Purpose: To evaluate the clinical relevance of circulating CEACAM5mRNA-positive cells in patients with operable colorectal cancer (CRC).

Methods: Peripheral blood was obtained from 265 patients with operable CRC before the initiation of adjuvant systemic therapy from 96 normal donors and RNA prepared from the Lovo and ARH-77 CRC and leukemic cell lines, respectively, was used as positive and negative controls. The detection of CEACAM5mRNA-positive cells was done using a real-time PCR assay. The association with known prognostic factors and the effect of CEACAM5mRNA-positive cells on patients' prognosis was investigated.

Results: The analytical detection limit of the method was found to correspond to 0.7 Lovo cell equivalence/5 μg RNA, with a sensitivity of 1 tumor cell/105 normal cells and a specificity of 97%. Ninety-eight (37%) patients had detectable circulating CEACAM5mRNA-positive cells. Detection of CEACAM5mRNA-positive cells was significantly associated with higher relapse rate (P < 0.001), decreased disease-free survival (DFS; P < 0.001), higher death rate (P = 0.017), and decreased median overall survival (P = 0.025). Multivariate analysis revealed that the detection of circulating CEACAM5mRNA-positive cells was an independent prognostic factor for decreased DFS [HR = 3.4; 95% CI: 2.0–5.9; P < 0.001].

Conclusions: Detection of peripheral blood CEACAM5mRNA-positive cells is an adverse prognostic factor correlated with poor clinical outcome in patients with operable CRC. Clin Cancer Res; 17(1); 165–73. ©2010 AACR.

Translation Relevance

Detection of circulating cancer cells (CTC) in blood samples could serve as a precious tool for prognosis determination and treatment effectiveness monitoring. In colorectal cancer (CRC), the most frequently analyzed marker is the carcinoembryonic antigen (CEA). After the initial studies had indicated that CEACAM5mRNA could be used as a specific tumor cell marker, several subsequent reports showed that low CEACAM5mRNA levels also detected in blood samples of normal individuals because of a splicing variant are also expressed in normal WBCs.

On the basis of this, we established an assay for the detection of CEACAM5mRNA-positive cells and we confirmed the specificity, the repeatability, and the reproducibility of the method. Finally, we showed that detection of CEACAM5mRNA-positive cells was an adverse prognostic factor correlated with poor clinical outcome in early-stage CRC patients. If the results of this assays will be confirmed independently, then it could be used for patient selection and stratification in clinical trials.

Colorectal cancer (CRC) represents approximately 10% of all cancer cases (1). Although curative resection is possible in 2 of 3 of patients at diagnosis, almost one third of them will eventually develop relapse and will die of the disease (2).

Dissemination of tumor cells is an obligatory step toward progression to metastatic disease. Disseminated CRC cells can be identified in various body compartments using immunophenotyping and reverse transcription-polymerase chain reaction (RT-PCR) techniques (3, 4). In patients with early-stage CRC, the detection of malignant cells in the bone marrow, the peritoneal lavage, and the regional lymph nodes is associated with poor survival (5, 6)

Detection and quantification of circulating tumor cells CTCs in blood samples could serve as a unique and easy diagnostic tool to determine prognosis and monitor treatment effectiveness (7). Quantitative real-time RT-PCR (RT-qPCR) has been shown to provide the sensitivity and the practicability that is necessary to detect rare CTCs in patients' blood. (8) In CRC, the most frequently analyzed marker is the carcinoembryonic antigen (CEA). The initial clinical studies have analyzed the usefulness of CEACAM5mRNA for the detection of CTCs in blood samples of patients with CRC, but none of them have quantified the PCR product (4, 9–11). Nevertheless, a low level of CEACAM5mRNA expression has been detected in normal subjects, suggesting an illegitimate expression of the CEACAM5 gene in hematopoietic cells (12, 13) whereas other studies have failed to show significant differences of CEACAM5mRNA expression between cancer patients and healthy individuals (14). These studies have been conducted using sets of primers that amplify a splice variant of CEACAM1 expressed in normal white blood cells (WBC), in which an “intron” sequence replaces part of the exon 10 (12), thus explaining the findings of CEACAM5mRNA expression in normal blood samples (13) or in patients with inflammatory bowel disease (14) and in cultured WBCs after induction with G-CSF (3). On the basis of the aforementioned results, the overall usefulness of CEACAM5 as a PCR-based tumor cell detection marker remains questionable.

The aim of this study was to investigate the presence of CEACAM5mRNA-positive CTCs using a new RT-qPCR assay, which is designed to avoid the amplification of the spice variant expressed in normal WBCs and to determine their prognostic significance in patients with operable (stages II–III) CRC.

