6) as well as in the

entire study group (HR 5.6; 95% CI 2.6–12.0). CONCLUSIONS: Preoperative CTC detection is a strong and independent prognostic marker in non-metastatic CRC. SIMILAR CONTENT BEING VIEWED BY

OTHERS THE UTILITY OF CTDNA IN DETECTING MINIMAL RESIDUAL DISEASE FOLLOWING CURATIVE SURGERY IN COLORECTAL CANCER: A SYSTEMATIC REVIEW AND META-ANALYSIS Article Open access 08 November 2022

CIRCULATING TUMOUR DNA AND ITS CLINICAL UTILITY IN PREDICTING TREATMENT RESPONSE OR SURVIVAL IN PATIENTS WITH METASTATIC COLORECTAL CANCER: A SYSTEMATIC REVIEW AND META-ANALYSIS Article 19

April 2022 CEA, CA19-9, CIRCULATING DNA AND CIRCULATING TUMOUR CELL KINETICS IN PATIENTS TREATED FOR METASTATIC COLORECTAL CANCER (MCRC) Article 10 June 2021 MAIN Although tremendous efforts

in diagnosis and treatment have been made, colorectal cancer (CRC) remains the fourth most common cause of cancer-related death with 1 million new cases and 500 000 deaths worldwide each

year (Weitz et al, 2005; Cunningham et al, 2010; Brenner et al, 2014). In theory, early stage CRC is cured by surgical resection alone and UICC stage I and II patients are only offered

adjuvant chemotherapy in case of risk factors (e.g., intraoperative tumour perforation). UICC stage III patients are routinely recommended adjuvant chemotherapy according to current

guidelines (Engstrom et al, 2009; Labianca et al, 2010). However, up to 25% of these patients will develop recurrent disease and current selection criteria for high-risk patients remain

insufficient (Weitz et al, 2005; Engstrom et al, 2009; O’Connor et al, 2011). The prognostic impact of circulating tumour cells (CTC) in the blood of patients with CRC has been studied

extensively and was recently confirmed by a meta-analysis (Rahbari et al, 2010). However, different non-standardised detection methods of CTC have made inter-study comparisons difficult, and

only limited data exists about the prognostic role of CTC in patients with early stage CRC (Ito et al, 2002; Iinuma et al, 2006, 2011; Lloyd et al, 2006; Allen-Mersh et al, 2007; Sadahiro

et al, 2007; Wang et al, 2007; Uen et al, 2008; Maestro et al, 2009; Wong et al, 2009; Lu et al, 2011; Thorsteinsson and Jess, 2011; Thorsteinsson et al, 2011). The Food and Drug

Administration has approved the CellSearch system for CTC monitoring in patients with metastatic colorectal, prostate and breast cancers (Cohen et al, 2006, 2008, 2009; Miller et al, 2010;

Sastre et al, 2012). Introduction of this semi-automatic assay allows for detection and enumeration of CTC in a standardised fashion. However, data on CTC as a predictive and prognostic

marker in CRC patients have so far been exclusively available for patients with metastatic disease. Recently, it has been reported that detection of CTC in the blood of patients with

non-metastatic breast cancer is an independent prognostic marker for overall and progression-free survival (OS and PFS, respectively) (Lucci et al, 2012). It was the aim of the present

prospective study to evaluate the prognostic value of CTC measured by the CellSearch system in patients with potentially curable disease, focussing on non-metastatic CRC. METHODS Reporting

of the present study was in accordance with the REMARK guidelines (McShane et al, 2005). PATIENTS Patients with UICC stage I–IV CRC who underwent surgery with curative intent at the

Department of General, GI and Transplant Surgery, University Hospital Heidelberg, Heidelberg, Germany between May 2009 and August 2012 were included in this study. In addition to patients

with non-metastatic CRC (UICC I–III group), patients with limited, synchronous metastases were eligible for inclusion if their operation was done with curative intent and no macroscopic

tumour (primary CRC and metastases) remained after surgery (UICC I–IV group). Patients were deemed ineligible if they refused participation, presented with unresectable CRC and/or liver

metastases or had a history of any other malignancy during the past 5 years. Written informed consent was obtained prior to surgery. The study was approved by the ethics committee of the

