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Clinical Cancer Research Vol. 12, 5972-5977, October 15, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Carcinoembryonic Antigen Messenger RNA Expression in Blood Predicts Recurrence in Esophageal Cancer

Tetsuro Setoyama, Shoji Natsugoe, Hiroshi Okumura, Masataka Matsumoto, Yasuto Uchikado, Sumiya Ishigami, Tetsuhiro Owaki, Sonshin Takao and Takashi Aikou

Authors' Affiliations: Department of Surgical Oncology and Digestive Surgery, Field of Oncology, Course of Advanced Therapeutics, Kagoshima University, Graduate School of Medical and Dental Science, Kagoshima, Japan

Requests for reprints: Tetsuro Setoyama, Department of Surgical Oncology and Digestive Surgery, Kagoshima University, Graduate School of Medical and Dental Science, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. Phone: 81-99-275-5361; Fax: 81-99-265-7426; E-mail: setoyam2{at}m.kufm.kagoshima-u.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The clinical significance of isolated tumor cells (ITC) in blood has not been clearly established, particularly during follow-up in cancer patients. We conducted a longitudinal analysis of the relationship between ITC in blood during follow-up and clinicopathologic findings in patients with esophageal squamous cell carcinoma.

Experimental Design: Blood samples obtained from 106 patients were examined by real-time RT-PCR assay targeting carcinoembryonic antigen (CEA) mRNA. Follow-up examination every 3 months after surgery included testing for CEA mRNA and tumor markers, as well as imaging.

Results: Thirty-nine (36.8%) patients were positive for CEA mRNA expression. CEA mRNA positivity significantly correlated with tumor depth, lymph node metastasis, stage, and venous invasion. Recurrent disease was found in 34 of 106 (32.1%) cases. CEA mRNA was found in 28 (76.5%) patients experiencing relapse. Of these 28 patients, the number positive of CEA mRNA before detection by imaging, at the same time of detection by imaging, and after detection by imaging was 18 (52.9%), 8 (23.5%), and 2 (5.9%), respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for CEA mRNA were higher than those for serum CEA or squamous cell carcinoma. Patients positive for CEA mRNA experienced significantly shorter disease-free interval than those with negative CEA mRNA (P < 0.001). According to multivariate analysis, CEA mRNA positivity was an independent factor for disease-free interval.

Conclusions: Examination of CEA mRNA in peripheral blood during follow-up is useful for early detection of occult recurrence. We believe that CEA mRNA in blood will be a new marker for recurrence in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis even after curative Resection (1). Therefore, early and accurate detection of recurrence is one of the main issues in ESCC. To detect recurrent disease, detection of serum tumor markers (2–7) and imaging diagnosis, including radiography, gastrointestinal endoscopy, magnetic resonance imaging, computed tomography, and ultrasonography (8–11), are generally used during the follow-up period. Isolated tumor cells (ITC) have recently been detected in the blood, bone marrow, lymph nodes, and peritoneal lavage fluid of cancer patients (12–14). We previously reported that ITC in blood during surgery correlated well with recurrence in patients having carcinomas of the esophagus (15), stomach (16), and pancreas (17) using nested reverse transcription-PCR (RT-PCR) targeting carcinoembryonic antigen (CEA). In esophageal cancer, some reports have examined ITC in blood before or during surgery by nested RT-PCR with primers of squamous cell carcinoma (SCC) antigen (18) or {Delta}Np63 (19). In recent years, real-time fluorescence PCR monitoring systems have been introduced (20–23) and ITC were detected more specifically when compared with conventional nested RT-PCR (24, 25).

If tumor relapse can be detected more rapidly, patient prognosis after recurrence may be prolonged by early initiation of treatment. Few reports have examined the relationship between ITC in blood and clinical factors during the follow-up period in ESCC. In the present study, we retrospectively investigated the clinical significance of the ITC in blood during the postoperative period after curative esophagectomy (R0 resection). Furthermore, we compared the detection of recurrence by imaging diagnoses and identification of serum tumor markers, such as CEA and SCC, and we analyzed whether ITC in blood was useful for detection of occult recurrence that was not apparent on imaging.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. A total of 106 consecutive patients with ESCC who underwent R0 resection at Kagoshima University Hospital from July 1999 to June 2004 were enrolled in this study. The ages of the 93 males and 13 females ranged from 39 to 87 years (mean, 63.3 years). Clinicopathologic findings were based on the tumor-node-metastasis classification for esophageal carcinoma from the International Union Against Cancer (26). Histologically, 27 patients had well-differentiated (G1), 59 moderately differentiated (G2), 16 poorly differentiated (G3), 1 undifferentiated (GX), and 3 adenosquamous carcinoma. Twenty-one tumors were located in the upper third, 51 in the middle third, and 34 in the lower third of the esophagus.