Patients and specimens

Since January 1998, peripheral blood (15 mL in EDTA) was obtained from consecutive patients with CRC at the time of the first visit (usually 3–8 weeks after surgery) in the Department of Medical Oncology of the University Hospital of Heraklion, on a routine basis after obtaining the patient's consent. Totally, blood samples were obtained from 269 patients with stage II–III CRC. Four patients were excluded from the analysis due to incomplete clinical data (1 case) or inadequate follow-up (3 cases). In this analysis, 265 patients with operable CRC (stages II–III) with a minimum follow-up of 24 months were included. Histopathologic data were obtained from the pathology reports and included the extramural vascular invasion status, tumor differentiation, maximum depth of invasion (pT), and lymph node involvement (pN). For the evaluation of the analytical sensitivity and specificity of the method, blood samples from 96 healthy blood donors, 15 patients with colonic adenomas, 21 patients with inflammatory bowel disease (IBD), and 100 patients with metastatic CRC (mCRC) were analyzed. All blood samples were obtained at the middle-of-vein puncture after the first 5 mL of blood were discarded to avoid contamination with epithelial cells from the skin. Adjuvant chemotherapy was administered in 257 (97%) patients, and in 102 of the cases (39%) an oxaliplatin-based regimen was used. Blood samples from 104 patients were also obtained within 1 month after the completion of the treatment. Patients were followed with clinical examination and laboratory tests every 3 months for the first 3 years and every 6 months for the next 2 years, and yearly thereafter. Chest and abdominal pelvic computed tomography was done yearly for the first 3 years and subsequently at the discretion of the physician. The study has been approved by the Ethics and Scientific Committees of our Institution, and all patients and donors signed informed consent.

Specimen characteristics and assay methods

Peripheral blood mononuclear cells (PBMC) were obtained by gradient density centrifugation and RNA extraction was done as previously described (7). RNA concentration was measured using the NanoDrop (Thermo Scientific) equipment. Amplification of the β-actin as internal reference gene was done to verify the RNA integrity. RNA prepared from the Lovo and ARH-77 CRC and leukemic cell lines, respectively, was used as positive and negative controls.

The SuperScript III PlatinumTranscriptase (Invitrogen) was used to prepare cDNA from 5 μg of total RNA, according to the manufacturer protocol. Also, 1 μg of colonic commercial RNA control (Stratagene) was used in serial dilutions (1:10) to verify the efficacy and reproducibility of the reaction.

The RT-qPCR reaction for β-actin and CEACAM5 was carried out using 2.5 μL of cDNA template and 6.25 μL of TaqMan Universal Master Mix (AB; Applied Biosystems), 1.25 μL– of specific primers (3 μmol/L), and 0.5 μL of hydrolysis probe (2.5 μmol/L) for each gene and adjusted with DPEC water to a final volume of 12.5 μL/reaction. Quantification of gene expression was performed using the ABI Prism 7900HT Sequence Detection System (AB). All experiments were performed in triplicates. Only triplicates with a standard deviation of the quantification cycle (Cq) value less than 0.25 were accepted.

Quantification was based on an external calibration curve that was obtained by using external standard cDNAs. Total RNA was prepared from 1 × 106 Lovo cells. cDNA synthesis of serial dilutions of this RNA, corresponding to 1–105 Lovo cells, was also analyzed in each run. This calibration curve was created by plotting the number of Lovo cells corresponding to each external standard cDNA versus the value of its Cq. The number of CEACAM5mRNA-positive cells for all of the tested samples was expressed as Lovo cell equivalents/5 μg of total RNA according to the external standard calibration curve. In addition, to determine the number of Lovo cells that could be recovered, we added decreasing number of Lovo cells (from 103 to 1) in 106 negative PBMCs in 5 different experiments. The SDS 2.3 software was used for the analysis of the results.

The primers and probe sets were designed using Primer Express 2.0 Software (AB). The β-actin primers and probe set have been previously described (15) whereas those for CEACAM5 (Supplementary Fig. 1) were designed according to the Ref Seq NM_004363.2 (http://www.ncbi.nlm.nih.gov/LocusLink). To avoid the amplification of the splice variant, which is expressed in normal WBCs, the probe was designed to spread across the junction of exon 9 with exon 10. BLAST search confirmed the specificity of this set of primers, as no homology to pseudogenes, splicing variants, or unexpected targets was found. As CEACAM1 (Ref Seq NC_001024912.1) is approximately 90% homologous with CEACAM5, we designed primers and probes for this family member and explored the specificity of the assay both in HL60 leukemic cell line and in PBMCs of a healthy volunteer (HV) for the expression of CEACAM1 (16, 17) and the MCF7 cell lines for that of CEACAM5 (18). Finally, genomic DNA contamination was excluded, as no RNA transcripts could be detected in each analyzed sample in the absence of reverse transcriptase.