University of Heidelberg (323/3004). Details about our management of patients with primary CRC and colorectal liver metastases have been reported elsewhere (Reissfelder et al, 2009; Rahbari

et al, 2012). Adjuvant treatment was done according to current treatment guidelines after obtaining interdisciplinary consensus for each patient. BLOOD SAMPLE COLLECTION AND CTC DETECTION

TNM cancer staging was in accordance with the criteria set by the American Joint Commission on Cancer (AJCC, 2010). Blood samples from the central venous blood or from the peripheral blood

were taken immediately prior to surgical incision after induction of general anaesthesia for all patients in the operating room. CTC detection in the central venous blood compartment is

similar to the cell count obtained from peripheral blood, which has been validated by us in previous studies. Blood samples were drawn into and immediately transferred to cell preservative

tubes (CellSave, Veridex LLC, Raritan, NJ, USA). Isolation of CTC with the CellTracks Autoprep System (Veridex LLC) and semi-automatic analysis via the CellTracks Analyzer II system (Veridex

LLC) have been described previously (Allard et al, 2004; Rahbari et al, 2012). Two trained operators blinded to patient’s data screened results independently, and differences in CTC count

were resolved by discussion. All results for individual patients were masked from the analysing investigators by using a number system as a unique patient identifier. Operators of the

CellSearch system were blinded for clinicopathological data of patient samples. Blood samples were only analysed for patients, and no control group with healthy volunteers or patients with

benign disease were included in this trial. CTC count in healthy volunteers has been studied extensively for the validation purposes of the CellSearch system (Allard et al, 2004;

Cristofanilli et al, 2004; Miller et al, 2010). CTC detected by the CellSearch technique are extremely rare in healthy volunteers (<3.5% for a threshold ⩾1 CTC per 7.5 ml blood) and

patients with benign disease (<7.5% for a threshold of ⩾1 CTC per 7.5 ml blood) (Miller et al, 2010). A control group was not included as it would not have added any substantial

information to the aim of the present study. STATISTICAL ANALYSES All analyses were carried out separately for patients with non-metastatic CRC (UICC I–III group) and the entire cohort (UICC

I–IV group). Patients with complete pathological response (T0, N0, M0) were included in the UICC I–III group. Categorical data were presented as absolute and relative frequencies.

Continuous data were presented as median and range. For CTC data, the arithmetic mean and s.e.m. (s.d.) were reported. The association of CTC detection with clinicopathological variables was

evaluated using the _χ__2_-test. The primary end point of the present study was OS, which was calculated from the date of surgery to the date of death due to any cause or the date of last

follow-up information. Progression-free survival was defined as time from the date of surgery until objective tumour progression or death. Survival curves were constructed according to the

Kaplan–Meier method and compared using the log-rank test. As limited data were available on cutoff levels for the number of CTC to determine tumour cell-positive patients, all analyses were

carried out for three cutoffs, that is, ⩾1 CTC, ⩾2 CTC and ⩾3 CTC. Variables that had significant associations with CTC detection on univariate analyses were included in multivariate

analyses using Cox proportional hazards regression methodology. A _P-_value ⩽0.05 was considered to indicate a statistically significant difference. All _P_-values were two-sided.

