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. 2021 Jun 10;16(6):e0252897. doi: 10.1371/journal.pone.0252897

Prognostic value of preoperative circulating tumor cells counts in patients with UICC stage I-IV colorectal cancer

Thaer S A Abdalla 1,*, Jan Meiners 1, Sabine Riethdorf 2, Alexandra König 3, Nathaniel Melling 1, Tobias Gorges 2, Karl-F Karstens 1, Jakob R Izbicki 1, Klaus Pantel 2, Matthias Reeh 1
Editor: Dominique Heymann4
PMCID: PMC8191913  PMID: 34111181

Abstract

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. There is an urgent need to identify prognostic markers for patients undergoing curative resection of CRC. The detection of circulating tumor cells in peripheral blood is a promising approach to identify high-risk patients with disseminated disease in colorectal cancer. This study aims to evaluate the prognostic relevance of preoperative CTCs using the Cellsearch® system (CS) in patients, who underwent resection with curative intent of different stages (UICC I-IV) of colorectal cancer. Out of 91 Patients who underwent colorectal resection, 68 patients were included in this study. CTC analysis was performed in patients with CRC UICC stages I-IV immediately before surgery. Data were correlated with clinicopathological parameters and patient outcomes. One or more CTCs/7.5 mL were detected in 45.6% (31/68) of patients. CTCs were detected in all stages of the Union of International Cancer Control (UICC), in stage I (1/4, 25%), in stage II (4/12, 33.3%), in stage III (5/19, 26.3%) and in stage IV (21/33, 63.6%). The detection of ≥ 1 CTCs/ 7.5ml correlated to the presence of distant overt metastases (p = 0.014) as well as with shorter progression-free (p = 0.008) and overall survival (p = 0.008). Multivariate analyses showed that the detection of ≥ 1 CTCs/ 7.5ml is an independent prognostic indicator for overall survival (HR, 3.14; 95% CI, 1.18–8.32; p = 0.021). The detection of CTCs is an independent and strong prognostic factor in CRC, which might improve the identification of high-risk patients in future clinical trials.

Introduction

Colorectal cancer (CRC) is the 3rd most diagnosed cancer and the 4th leading cause of death in the world with 1.3 million new cases annually [1]. Two-thirds of the patients with CRC present with localized and potentially curable disease at diagnosis (TNM Stage I-III) [2]. In these patients, surgery remains the most important treatment modality.

Currently, the overall 5-year survival is 65% [1, 2]. This increased over the last four decades after the introduction of screening programs and the concept of adjuvant chemotherapy [3, 4]. The surgical technique also evolved with the introduction of total mesorectal excision (TME) for rectal cancer and central venous ligature (CVL) with complete mesocolic excision (CME) for colon cancer, where sharp dissection in the embryological planes increases lymph node yields, subsequently improving staging and survival [5, 6]. Nevertheless, tumor recurrence or spread to distant sites and formation of metastases still occur in 20% of the patients and is the leading cause of death in these patients [7]. Even in patients with apparently early stages TNM (I-II), local recurrence or distant metastases occur despite proper treatment [8].

Tumor persistence and progression occur mainly due to circulating tumor cells (CTCs) which are seeded from the primary tumor and target distant organs, where they eventually mature and cause secondary metastasis [9]. Preoperative identification of CTCs through liquid biopsy is of proven utility in predicting prognosis in breast, colon, and prostate cancer [10–12].

The 8th AJCC Cancer Staging Manual expanded the definitions of Tis, T4a, and M1 and nodal micrometastasis in CRC [13]. However, unlike in breast cancer, new staging categories like M0 (i+), in which CTC or disseminated tumor cells in the bone marrow are detected, are still lacking [14].

Identifying CTCs preoperatively in liquid biopsies could provide information to solve common dilemmas in CRC [15], like patient selection for adjuvant chemotherapy in stage II CRC after tumor resection.

Isolation and molecular analysis of CTCs in peripheral blood are promising approaches to identify disseminated disease, to upgrade or downgrade the multimodal therapy. The CellSearch® (CS) (Menarini, Silicon Biosystems, Bologna, Italy), a known method for quantification of CTCs based on the expression of the epithelial cell adhesion molecule (EpCAM) and of keratin, is the first standardized system approved by the U.S. Food and Drug Administration for capturing and detection of CTCs derived from metastatic breast and prostate cancer as well as metastatic CRC [10, 16].