Study design and statistics

The aim of this study was to explore the prognostic significance of CEACAM5mRNA-positive CTCs in patients with stage II–III CRC. Disease-free survival (DFS) and overall survival (OS) were calculated from the day of surgery to the first documented relapse or death, respectively. Relapse was defined as the finding of metastatic disease, local recurrence, or a second primary tumor. The evaluation for the presence of CEACAM5mRNA-positive cells was done blindly to clinical data. The potential association between baseline characteristics, relapse, and CEACAM5mRNA-positive cells was compared with the 2-sided Fisher exact test for categorical variables. The association of risk factors with time-to-event endpoints was analyzed with the log-rank test, and the Kaplan–Meier method was used to plot the corresponding DFS and OS curves. Univariate and multivariate Cox proportional hazards regression models with hazard ratios and 95% CIs were used to assess the association between potential prognostic factor and DFS or OS. Statistical significance was set at P ≤ 0.05.

RT-qPCR assay optimization

cDNA obtained from Lovo cells was analyzed to determine the analytical sensitivity and linearity of the RT-qPCR assay. The calibration curves of these experiments proved the linearity of the assay over the entire quantification range (1–105 Lovo cells) with a correlation coefficient of 0.994 (Supplementary Fig. 2). The RT-qPCR efficiency expressed as E = 10(−1/slope)−1 (19) was 0.97 ± 0.029 SD [coefficient of variation (CV): 3.0%; n = 18) and the mean value of slope and intercept were 3.516 ± 0.11 (CV: 3.1; n = 18) and 30.28 ± 0.78 (CV: 2.6%; n = 18 experiments), respectively. The limit of detection (LOD) of the assay was found to correspond to 0.7 Lovo cell equivalents/5 μg of RNA (LOD = 3.3 SD/slope, where SD is the standard deviation of the Cq for 1 Lovo cell equivalent; refs. 8, 20).

For the determination of the repeatability of the assay, cDNA samples corresponding to 1 to 10.000 Lovo cells were assayed in the same run in 6 parallel determinations, using the same calibration curve (21). The SDs of the Cq values and the CVs for Lovo cells are shown in Supplementary Table 1, which shows the repeatability of the assay. The results obtained from the same RNA, which was frozen in aliquots, and analyzed over a period of 6 weeks in 12 different experiments done in different days confirmed the reproducibility of the assay (22). In addition, as summarized in Supplementary Table 2, in 5 different experiments we were able to recover 10 Lovo cells in 106 normal PBMCs (1:105).

Only CEMCAM1 was amplified in HL60 cells, whereas only CEACAM5 was amplified in MCF7 cells with the use of conventional PCR using 100 ng of genomic DNA/reaction (Supplemental Fig. 3A). These results were also confirmed in the RT-qPCR starting from 5 μg of RNA for each gene (Supplemental Fig. 3B). In addition, sequencing of the RT-qPCR product confirmed the specificity of the assay (Supplemental Fig. 3C).

CEACAM5mRNA-positive cell detection in patients and control groups

The incidence of detection of CEACAM5mRNA-positive cells in the controls and patients is presented in Table 1. According to the cutoff point (>0.7 Lovo cells equivalents/5 μg of RNA), only 2 (2.2%) samples from the 96 healthy volunteers (HV) tested positive, suggesting a specificity rate of 97.8%. Table 1 also indicates that 1 (4.6%) patient with IBD but none with colonic adenomas had detectable CEACAM5mRNA-positive cells. In contrast, CEACAM5mRNA-positive cells could be detected in 98 (37%) and 44 (44%) patients with stage II–III and IV CRC, respectively (P = 0.4; Table 1). The median number of CEACAM5mRNA-positive CTCs was 1.72 (range, 0.72–26.5) and 4.18 (range, 0.94–218.2) in patients with stage II–II and metastatic disease, respectively (P = 0.4; Table 1). There was no significant association for the detection of CEACAM5mRNA-positive CTCs with any of the examined clinicopathologic features in patients with stage II–III disease (Table 2). In contrast, in patients with metastatic disease, liver involvement was associated with significantly higher CEACAM5mRNA-positive CTCs detection (52% vs. 31% in patients with or without liver metastasis; P = 0.021; Table 3).