Statistical analyses were done with the SPSS software version 19 (SPSS, Chicago, IL, USA) and JMP program version 7 (SAS Institute Inc., Cary, NC, USA). RESULTS A total of 287 Patients with

resectable CRC who underwent potentially curative therapy were included in this study, including 239 patients with non-metastatic disease (UICC stage I–III). Forty-eight patients underwent

resection for CRC with synchronous metastases with curative intent. In the UICC stage I–III group, 157 male (65.7%) and 82 female (34.3%) patients were included; in the UICC stage I–IV

group, there were 186 male (64.5%) and 101 female patients (35.2%). The majority of patients with metastatic disease had liver metastases, whereas metastases in the lungs and other locations

were present in 6 (12.5%) and 7 (14.6%) patients, respectively. Three (6.2%) patients had multiple sites of metastases (Table 1). DETECTION RATE OF CTC IN CRC IS STAGE DEPENDENT In the

analysis of preoperative blood samples, ⩾1 CTC per 7.5 ml of blood were found in 30 patients (10.5%), ⩾2 CTC in 9 patients (3.1%) and ⩾3 more CTC in 5 patients (1.7%) (Supplementary Table

1). Detection rate of CTC was significantly correlated with the stage of disease comparing non-metastatic and metastatic patients with 3 (4.9%), 9 (10.5%), 7 (8.3%) and 9 (18.8%) patients

with ⩾1 detected CTC in UICC stages I, II, III and IV, respectively (_P_=0.03). The stage-dependent CTC detection with increased detection rates for patients with UICC IV disease was

confirmed for detection of ⩾2 (_P_=0.001) or ⩾3 (_P_=0.008) CTC per 7.5 ml of blood (Figure 1). Furthermore, patients’ stage of disease was associated significantly with the number of CTC in

peripheral blood detected intraoperatively. Postoperative blood samples with CTC analyses on postoperative days 3 and 7 were carried out in 51 and 28 patients, respectively. In the UICC

I–III group ⩾1 CTC was detected on postoperative days 3 and 7 in 4 (10.0%) and 3 (14.3%) patients, whereas in the UICC stage I–IV group 9 (17.6%) and 6 (21.4%) patients had detectable CTC.

LACK OF ASSOCIATION OF PRIMARY TUMOUR CHARACTERISTICS WITH CTC DETECTION IN NON-METASTATIC CRC The association of various clinicopathological characteristics with preoperative presence of

CTC in the systemic circulation was analysed for three different cutoff values of CTC detection. No primary tumour characteristics or clinicopathological parameters predicted the presence of

CTC for the non-metastatic (UICC stage I–III) group (Supplementary Table 2). There was, however, a trend towards higher detection rates in patients who had received any kind of neoadjuvant

therapy (_P_=0.07) and a trend towards increased CTC detection in patients with locally advanced tumours (T3/4 _vs_ T1/2; _P_=0.14). To further elucidate whether a certain kind of

neoadjuvant therapy caused an increase in CTC, we performed further subgroup analyses. These revealed a significantly higher CTC detection rate in non-metastatic patients with neoadjuvant

chemoradiotherapy (_P_=0.002). This association remained statistically significant for the entire study cohort, including patients with stage IV disease (_P_=0.03). The analyses of the

entire study group confirmed the significantly higher presence of CTC in patients with metastatic disease compared with non-metastatic patients (_P_=0.04). In addition, preoperative CEA

level (_P_=0.03) and CA 19-9 level (_P_=0.01) were significantly associated with detection of ⩾1 CTC per 5 ml of blood in patients with UICC I–IV disease (Supplementary Table 2). The

association of T stage and CTC detection failed to reach statistical significance in these patients (_P_=0.08). PRESENCE OF CTC IS A STRONG AND INDEPENDENT PREDICTOR OF OS AND PFS IN