The aim of this study is to assess the preoperative value of CTCs in different stages of CRC on the overall survival and progression-free survival using CS.

Materials and methods

This prospective study was conducted at the University Hospital Hamburg-Eppendorf in Germany. The study was approved by the medical ethics committee of the Chamber of Physicians of Hamburg. All patients gave their written informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki.

The inclusion criteria were: 1) adult patients (≥ 18 years) and 2) presence of a primary resectable CRC. Patients with synchronous malignancy were excluded. Also, patients with resectable metachronous metastasis of CRC or local recurrence were excluded (Fig 1). Out of the 91 patients who underwent colorectal resection, 68 were eligible for inclusion in this study.

Fig 1. Flow chart.

Fig 1

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Process of patient selection.

Peripheral blood samples for CTC analysis were collected immediately before surgery and within the first 96 hours postoperatively. Follow up was conducted according to S3 German Guidelines [17]. Events considered were death, local recurrence, and distant metastasis. Overall survival was the time from operation to death or last follow-up, and progression-free survival was defined as the time from operation to the diagnosis of tumor recurrence.

CTC analysis

CTC analysis was performed using CS as previously described [18]. Blood samples (7.5 mL) were collected in CellSave preservative tubes, stored at room temperature, and processed within 96 hours after blood collection, according to the manufacturer’s instructions. The accuracy and reproducibility of CS have been described previously [18, 19]. The presence of a nucleus, cytokeratin expression, round or oval cell morphology of cells with a diametre of at lest 4 μm, and absence CD45 expression served as the criteria for CTCs [18].

Statistical analysis

SPSS statistic software version 25 was used. Histological characteristics were expressed as descriptive statistics. The x2 was used to investigate the association between CTCs and histopathological parameters. Survival rates were determined using the Kaplan–Meier method and were compared using the log‐rank test. A multivariate analysis of factors that might influence OS was performed using the Cox proportional hazards regression model. The results were presented as hazard ratios with 95% CI. All comparisons were two‐tailed. A p-value of less than 0.05 was considered statistically significant. All authors had access to the study data and had reviewed and approved the final manuscript.

Results

Patient characteristics and CTC detection

CTC analysis was performed in blood samples from 68 patients preoperatively. 38 patients had CTC analysis within the first 96 hours after the procedure. Forty-three patients were nodal positive. Distant metastasis was present in 34 patients at the time of operation (Table 1).

Table 1. Site of metastasis.

Site of Metastasis Number of Patients
Lungs 4
Liver 21
Peritoneum 9
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The median age of patients was 64.7 years (range, 18–88 years). Applying a cut-off of ≥ 1 CTC, 31 out of 68 (45%) patients were CTC-positive preoperatively and 13 out of 38 (19.1%) patients were CTC-positive postoperatively. In 8 out of 13 patients, CTCs were also present preoperatively.

We assessed the correlation between CTC detection preoperatively with sex, age and the following histopathologic parameters: Grade of differentiation (G), tumor Invasion (T), nodal status (N), metastases (M), Union of International cancer control (UICC-Stage), HER2, KRAS, MSI mutations and tumor location (Table 2). The detection of CTCs was related to the presence of distant metastases (p = 0.014) and showed a tendency towards the Union of International cancer control stage (p = 0.065). Other parameters did not significantly correlate with CTC positivity preoperatively. Postoperatively, CTC detection did not correlate with any of the above-mentioned parameters.

Table 2. Patient characteristics and correlation of CTCs at baseline with clinicopathological parameters.