Table 1.

Detection of CEACAM5mRNA-positive cells in different groups of patients and healthy individuals

No. of samplesNo. of +ve samples (%)Mean no. of cellsRange (min–max)SD (±)
Healthy blood donors 96 2 (2.2) 0.21 0.0–0.87 0.23 
IBD 21 1 (4.7) 0.47a 0.16–0.85 0.34 
Adenomas 15 0 (0.0) 0.32b 0.0–0.64 0.29 
Stage II–III CRC 265 98 (37) 0.68 0.1–26.5 0.7 
 CEA mRNA (+) 98  1.72c 0.72–26.5 1.8 
 CEA mRNA (−) 167  0.52 0.1–0.68 0.71 
Metastatic CRC 100 44 (44) 0.74 0.2–218.2 0.0 
 CEA mRNA (+) 44  4.18d 0.94–218.2 6.21 
 CEA mRNA (−) 56  0.58 0.2–0.69 0.65 
No. of samplesNo. of +ve samples (%)Mean no. of cellsRange (min–max)SD (±)
Healthy blood donors 96 2 (2.2) 0.21 0.0–0.87 0.23 
IBD 21 1 (4.7) 0.47a 0.16–0.85 0.34 
Adenomas 15 0 (0.0) 0.32b 0.0–0.64 0.29 
Stage II–III CRC 265 98 (37) 0.68 0.1–26.5 0.7 
 CEA mRNA (+) 98  1.72c 0.72–26.5 1.8 
 CEA mRNA (−) 167  0.52 0.1–0.68 0.71 
Metastatic CRC 100 44 (44) 0.74 0.2–218.2 0.0 
 CEA mRNA (+) 44  4.18d 0.94–218.2 6.21 
 CEA mRNA (−) 56  0.58 0.2–0.69 0.65 

aStage II–III CRC patients versus patients with IBD (P < 0.001) mCRC (P = 0.4).

bStage II–III CRC patients versus patients with colonic adenomas (P < 0.001).

cStage II–III CRC patients versus healthy donors (P < 0.001).

dStage II–III CRC patients versus patients with metastatic CRC (P = 0.4).

Table 2.

Characteristics of patients with stages II–III according to the presence of CEACAM5mRNA-positive CTCs

CEA mRNA status
No. of patients(N = 98)Negative (N = 167)
N%N%P
Gender 
 Male 167 67 40 100 60 0.2 
 Female 98 31 32 67 68  
Age 
 ≤70 y 185 69 37 116 63 0.9 
 >70 y 80 29 36 51 64  
Tumor location 
 Colon 184 69 37 115 63 0.9 
 Rectum 81 29 36 52 63  
Stage at diagnosis 
 II 105 41 39 65 61 0.6b 
 High-risk stage IIa (85) (34) (40) (51) (60)  
 III 160 57 36 103 64  
Histologic grade 
 I–II 194 73 37 121 63 0.8 
 III 71 29 43 42 57  
Mucinous 
 Yes 53 20 37 33 63 1.0 
 No 212 78 37 134 63  
Neural invasion 
 Yes 78 29 37 49 63 1.0 
 No 187 69 37 118 63  
Vessels invasion 
 Yes 118 44 37 74 63 1.0 
 No 147 54 37 93 63  
Vessels cancer emboli 
 Yes 67 30 45 37 55 0.1 
 No 198 68 34 130 66  
CEA mRNA status
No. of patients(N = 98)Negative (N = 167)
N%N%P
Gender 
 Male 167 67 40 100 60 0.2 
 Female 98 31 32 67 68  
Age 
 ≤70 y 185 69 37 116 63 0.9 
 >70 y 80 29 36 51 64  
Tumor location 
 Colon 184 69 37 115 63 0.9 
 Rectum 81 29 36 52 63  
Stage at diagnosis 
 II 105 41 39 65 61 0.6b 
 High-risk stage IIa (85) (34) (40) (51) (60)  
 III 160 57 36 103 64  
Histologic grade 
 I–II 194 73 37 121 63 0.8 
 III 71 29 43 42 57  
Mucinous 
 Yes 53 20 37 33 63 1.0 
 No 212 78 37 134 63  
Neural invasion 
 Yes 78 29 37 49 63 1.0 
 No 187 69 37 118 63  
Vessels invasion 
 Yes 118 44 37 74 63 1.0 
 No 147 54 37 93 63  
Vessels cancer emboli 
 Yes 67 30 45 37 55 0.1 
 No 198 68 34 130 66  

aHigh-risk stage II: T4 tumor or grade III or perforation-obstruction at diagnosis, or <12 lymph node dissected.

bComparison between stages II and III.