With regard to depth of tumor invasion, the number of patients with pT1, pT2, pT3, and pT4 tumors was 50 (47.2%), 10 (9.4%), 42 (39.6%), and 4 (3.8%), respectively. Lymph node metastasis (pN1) occurred in 55 of 106 (51.9%) patients. All seven pM1 tumors were due to distant lymph node metastases. Lymphatic and venous invasion were found in 66 (62.3%) and 56 (52.8%) of patients, respectively. Twelve patients underwent neoadjuvant chemoradiation therapy using low-dose cisplatin (7 mg/m2) plus 5-fluorouracil (350 mg/m2) and 40-Gy radiation. After discharge, all patients were followed up with radiography and serum tumor marker (SCC and CEA) examination, computed tomography every 3 months, and ultrasonography every 6 months. Bronchoscopic and endoscopic examination and bone scintigraphy were done when necessary. Usually, most recurrent diseases were detected by computed tomography examination. Cervical nodal recurrence is useful for ultrasound, local recurrence for bronchoscopic and endoscopic examination, and scintigraphy for bone metastasis. Thus, because most recurrences such as mediastinal lymph node, lung, or liver recurrence were detected by computed tomography, there was little effect of ultrasound examination on recurrent disease. Biopsy examination was not routinely done to determine the histologic conformation. New lesions detected by imaging means were regarded as relapse in comparison with previous examination. All imagings were evaluated by two or three independent observers, including radiologists. The median follow-up period was 27.9 months (range, 5-72.0 months). Written informed consent was obtained from patients before surgery and on the use of obtained samples for research, in accordance with the institutional guidelines of our hospital.

Blood samples. In the present study, we investigated CEA mRNA expression of patients after surgery in the outpatient clinic during follow-up. Blood samples were obtained from the peripheral vein every 3 months. The first 6 mL of blood were discarded to prevent epidermal contamination.

Determination of CEA and SCC antigen in serum samples. Serum samples were also used for assay of tumor markers CEA and SCC. Serum levels of SCC and CEA were determined with a commercial enzyme immunoassay kit (SCC antigen and CEA; Dainabot Co., Ltd., Tokyo, Japan). Patients whose serum levels were >1.5 ng/mL SCC, or 5 ng/mL CEA, were usually considered to be SCC positive or CEA positive, respectively.

Cell lines. To prepare for CEA-specific RT-PCR, three cell lines, TE-2 (esophageal cancer cell line), MKN45 (gastric cancer cell line), and MCF-7 (breast cancer cell line), were used. Lymphocytes were collected from healthy volunteers without epithelial malignancy. After lymphocytes were isolated from peripheral blood by gradient centrifugation, the mononuclear cell layer was collected. Cell lines were serially diluted (10-fold) in 2 x 107 to 5 x 107 lymphocytes to give carcinoma cell/lymphocyte ratios ranging from 1:10 to 1:107.

RNA extraction and cDNA synthesis. Five-milliliter blood samples including EDTA were diluted with 5 mL of 0.05 mol/L PBS (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). Blood cells were isolated as peripheral blood mononuclear cells with lymphocyte separation medium (ICN Biomedicals, Inc., Aurora, OH) and were subjected to density gradient centrifugation at 420 x g at room temperature for 20 minutes. The layer of cells was collected and washed with 30 mL of sterile PBS. After centrifugation at 1,350 x g at 4°C for 15 minutes, cell pellets were suspended in 1 mL of Isogen (Nippon Gene, Toyama, Japan) and were stored at –80°C until use. Samples frozen in Isogen were thawed and total RNA (12) was extracted by the guanidium-isothiocyanate-phenol-chloroform–based method. Concentration of total RNA was determined by absorption measurements at absorbances of 260 and 280 nm using a UV-visible spectrophotometer (BioSpec-1600, Shimadzu, Kyoto, Japan).

Before the synthesis of cDNA, 1 µL of DNase I and 1 µL of 10x reaction buffer were added to 1 µg of total RNA with diethyl pyrocarbonate water in a total volume of 10 µL. The reaction mixture was incubated for 10 minutes at 37°C and 1 µL of 25 mmol/L EDTA was added. An 11-µL aliquot of reaction mixture was incubated for 10 minutes at 65°C and quickly chilled on ice, after which cDNA was synthesized using All Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA) according to the instructions of the manufacturer.