Variables Preoperative ≥ 1 CTC
All CTC-Positive p-value
All 68 31 (45.6%)
Age 0.812
<65 20 8 (40%)
65–74 28 13 (46.4%)
≥75 20 10 (50%)
Sex 0.320
Male 44 18 (40.9%)
Female 24 13 (54.2%)
Grade 0.636
G1 1 0 (0%)
G2 48 23 (49.9%)
G3 13 6 (46.2%)
No Grading* 6
Tumor size 0.270
T1 3 0 (0%)
T2 8 4 (50%)
T3 38 16 (42.1%)
T4 19 11 (57.9%)
Nodal status 0.707
N0 25 12 (48%)
N1 14 5 (35.7%)
N2 29 14 (48.3%)
Metastatic stage 0.014
M0 34 10 (29.4%)
M1 34 21 (61.8%)
UICC Stage 0.065
Stage I 4 1 (25%)
Stage II 12 4 (33.3%)
Stage III 18 5 (27.8%)
Stage IV 34 21 (61.8%)
Tumor site 0.291
Right side 22 9 (40.9%)
Left side 14 8 (57.1%)
Rectum 32 18 (56.2%)
HER2-Mutation 0.483
Negative 44 22(50%)
Positive 4 2 (50%)
Missing 20 7 (35%)
KRAS-Mutation 0.345
Negative 24 10 (41.7%)
Positive 29 16(55.2%)
Missing 15 5 (33.3%)
MSI 0.718
Present 5 2 (40%)
not present 25 13 (52%)
Missing 38 16 (42.1%)
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P-value Indicates significance according to the χ2 test when CTC-negative patients are compared with CTC-positive patients. Round parentheses indicate percentages. No Grading* after radiotherapy for rectal cancer, instead of Dworak’s system of tumor regression

Univariate and multivariate analysis of survival

The median survival time was 32 months. Kaplan-Meier’s univariate analysis showed that patients with ≥ 1 CTC had significantly shorter progression-free (p = 0.008) as well as overall survival (p = 0.008) compared to CTC-negative patients (Fig 2). Also, the presence of distant metastases at baseline was associated with shorter OS (p-value 0.002) and shorter PFS (p-value < 0.001) (Fig 3). However, postoperative CTCs did not correlate with OS (p-value = 0.829) and PFS (p-value 0.876) (Fig 4).

Fig 2. Kaplan-Meier survival analysis for overall survival and progression-free survival according to the preoperative CTCs.

Fig 2

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(a): Overall survival in patients with CRC, positive vs. negative preoperative CTCs. (b): Progression-free survival in patients with CRC, positive vs. negative preoperative CTCs. P-value Indicates significance according to Log-Rank (Mantel-Cox) test when CTC-negative patients are compared with CTC-positive patients preoperatively.

Fig 3. Kaplan-Meier analysis for overall survival and progression-free survival according to metastatic status.

Fig 3

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Fig (a): Overall survival in patients with CRC, metastatic vs. non-metastatic disease. Fig (b): Progression-free survival in patients with CRC, metastatic vs. non-metastatic disease. P-value Indicates significance according to Log-Rank (Mantel-Cox) test when mCRC is compared to non-mCRC.

Fig 4. Kaplan-Meier analysis for overall survival and progression-free survival according to the postoperative CTCs.

Fig 4

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Fig (a): Overall survival in patients with CRC, positive vs. negative postoperative CTCs. Fig (b): Progression-free survival in patients with CRC, positive vs. negative postoperative CTCs. P-value Indicates significance according to Log-Rank (Mantel-Cox) test when CTC-negative patients are compared with CTC-positive patients postoperatively.

Nine clinicopathological factors were analyzed using Cox-Regression analysis. Only 4 factors correlated with survival in univariate analysis (Table 3). These included age, metastatic disease, UICC stage, preoperative CTC detection (p < 0.05). Gender, postoperative CTC detection, and other factors were not related to survival in the univariate analysis. The four factors significantly related to survival in univariate analysis were evaluated in the multivariate analysis, which showed that only advanced age and preoperative CTC detection were independent prognosticators of an unfavorable OS (Table 3).

Table 3. Univariate and multivariate analysis of overall survival in patients with CRC.

Univariate Analysis Multivariate Analysis
HR 95% CI p HR 95% Cl p
Age, <65,65–74, ≥75 2.95 1.50–5.80 0.002 2.85 1.51–5.38 0.001
Sex, male vs female 0.833 0.37–1.87 0.650 1.12 0.49–2.52 0.784
Grade of Differentiation, G1-3 1.30 0.51–3.33 0.585
T, T1-T4 1.26 0.73–2.19 0.392
N, N0- N2 1.09 0.67–1.78 0.710
Metastatic stage, M0 vs M1 3.01 1.11–8.20 0.019 2.54 0.30–21.16 0.388
UICC Stage 2.20 1.15–4.18 0.016 1.04 0.33–3.33 0.938
Preoperative CTCs, neg vs pos 2.85 1.25–6.46 0.012 3.14 1.18–8.32 0.021
Postoperative CTCs, neg vs pos 1.53 0.43–5.51 0.515
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p Indicates significance according to cox regression analysis comparing the specified variables. HR indicates hazard ratio.