Table 3.

Characteristics of patients with stage IV according to the presence of CEACAM5mRNA-positive CTCs

CEACAM5mRNA status
Positive (N = 44)Negative (N = 56)
No of patientsN%N%p
Gender 
 Male 58 27 47 31 53 0.6 
 Female 42 17 40 25 60  
Age, y 
 ≤70 71 32 46 39 54 0.8 
 >70 29 12 41 17 59  
Tumor location 
 Colon 67 28 41 39 59 0.3 
 Rectum 33 16 48 17 52  
Histologic grade 
 I–II 61 25 41 36 59 0.3 
 III 39 19 49 20 51  
Liver involvement 
 Yes 64 33 52 31 48 0.021 
 No 36 11 31 25 69  
CEACAM5mRNA status
Positive (N = 44)Negative (N = 56)
No of patientsN%N%p
Gender 
 Male 58 27 47 31 53 0.6 
 Female 42 17 40 25 60  
Age, y 
 ≤70 71 32 46 39 54 0.8 
 >70 29 12 41 17 59  
Tumor location 
 Colon 67 28 41 39 59 0.3 
 Rectum 33 16 48 17 52  
Histologic grade 
 I–II 61 25 41 36 59 0.3 
 III 39 19 49 20 51  
Liver involvement 
 Yes 64 33 52 31 48 0.021 
 No 36 11 31 25 69  

Prognostic significance of the CEACAM5mRNA-positive cell detection

Disease relapse

After a median follow-up period of 34.1 months (range, 24.2–122.4 months), 57 (22%) relapses were observed among the 265 patients with operable CRC. All relapsed patients had distant metastases and 7 also presented local relapse. The incidence of relapses was significantly higher in patients with CEACAM5mRNA-positive CTCs than in patients without (37% vs. 12%; P < 0.001; Table 4). The median DFS has not been reached, but it was significantly higher in patients without detectable circulating CEACAM5mRNA-positive cells compared with patients with (P < 0.001; Fig. 1A). In addition, the probability of 3-year relapse-free survival was 83% and 58% in patients with and without detectable CEACAM5mRNA-positive CTCs, respectively (P = 0.001). Moreover, patients with undetectable CEACAM5mRNA-positive CTCs before and after the administration of the adjuvant treatment (n = 54) experienced significantly increase DFS in comparison with those (n = 20) with detectable CEACAM5mRNA-positive CTCs before and after adjuvant treatment (P = 0.017) or with those (n = 9) with detectable CEACAM5mRNA-positive CTCs only after the completion of the adjuvant treatment (P = 0.005; Fig. 1C). Also, in the patients with metastatic disease, detection of CEACAM5mRNA-positive CTCs was correlated with significantly decreased progression-free survival (median = PFS 6.6 and 9.2 months in patients with and without detectable CEACAM5mRNA-positive CTC, respectively; P = 0.043).

Figure 1.

A, DFS according to the detection of CEACAM5mRNA-positive cells. Detection of CEACAM5mRNA-positive cell was associated with significantly decreased DFS. B, median OS according to the detection of CEACAM5mRNA-positive cells. Patients without CEACAM5mRNA-positive cells presented significantly higher OS than those with detectable CEACAM5mRNA-positive cells. C, DFS according to the detection of CEACAM5mRNA-positive cells before and/or after the administration of adjuvant chemotherapy. Detection of CEACAM5mRNA-positive cell before the initiation and at the end of adjuvant chemotherapy was correlated with significantly decreased DFS. D, median OS according to the detection of CEACAM5mRNA-positive cells before and/or after the administration of adjuvant chemotherapy. Detection of CEACAM5mRNA-positive cell before the initiation and at the end of adjuvant chemotherapy was correlated with significantly decreased OS.

Figure 1.

A, DFS according to the detection of CEACAM5mRNA-positive cells. Detection of CEACAM5mRNA-positive cell was associated with significantly decreased DFS. B, median OS according to the detection of CEACAM5mRNA-positive cells. Patients without CEACAM5mRNA-positive cells presented significantly higher OS than those with detectable CEACAM5mRNA-positive cells. C, DFS according to the detection of CEACAM5mRNA-positive cells before and/or after the administration of adjuvant chemotherapy. Detection of CEACAM5mRNA-positive cell before the initiation and at the end of adjuvant chemotherapy was correlated with significantly decreased DFS. D, median OS according to the detection of CEACAM5mRNA-positive cells before and/or after the administration of adjuvant chemotherapy. Detection of CEACAM5mRNA-positive cell before the initiation and at the end of adjuvant chemotherapy was correlated with significantly decreased OS.