Real-time RT-PCR. A CEA-specific oligonucleotide primer was designed based on the report by Gerhard et al. (13). The sequences were 5'-TGTCGGCATCATGATTGG-3' (sense) and 5'-GCAAATGCTTTAAGGAAGAAGC-3' (antisense). Fluorescent and LC-Red probe sequences used for CEA identification were 5'-CCTGAAATGAAGAAACTACACCAGGGC-fluorescein and 5'-LC-Red 640-GCTATATCAGAGCAACCCCAACCAGC-phosphate. Amplification of CEA by PCR with a quantitative fluorescence LightCycler (Roche Diagnostics, Mannheim, Germany) was carried out in a 20-µL reaction mixture containing 2 µL of LightCycler FastStart DNA Master Hybridization Probes (Roche) as a master mixture, 3.0 mmol/L MgCl2, 0.5 µmol/L each of sense and antisense primers, 0.4 µmol/L fluorescent probe, 0.2 µmol/L LC-Red probe, and 5 µL of template cDNA in a LightCycler capillary. Before amplification, 0.32 µL of anti-Taq DNA polymerase antibody (TaqStart antibody, Clontech) was added to the reaction mixture, which was then incubated at room temperature for 5 minutes to avoid primer elongation. The temperature profile used for amplification was as follows: denaturation for 1 cycle at 95°C for 10 minutes and 40 cycles at 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 5 seconds. Real-time PCR monitoring was achieved by measuring the fluorescence signal at the end of annealing phase of each cycle. To prove the integrity of isolated RNA, a PCR assay with primers and probes specific for glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA was carried out under the same conditions as described above. The primer sequences used for GAPDH amplification were 5'-TGAACGGGAAGCTCACTGG-3' (sense) and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense). The probe sequences used for GAPDH identification were 5'-TCAACAGCGACACCCACTCCT-fluorescein and 5'-LC-Red 640-CACCTTTGACGCTGGGGCT-phosphate. Optimal reagent concentrations and PCR cycling conditions were established by Nihon Gene Research Laboratories (Sendai, Japan). External standards for CEA and GAPDH mRNA were prepared by 10-fold serial dilutions of cDNA synthesized by MKN45. The CEA mRNA values were adjusted against GAPDH mRNA values and are presented as relative CEA mRNA scores [(CEA mRNA / GAPDH mRNA) x 106; cutoff, 9].

Statistical analyses. Statistical analysis of group differences was done with {chi}2 test and Student's t test. The Kaplan-Meier method was used for clinical outcome and differences were estimated with the log-rank test. The prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards regression model). P < 0.05 was considered to be statistically significant. All statistical analyses were done with the software package JMP 5 for Windows software (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Detection sensitivity of CEA mRNA by real-time RT-PCR. CEA mRNA was detected in TE-2, MKN45, and MCF-7 cell lines. The lower limit of detection was a concentration of 10 tumor cells per 107 lymphocytes. The sensitivity of the RT-PCR product was confirmed by conventional nested RT-PCR. Of 106 patients with ESCC, CEA mRNA expression was detected in 67 (63.2%) patients. The mean corrected CEA mRNA score [(CEA mRNA / GAPDH mRNA) x 106] was 34.6 (range, 0-6,344.1).

Cutoff value of CEA mRNA expression in blood. CEA mRNA expression was detected in 10 of 28 (35.7%) healthy volunteers and the mean corrected CEA mRNA score was 0.2 (range, 0-1.6). In 22 patients with inflammatory bowel disease (11 Crohn's disease and 11 ulcerative colitis), CEA mRNA was detected in 5 (22.7%) patients and the mean corrected CEA mRNA score was 1.71 (range, 0-8.4). In 20 patients with benign disease who underwent laparotomy (7 cholecystectomy, 4 myoma uteri, 2 abdominal aortic aneurysm, 6 ileus, and 1 ischemic colitis), CEA mRNA was detected in 6 (30%) patients and the mean corrected CEA mRNA score was 2.15 (range, 0-8.6). Because the maximum value of CEA mRNA in patients without malignancy was 8.6, a cutoff value of 9.0 was used in the present study. Using this cutoff, 39 (36.8%) patients were diagnosed as being positive for CEA mRNA expression. Ten patients had CEA mRNA level between 9 and 20; CEA mRNA level was >20 in the remaining patients. In 39 positive CEA mRNA patients during follow-up, 26 (66.7%) patients had positive CEA mRNA value at the first visit postoperatively.

Of these 39 patients, 19 patients had normal CEA mRNA level at least once and 10 patients had recurrent disease. On the other hand, the remaining 20 patients had continuously high level CEA mRNA and 18 patients had recurrence. Of 12 patients with preoperative chemoradiation therapy, 5 of 7 patients with positive CEA mRNA during follow-up had recurrent disease. The remaining 5 patients had negative CEA mRNA and no recurrence. Preoperative chemoradiation therapy did not affect the positivity of CEA mRNA expression (P = 0.08). In this series, patients did not receive routinely adjuvant therapy after operation according to the result of CEA mRNA.