PATIENTS WITH NON-METASTATIC CRC The mean follow-up time was 28 (0–53) months. During the follow-up period, 22 (9.2%) patients died and 23 (9.6%) patients were diagnosed with disease

progression in the UICC I–III group. In the complete study group, 40 (13.9%) patients died and 45 (15.7%) patients had disease progression during follow-up (Figure 2). To evaluate the

prognostic value of preoperative CTC detection on OS and PFS in patients with non-metastatic CRC, univariate analyses were performed initially to evaluate known prognosticators in the

present study cohort. In patients with non-metastatic disease, age (_P_=0.036) was associated with significantly shorter OS, whereas T stage (_P_=0.029) and N status (_P_=0.002) were

predictors of poor PFS. Patients with non-metastatic disease who were found to have ⩾1 CTC per 7.5 ml blood preoperatively had significantly shorter OS (_P_<0.001) and PFS (_P_<0.001)

on univariate analyses (Table 2). This association was also confirmed for the preoperative detection of ⩾2 and ⩾3 CTC (data not shown). Furthermore, the adverse prognostic impact of

detection of ⩾1 CTC on OS (_P_<0.001) and PFS (_P_<0.001) was confirmed for the cohort of 158 patients who had no neoadjuvant treatment (Supplementary Figure 1). Identification of

patients with node-negative disease who are at risk for disease recurrence remains a clinical challenge. Our study population included 155 patients with node-negative CRC who had no evidence

of distant metastases at the time of diagnosis. Of these, 14 (9%) had ⩾1 CTC per 7.5 ml blood preoperatively. These patients had significantly worse OS (34.1 _vs_ 50.4 months; _P_=0.001)

and PFS (30.8 _vs_ 50.6 months; _P_<0.001). On multivariate analyses, preoperative detection of ⩾1 CTC per 7.5 ml blood was confirmed as a strong and independent predictor of OS (HR 5.5;

95% CI 2.3–13.6; _P_<0.001) and PFS (HR 12.7; 95% CI 5.2–31.1; _P_<0.001) in patients with UICC stage I–III disease. The analyses of the entire study cohort, including patients with

UICC stage I–IV disease, revealed patients’ age (_P_=0.04), N status (_P_=0.001), presence of distant metastases (_P_<0.001), elevated preoperative CEA level (_P_=0.001) and elevated

preoperative CA 19-9 level (_P_<0.001) to be associated with significantly impaired OS, whereas a significant association with shortened PFS was found for patients’ T stage (_P_=0.005), N

stage (_P_<0.001), presence of distant metastases (_P_<0.001), elevated preoperative CEA level (_P_=0.001) and elevated preoperative CA 19-9 level (_P_<0.035) (Table 3). Patients

with ⩾1 CTC per 7.5 ml blood preoperatively had significantly worse OS (_P_<0.001) and PFS (_P_<0.001). A strong association of preoperative CTC detection and OS well as PFS was also

found for a cutoff of ⩾2 and ⩾3 CTC per 7.5 ml blood preoperatively. After exclusion of patients with neoadjuvant therapy, CTC detection was still associated with poor OS (_P_<0.001) and

PFS (_P_<0.001). In these patients, ⩾1 CTC per 7.5 ml blood was associated with poor OS (_P_<0.001) and PFS (_P_<0.001). This association was confirmed for the preoperative

detection of ⩾2 and ⩾3 CTC per 7.5 ml blood. In addition, age (_P_=0.036) was associated with poor OS, whereas T stage (_P_=0.029) and N status (_P_=0.002) were predictors of poor PFS in

these patients with non-metastatic disease. On multivariate analyses, including all variables with significant associations on univariate analyses, presence of ⩾1 CTC was revealed as an

independent predictor of OS (HR 5.6; 95% CI 2.6–12.0; _P_<0.001) and PFS (HR 7.8; 95% CI 3.9–15.5; _P_<0.001). Remarkably, the prognostic impact of preoperative CTC detection was

independent of the presence of distant metastases, which was revealed as a further independent prognostic factor of OS (HR 2.3; 95% CI 1.0–5.0; _P_=0.035) and PFS (HR 7.8; 95% CI 3.9–15.5;