Discussion

Despite advances in multimodal treatment, CRC is the second most common cause of cancer death worldwide [1]. CTCs play a pivotal role in disease progression, metastasis, and recurrence [20]. Aside from TNM classification and status of resection margin, novel tools and staging systems are needed for adequate prognostic staging and for guiding multimodal therapy [21]. CTC identification has proven to be an important prognosticator in breast cancer [22]. For this reason, a decade ago, the 7th AJCC Cancer Staging Manual introduced the new category M0(i) for breast cancer, which is defined by the presence of circulating or disseminated tumor cells not exceeding 0.2 mm detectable in bone marrow, circulating blood, or other non-regional tissues of non-metastatic patients. In contrast, such a category is still lacking in the current CRC Staging system [23], this is because the assessment of CTCs in CRC is still controversial [16] and different technical platforms have provided conflicting results [24–26]. Here, we applied the FDA-cleared Cellsearch® system, which uses immunomagnetic enrichment (EpCAM) and immunocytochemical (cytokeratin, CD45, DAPI) fluorescence analysis. The epithelial cell adhesion molecule (EpCAM) was first identified in colon cancer in 1979 [27]. It is expressed by a variety of epithelial cells, is highly expressed in colorecal cancer cells [28] and is known to promote tumor expansion and oncogenesis [29]. Thus EpCAM is widely used to capture and detect CTCs. During the process of dissemination and metastasis, tumor cells undergo epithelial-mesenchymal transition (EMT) and might lose their epithelial cell features including EpCAM and/or keratin expression. CTCs that have completely downregulated these epithelial properties are not detectable with the CellSearch system (CS) and require marker-free enrichment methods such as size-, plasticity-, or density-based approaches (Alix-Panabieres C and Pantel K Cancer Discovery 2021) for their identification [30, 31]. Nevertheless, the CellSearch system (CS) which based on EpCAM-related capture of CTCs is standardized, time-efficient and provides clinically relevant results for CTC detection in a wide range of carcinoma patients [31–33]. Several studies have addressed the detectability of CTCs and their prognostic impact in mCRC using CS. According to Cohen et al., CTCs detected using CS provide additional information about tumor burden to imaging studies and therefore have gained a prognostic and predictive value in guiding treatment of mCRC [12]. In addition, Bork et al. showed that CTCs were detectable using CS in patients with CRC (I-III). CTCs were detectable even in patients without nodal or distant metastasis at time of operation (UICC I-II), which was associated with worse survival outcome [34]. In our cohort CTCs were detectable in 45.6% of the patients before surgical resection and were present in all stages of CRC (I-IV). Even in early-stage CRC, UICC I (T1-2, N0, M0), 25% of patients were CTC-positive.

Up to now, there is no consensus regarding the threshold used to define CTC positivity in CRC [16]. Cohen et al have used the cut-off of ≥ 3 CTC/7.5 ml for defining CTC positivity in metastatic CRC [12], while others have shown that cut-offs ≥1 CTC/7.5 ml and ≥ 2 CTC/7.5 ml were also associated with poor prognosis in CRC [35, 36]. We used a strict cut-off of ≥1 CTC/7.5 ml. CTC detection before surgery was associated significantly with a shorter progression-free (P = 0.008) and overall survival (P = 0.008). Multivariate analyses identified CTCs as a strong, independent, prognostic indicator for overall survival.

Many studies have shown inferior survival for right-sided tumors [37, 38], which has prompted us to assess the effect of tumor location on CTC detection. Right-sided colon cancer (RCC), left-sided colon cancer (LCC), and rectal cancer (RC) were considered independently [39–41]. Although more CTCs were present in LCC compared to RCC and RC, there was not a statistically significant correlation. A previous publication from Nicolazzo et al. showed similar results [39]. However, larger cohorts are needed to deliver a robust conclusion about tumor sidedness and CTC detection.

From a clinical perspective, using CTCs in peripheral blood allows assessing cancer prognosis in a non-invasive and easily applicable method [16] that allows real-time monitoring of tumor dynamics [42]. Our present study supports the assumption that preoperative CTC detection in CRC identifies patients with shorter OS and PFS, which might contribute to an improved stratification of high-risk CRC patients. Nevertheless, larger validation studies are required before the implementation of CTC detection into tumor staging classification of CRC.