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Table 4.

Incidence of disease relapse and death according to the detection of CEACAM5mRNA-positive circulating tumor cells

Relapses (n = 57)Deaths (n = 45)
CEA mRNA statusNo. of patientsn%Pan%Pa
 Positive 98 36 37 <0.001 24 24 0.017 
 Negative 167 21 12  21 12  
Relapses (n = 57)Deaths (n = 45)
CEA mRNA statusNo. of patientsn%Pan%Pa
 Positive 98 36 37 <0.001 24 24 0.017 
 Negative 167 21 12  21 12  

aFisher's exact test.

Survival

During the follow-up period, 45 of 265 patients (17%) died because of disease progression (Table 4). Twenty-four (24%) deaths occurred in patients with detectable CEACAM5mRNA-positive CTCs and 21 (12%) in patients without (P = 0.017; Table 4). The median OS of patients without detectable CEACAM5mRNA-positive CTCs was significantly higher than that of patients with detectable CEACAM5mRNA-positive CTCs (P = 0.025; Fig. 1B). The 3- and 5-year probability of survival was 79% and 57%, respectively, for CEACAM5mRNA-positive patients in comparison with 88% and 78%, respectively, for CEACAM5mRNA-negative patients. Also, patients with undetectable CEACAM5mRNA-positive CTCs before and after the administration of the adjuvant treatment (n = 54) experienced significantly increased OS in comparison with those (n = 20) with detectable CEACAM5mRNA-positive CTCs before and after adjuvant treatment (P = 0.001) or with those (n = 9) with detectable CEACAM5mRNA-positive CTCs only after the completion of the adjuvant treatment (P = 0.001; Fig. 1D). Similarly, in patients with metastatic disease, the detection of CEACAM5mRNA-positive CTCs was associated with decreased survival (21.6 months vs. 17.3 months; P = 0.047).

Univariate and multivariate analyses

The univariate analysis showed that CEACAM5mRNA-positive patients presented higher risk of progression than CEACAM5mRNA-negative patients (HR = 3.4; P < 0.001; Table 5). Also, disease stage (III vs. II), tumor differentiation (poor vs. well/moderate), and presence of neural invasion and cancerous vessels emboli (CVE) were associated with decreased DFS (Table 5). Multivariate analysis confirmed that the detection of CEACAM5mRNA-positive CTCs was independent prognostic factor for decreased DFS (HR = 3.4; 95% CI: 2.0–5.9; P < 0.001; Table 6). Moreover, stage III (HR = 3.0; 95% CI: 1.5–5.9; P = 0.002) and the presence of poorly differentiated tumors (HR = 1.9; 95% CI: 1.1–3.3; P = 0.022) also emerged as independent prognostic factors associated with decreased DFS (Table 6).

Table 5.

Characteristics of enrolled patients and univariate analysis for disease-free survival and overall survival