_P_=0.002) (Table 4). DISCUSSION In the present study, we investigated whether preoperative detection of CTC in the systemic circulation can serve as a prognostic biomarker in CRC patients

without distant metastases or with limited metastatic disease amenable to curative resection. Although we have previously demonstrated a prognostic value of CTC in patients with primary CRC

in a meta-analysis, the included studies varied considerably with respect to the methods of CTC detection. Indeed, the predictive and prognostic value of CTC using standardised assays has

only been demonstrated and validated for advanced metastatic CRC (Cohen et al, 2008, 2009; Seeberg et al, 2014). A recently published study from the MD Anderson Cancer Center revealed CTC

detected by the CellSearch system as an independent prognostic marker for OS and PFS in patients with non-metastatic breast cancer (Lucci et al, 2012). Here we show for the first time that

preoperative CTC detection using the standardised CellSearch system is a strong and independent prognostic factor for disease progression and survival in non-metastatic CRC patients.

Interestingly, the detection of CTC preoperatively did not correlate with any other clinicopathological factor in patients with non-metastatic disease. As we did not find any association

between CTC detection and clinical parameters, this suggests CTC detection as an independent indicator of aggressive tumour biology in our patients, in line with the findings of Lucci et al

(2012) in non-metastasised breast-cancer patients. In line with these findings, the detection of CTC in UICC stage I–III patients proofed to be the strongest predictor of OS and PFS,

considering known prognostic factors, such as age or lymph node status. Collectively, these data suggest CTC as a potentially useful prognostic and predictive biomarker in non-metastatic CRC

patients that may help to further stratify patient’s risk status within different stages of disease. Furthermore, this should prompt further studies to dissect the molecular mechanisms of

tumour cell dissemination. The CellSearch system enables the detection as well as enumeration of CTC. Previous studies have suggested different cutoff levels to determine ‘CTC positivity’.

Allard et al (2004) evaluated the CellSearch system in a study of 964 patients. Based on the mean CTC count in healthy subjects, these authors suggested the detection of ⩾2 CTC per 7.5 ml of

blood as abnormal. Using a training and validation set of patients with metastatic CRC, Cohen et al (2008, 2009) defined unfavorable prognostic groups by detection of ⩾3 CTC per 7.5 ml of

blood. All patients in these studies had metastatic disease, we report here the largest study of patients with non-metastatic CRC studied with the CellSearch system so far and the first to

evaluate its prognostic value in these patients. Our results show a significantly lower detection rate of CTC in patients with non-metastatic CRC. Based on the relatively low proportion of

patients with ⩾1 CTC per 7.5 ml blood who had a significantly worse outcome compared with patients without CTC, we recommend that risk stratification of non-metastatic CRC patients should be

done at a threshold of ⩾1 CTC. Adjuvant therapy in patients with node-negative CRC remains a controversial issue. Evidence from recent studies suggests that patients’ selection for adjuvant

therapy based on clinical factors might be inaccurate and molecular markers might be useful (O’Connor et al, 2011; Rahbari et al, 2011, 2014). Our study demonstrates that the CellSearch

system provides prognostic information in CRC patients with UICC stage I/II disease and confirms previous data using CTC detection by CK20 PCR (Koch et al, 2006). These data suggest that

detection of CTC using standardised assays should be considered to improve current staging of patients (i.e., cM0(i+)) and the selection of patients for adjuvant chemotherapy. However,

several issues need to be addressed before integration of CTC detection into the TNM classification and treatment guidelines. First, our results on patients with non-metastatic CRC need to

be validated in further cohorts from different institutions. Second, there is no proof so far that adjuvant therapy improves long-term outcome of patients with cM0(i+) disease. We recently

showed that preoperative risk status using clinical parameters can predict efficacy of adjuvant chemotherapy after resection of CRC liver metastases (Rahbari et al, 2014). A randomised

controlled trial would be needed to achieve level I evidence on the benefit of adjuvant therapy in CRC patients with cM0(i+) disease. However, such a trial will be almost impossible to

perform owing to the low CTC detection rate in this group of patients, and an international patient registry might offer an alternative strategy to provide valuable information. Third,

detection of CTC using the CellSearch technique is only investigating a subgroup of CM0(i+) patients. The CellSearch technique only detects CTC in the blood, which are positive for EPCAM and

cytokeratin. However, there is evidence that not all CTC are positive for these markers, and it remains subject of future studies to evaluate the prognostic and predictive value of other