Conclusion

Preoperative CTC detection is an important prognostic marker for survival in CRC, which can be further developed as an enrichment tool to study a high-risk population of CRC patients in clinical trials.

Supporting information

S1 Data. Patients histopathological and survival data.

(SAV)

Click here for additional data file. (8.1KB, sav)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Dominique Heymann

22 Apr 2021

PONE-D-21-08416

Prognostic value of preoperative circulating tumor cell counts in patients with UICC Stage I-IV colorectal cancer

PLOS ONE

Dear Dr. Abdalla,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In addition to the reviewer's comment, it is strongly recommended to comment and discuss: i) the size and shape of CTCs dectected (ie. presence of clsuters); ii)  the limit of the technique used based on EpCAM marker

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We look forward to receiving your revised manuscript.

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Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

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Reviewer #1: Yes

**********

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Reviewer #1: Yes

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Reviewer #1: The authors should state in their discussion that this is not the first study to investigate CTC in non-metastatic colorectal cancer and discuss related work in more detail. E.g. the article "Circulating tumour cells and outcome in non-metastatic colorectal cancer: a prospective study. Br J Cancer. 2015 Apr 14;112(8):1306-13. doi: 10.1038/bjc.2015.88. " should be cited and discussed, as this was the first trial to investigate non-metastatic CTC count in CRC with the Cell search system.

**********

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PLoS One. 2021 Jun 10;16(6):e0252897. doi: 10.1371/journal.pone.0252897.r002

Author response to Decision Letter 0

20 May 2021

Dear Prof. Heymann,

Thank you very much for giving us the opportunity to resubmit our manuscript in your highly respected journal.

In the following you will find point by point answers with regard to the editorial comments as well as the comments by the reviewers. Each table was revised in accordance to the journal’s guidelines. All changes are clearly marked in the revised manuscript.

We would greatly appreciate to publish our work in the “PLOS ONE” and ask for a revised review process.

Sincerely yours,

Thaer S. A. Abdalla

Editorial comments:

1. Comment and discuss the size and shape of CTCs dectected (ie. presence of clusters)

Thank you for your kind remarks. Keratin-positive nucleated cells of round or oval shape with a diameter of at least 4 µm and CD45 negativity were considered CTCs. Only in a minority of cases CTC clusters with 2 or 3 CTCs were detected.

2. Comment and discuss the limit of the technique used based on EpCAM marker

As requested, the limits of the anti-EpCAM methods have been discussed in the lines 174-185 pages 9-10.

3. Changes in the reference list

The list of references has been updated in order to answer the requested changes. The references 27-30 and 32-34 have been recently added.

Reviewer 1:

First of all we would like to give our thanks to the Reviewer 1 on his thoughtful comments and suggestions.

1. The authors should state in their discussion that this is not the first study to investigate CTC in non-metastatic colorectal cancer and discuss related work in more detail. E.g. the article "Circulating tumour cells and outcome in non-metastatic colorectal cancer: a prospective study. Br J Cancer. 2015 Apr 14;112(8):1306-13. doi: 10.1038/bjc.2015.88. " should be cited and discussed, as this was the first trial to investigate non-metastatic CTC count in CRC with the Cell search system.

Thank you for this remark. We absolutely agree that there are previous studies to investigate CTCs in non metastastic colorectal cancer like the one by Bork et al. Thus, we added a revised description on page 10 of the manuscript.

In our work we used the CellSearch® system for identifying CTCs in all stages of CRC scheduled for resection with curative intent and compared their detection preoperatively and within 96 hours postoperatively to the OS and PFS.

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file. (45KB, doc)

Decision Letter 1

Dominique Heymann

25 May 2021

Prognostic value of preoperative circulating tumor cells counts in patients with UICC Stage I-IV colorectal cancer

PONE-D-21-08416R1

Dear Dr. Abdalla,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Dominique Heymann

2 Jun 2021

PONE-D-21-08416R1

Prognostic value of preoperative circulating tumor cells counts in patients with UICC Stage I-IV colorectal cancer.

Dear Dr. Abdalla:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Pr. Dominique Heymann

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Data. Patients histopathological and survival data.

    (SAV)

    Click here for additional data file. (8.1KB, sav)
    Attachment

    Submitted filename: Response to Reviewers.doc

    Click here for additional data file. (45KB, doc)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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