FeatureN%Disease-free survivalOverall survival
HR (95% CI)PHR (95% CI)P
Median age (range), y 65 (22–86)     
 70 vs. 80 30 1.4 0.2 1.8 0.08 
 ≤70 185 70 (0.8–2.7)  (0.9–3.4)  
Gender 
 Male vs. 167 63 1.2 0.4 1.3 0.4 
 Female 98 37 (0.7–2.1)  (0.7–2.1)  
Stage at diagnosis 
 III vs. 160 60 3.4 0.001 2.6 0.005 
 II 105 40 (1.8–6.6)  (1.3–5.3)  
Tumor location 
 Colon vs. 184 69 1.2 0.4 1.6 0.1 
 Rectum 81 31 (0.7–2.1)  (0.9–2.7)  
Histologic grade 
 III vs. 71 27 2.7 <0.001 3.6 <0.001 
 I–II 194 73 (1.6–4.5)  (1.7–5.7)  
Mucinous 
 No vs. 212 80 0.7 0.3 0.6 0.2 
 Yes 52 20 (0.3–1.4)  (0.3–1.4)  
Vessels invasion 
 Yes vs. 118 45 1.4 0.1 1.4 0.3 
 No 147 55 (0.8–2.4)  (0.8–2.5)  
Neural invasion 
 Yes vs. 78 29 1.9 0.01 2.4 0.3 
 No 187 71 (1.1–3.3)  (1.3–4.5)  
Vessels cancer emboli 
 Yes vs. 67 25 1.9 0.01 3.1 0.001 
 No 198 75 (1.1–3.3)  (1.7–5.7)  
CEACAM5mRNA CTCs 
 Positive vs. 98 37 3.2 <0.001 1.9 0.03 
 Negative 167 63 (1.8–5.5)  (1.1–3.5)  
FeatureN%Disease-free survivalOverall survival
HR (95% CI)PHR (95% CI)P
Median age (range), y 65 (22–86)     
 70 vs. 80 30 1.4 0.2 1.8 0.08 
 ≤70 185 70 (0.8–2.7)  (0.9–3.4)  
Gender 
 Male vs. 167 63 1.2 0.4 1.3 0.4 
 Female 98 37 (0.7–2.1)  (0.7–2.1)  
Stage at diagnosis 
 III vs. 160 60 3.4 0.001 2.6 0.005 
 II 105 40 (1.8–6.6)  (1.3–5.3)  
Tumor location 
 Colon vs. 184 69 1.2 0.4 1.6 0.1 
 Rectum 81 31 (0.7–2.1)  (0.9–2.7)  
Histologic grade 
 III vs. 71 27 2.7 <0.001 3.6 <0.001 
 I–II 194 73 (1.6–4.5)  (1.7–5.7)  
Mucinous 
 No vs. 212 80 0.7 0.3 0.6 0.2 
 Yes 52 20 (0.3–1.4)  (0.3–1.4)  
Vessels invasion 
 Yes vs. 118 45 1.4 0.1 1.4 0.3 
 No 147 55 (0.8–2.4)  (0.8–2.5)  
Neural invasion 
 Yes vs. 78 29 1.9 0.01 2.4 0.3 
 No 187 71 (1.1–3.3)  (1.3–4.5)  
Vessels cancer emboli 
 Yes vs. 67 25 1.9 0.01 3.1 0.001 
 No 198 75 (1.1–3.3)  (1.7–5.7)  
CEACAM5mRNA CTCs 
 Positive vs. 98 37 3.2 <0.001 1.9 0.03 
 Negative 167 63 (1.8–5.5)  (1.1–3.5)  
Table 6.

Multivariate analysis for independent prognostic factors for DFS and OS

Hazard ratio95% CIP
DFS 
CEACAM5mRNA (positive vs. negative) 3.4 2.0–5.9 <0.001 
Stage (III vs. II) 3.0 1.5–5.9 0.002 
 Grade (3 vs. 1–2) 1.9 1.1–3.3 0.022 
 Neural invasion (yes vs. no) 1.2 0.6–2.7 0.552 
Vessels tumoral emboli (yes vs. no) 1.4 0.8–2.6 0.183 
OS 
CEACAM5mRNA (positive vs. negative 1.9 0.95–4.0 0.065 
Stage (III vs. II) 1.9 1.2–3.9 0.048 
 Grade (3 vs. 1–2) 1.9 0.98–3.7 0.056 
Vessels tumoral emboli (yes vs. no) 2.4 1.4–4.4 0.008 
Hazard ratio95% CIP
DFS 
CEACAM5mRNA (positive vs. negative) 3.4 2.0–5.9 <0.001 
Stage (III vs. II) 3.0 1.5–5.9 0.002 
 Grade (3 vs. 1–2) 1.9 1.1–3.3 0.022 
 Neural invasion (yes vs. no) 1.2 0.6–2.7 0.552 
Vessels tumoral emboli (yes vs. no) 1.4 0.8–2.6 0.183 
OS 
CEACAM5mRNA (positive vs. negative 1.9 0.95–4.0 0.065 
Stage (III vs. II) 1.9 1.2–3.9 0.048 
 Grade (3 vs. 1–2) 1.9 0.98–3.7 0.056 
Vessels tumoral emboli (yes vs. no) 2.4 1.4–4.4 0.008 

Similarly, patients with detectable CEACAM5mRNA-positive CTCs presented higher risk of death than patients without CEACAM5mRNA-positive cells (HR = 1.9; P = 0.028; Table 5). Disease stage, tumor differentiation, and presence of CVE were also significantly associated with OS. The multivariate analysis revealed that stage III (HR = 1.9; P = 0.048) and the presence of CVE (HR = 2.4; P = 0.008) were independent prognostic factors associated with decreased survival (Table 6).