CTC detection assays capturing a broader spectrum of CTC populations (Steinert et al, 2014). Furthermore, the clinical value of tumour cell detection in further body compartments such as the

bone marrow, regional lymph nodes and mesenteric blood needs to be clarified to determine which compartment provides the most accurate information on patients’ outcome (Rahbari et al, 2011,

2012). Interestingly, even though CTC detection _per se_ is a strong indicator of survival and disease burden, it remains unclear whether detected CTC are actually precursors of metastatic

lesions (and possess the ability to form distant metastases) or whether CTC originate from metastases (or the primary tumour) and are just a measurement of overall disease burden. The

molecular characterisation of single CTC will be an important step forward to answer these questions and to ultimately use CTC as liquid biopsies during different stages of disease

progression for treatment decisions and monitoring purposes (Torino et al, 2013; Coget et al, 2014). In conclusion, our study provides promising results for the use of CTC as a prognostic

and predictive biomarker in patients with non-metastatic CTC. The lack of association between CTC detection and clinical parameters suggests CTC as an independent indicator of aggressive

tumour biology. This should clearly be followed in larger trials with longer follow-up times. Our results warrant further validation within larger multi-institutional trials to test and

clarify whether detection of CTC in the blood should be included into current treatment guidelines of non-metastatic CRC. CHANGE HISTORY * _ 14 APRIL 2015 This paper was modified 12 months

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Res_ 15: 1005–1012. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Melanie Bernhardt for excellent technical assistance. This work was funded by German Research

Foundation (DFG; WE 3548/4-1), University Hospital Heidelberg, Heidelberg, Germany. AUTHOR CONTRIBUTIONS UB, NNR, JW, MWB and MK designed the study. UB, NNR, SS, CR and CK collected the

samples. UB and NNR analysed the data and NNR performed the statistical analyses. UB and NNR wrote the manuscript with assistance from MK and final approval from all authors. AUTHOR

INFORMATION Author notes * U Bork and N N Rahbari: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of GI, Thoracic and Vascular Surgery, University

Hospital Carl-Gustav-Carus Dresden, Dresden, Germany U Bork, N N Rahbari, S Schölch, C Reissfelder, J Weitz & M Koch * Department of General, GI and Transplant Surgery, University

Hospital Heidelberg, Heidelberg, Germany C Kahlert & M W Büchler * Department of Cancer Biology, Metastasis Research Center, University of Texas MD Anderson Cancer Center, Houston, TX,

USA C Kahlert Authors * U Bork View author publications You can also search for this author inPubMed Google Scholar * N N Rahbari View author publications You can also search for this author

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search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to U Bork. ETHICS DECLARATIONS COMPETING INTERESTS Moritz Koch has served as an external scientific

advisory board member of Veridex LLC. The other authors declare no conflict of interest. ADDITIONAL INFORMATION This work is published under the standard license to publish agreement. After

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visit http://creativecommons.org/licenses/by-nc-sa/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Bork, U., Rahbari, N., Schölch, S. _et al._ Circulating tumour cells and

outcome in non-metastatic colorectal cancer: a prospective study. _Br J Cancer_ 112, 1306–1313 (2015). https://doi.org/10.1038/bjc.2015.88 Download citation * Received: 22 September 2014 *

Revised: 13 January 2015 * Accepted: 09 February 2015 * Published: 31 March 2015 * Issue Date: 14 April 2015 * DOI: https://doi.org/10.1038/bjc.2015.88 SHARE THIS ARTICLE Anyone you share

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Nature SharedIt content-sharing initiative KEYWORDS * CTC * DTC * circulating tumour cells * colorectal cancer * minimal residual disease