The aims of this study was to develop a reliable and reproducible RT-qPCR assay, using CEACAM5mRNA as a detection marker for CTC quantification in patients with CRC and to evaluate their detection as a prognostic factor. In breast cancer, after “curative resection,” the rates of CTC detection are decreased after few weeks in a majority of the cases and the persistence of CTCs after have prognostic significance. (23) For this reason, we decided to obtain the blood samples just before the initiation of adjuvant chemotherapy (for stage III or high-risk stage II patients) or follow-up.

The results of our study should not be compared with those of previous studies in which PCR-based assays have been used for the detection of CEACAM5mRNA-positive cells. Most of these studies used qualitative assays (4, 6, 11, 24) and, most likely, included “false-positive” results due to the amplification of a splicing variant of CEACAM1, which is expressed in WBCs (12). On the basis of this assumption, we designed a specific set of primers and probe that did not amplify this variant and we proved the high specificity and efficiency of the method. Subsequently, we observed a significant decreased DFS (P < 0.001) and OS (P = 0.017) in patients with operable CRC and detectable CEACAM5mRNA-positive cells, whereas the detection of CEACAM5mRNA-positive cells was revealed as an independent prognostic factor for decreased DFS (HR = 3.4; P < 0.001). Also, a trend for increased risk of death (HR = 1.9; P = 0.065) was recorded that did not reach statistical significance probably due to the limited number of events occurring during the short follow-up period. In contrast, the presence of CVE was revealed as an independent prognostic factor for decreased OS in agreement with several previous reports (25, 26). In addition, in the subset of patients with available specimen at the completion of the adjuvant chemotherapy, the persistence of CEACAM5mRNA-positive cells was a strong prognostic factor of decreased DFS and OS. This preliminary finding provides a rational for the study of whether the detection CEACAM5mRNA-positive cells could be used as a surrogate marker for the efficacy of the adjuvant treatment.

There was no significant difference in the percentage of patients with detectable CEACAM5mRNA-positive cells between stage II/III and IV patients, even though the higher actual number of CTCs in stage IV patients. Of notice that within stage IV patients, those with liver involvement had a significantly higher percentage of CEACAM5mRNA-positive cells (P = 0.021). These results are in agreement with those reported in studies using cell-based techniques. Indeed, it has been reported that by using the CellSearch platform (Veridex LLC), ≥2 CTCs/7.5 mL blood could be detected only in patients with malignant epithelial tumors (27) whereas the CTC detection rate in CRC patients was 36.2% and the quantification of CTCs could be a valuable prognostic factor (28). A multicenter prospective study showed that CTCs could be detected in 60% of patients before treatment and was the strongest predictive factor for PFS and OS and that liver involvement was associated with significantly higher percentage of CTCs detection (29). Another interpretation of these results could be that none of the methods used since now are capable of detecting all the CTCs in circulation, and preliminary results from studies using advanced techniques indicated that CTCs could be detected in almost all patients with CRC (30).

Also, there was no difference in the percentage of patients with detectable CTCs between stage II (39%) and stage III (36%). The fact that 81% of the stage II patients were “high” risk stage II patients may partially explain this finding, as these patients had similar prognosis with those with stage IIIa or even IIIb disease. Also, the multivariate analysis revealed that the detection of CEACAM5mRNA-positive cells was prognostic factor for decreased DFS independent of stage (Table 6), whereas Sastre et al. reported almost identical detection rates in stage II (20.7%) and III (24.1%) patients (28). Moreover, it has been shown that CK-19mRNA–positive cells could be detected in 31% of breast cancer patients with negative axillary lymph nodes (31), indicating that dissemination of tumoral cells could be independent from lymph nodes infiltration.

However, as the presence of CTCs is a necessary (although not sufficient) step in the metastatic process, these cells probably define a subset of tumor cells that may lead to the development of metastasis. Metastatic cells might present very different molecular profiles from those of the primary tumor (30). Therefore, it is conceivable that the identification of CTCs might be used as a molecular signature of the metastatic ability of a specific tumor over time (32). The isolation and molecular characterization of CEACAM5mRNA-positive CTCs could address these important considerations and may allow for noninvasive genotyping of cancer cells, which could be monitored at specific therapeutic decision-making points during the patient treatment period (33).

Because of the weaknesses in the design of this study (not uniformally treated patients, lack of a validation patients' cohort), the results should be evaluated with caution. Future studies are warranted to evaluate whether the monitoring of CEACAM5mRNA-positive CTCs during the treatment of patients with early or metastatic disease could be of any clinical relevance influencing the therapeutic decision.

No potential conflicts of interest were disclosed.

This work was partly supported by research grants from the Cretan Association for Biomedical Research (CABR). Z.S. is a recipient of a CABR postdoctoral fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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