Relationship between CEA mRNA expression and clinicopathologic findings. When comparing the relationship between CEA mRNA positivity and clinicopathologic findings, CEA mRNA positivity was significantly correlated with gender, tumor depth, lymph node metastasis, distant lymph node metastasis, stage, lymphatic invasion, venous invasion, and histology (P = 0.02, P < 0.001, P < 0.001, P = 0.04, P < 0.001, P < 0.001, and P = 0.006, respectively; Table 1 ).


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  		Table 1. CEA mRNA positivity during the follow-up period and clinicopathologic findings

 
Relationship between recurrence and CEA mRNA expression, serum CEA, and SCC during the follow-up period. Recurrent disease was found in 34 of 106 (32.1%) cases. Modes of recurrence based on imaging included 7 hematogeneous, 6 simultaneous lymph node and hematogeneous, 17 lymph node, and 4 dissemination or local recurrence.

The time between the first positive finding of CEA mRNA and the detection of recurrence by imaging in the 34 patients with recurrent disease was investigated. CEA mRNA was found in 28 (82.3%) of these patients, among whom the number with positive CEA mRNA values before imaging, at the same time as imaging, and after imaging was 18 (52.9%), 8 (23.5%), and 2 (5.9%), respectively. The mean time difference between positive CEA mRNA and recurrence was 6.1 ± 8.2 months (mean ± SD) in 28 relapse of 34 patients with CEA positive result. On the other hand, the mean percentages of positive values for serum CEA and SCC before recurrence detected by imaging were 20.6% (7 patients) and 14.7% (5 patients), respectively. The mean time difference between CEA and SCC positivities and recurrence was 5.1 ± 5.8 and 2.3 ± 9.7 months, respectively. CEA mRNA values were elevated earlier before imaging-confirmed recurrence when compared with serum CEA and SCC (Table 2 ). Eleven of 13 patients with hematogeneous recurrence had positive CEA mRNA results. Recurrent disease was found in eight patients with negative CEA mRNA results. Of these eight patients, six had lymph node recurrence and the remaining two patients had lesions including hematogeneous recurrence.


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  		Table 2. Time difference between imaging diagnosis and CEA mRNA levels and serum tumor marker levels during the follow-up period in recurrent cases (n = 34)

 
CEA mRNA positivity was significantly correlated with incidence of recurrence (P < 0.001). Although serum SCC levels were significantly related to recurrence (P = 0.04), serum CEA levels were not. Sensitivity and specificity using CEA mRNA, serum CEA, and SCC levels were 76.5% and 84.7%, 35.3% and 79.2%, and 35.3% and 83.3%, respectively. In addition, the positive predictive and negative predictive values using CEA mRNA, serum CEA, and SCC levels were 70.3% and 88.4%, 44.4% and 72.2%, and 50.0% and 73.2%, respectively. Thus, sensitivity, specificity, positive predictive value, and negative predictive value for CEA mRNA were highest among the variables tested (Table 3 ).


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  		Table 3. Comparison between imaging diagnosis and CEA mRNA and serum tumor markers during the follow-up period

 
Univariate and multivariate analyses of disease-free interval. Factors related to disease-free interval were evaluated by univariate and multivariate analyses. According to univariate analysis, tumor depth, nodal metastasis, distant lymph node metastases, stage, lymphatic invasion, venous invasion, and CEA mRNA positivity during follow-up and serum SCC elevation during follow-up were significantly related to disease-free interval. Multivariate regression analysis revealed that tumor depth and CEA mRNA positivity were independent factors for disease-free interval (Table 4 ). When analyzing disease-free interval according to positivity for CEA mRNA, disease-free interval in patients positive for CEA mRNA was significantly lower than in those negative for CEA mRNA (29.7% versus 88.4%, P < 0.001; Fig. 1 ).


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  		Table 4. Univariate and multivariate analyses for disease-free interval

 

Figure 1
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  		Fig. 1. Disease-free interval according to CEA mRNA expression during the follow-up period. Disease-free interval in patients positive for CEA mRNA was significantly lower than in those negative for CEA mRNA (29.7% versus 88.4%; P < 0.001, log-rank test).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we evaluated the clinical significance of real-time RT-PCR detection of CEA mRNA expression by ITC in blood during the follow-up period after ESCC surgery. To our knowledge, this is the first longitudinal analysis of patients using CEA RT-PCR and the first inclusive analysis comparing RT-PCR values for CEA with those of serum CEA or SCC levels, which are used to monitor ESCC patients at various stages of the disease.

Previous studies have reported the relationship between postoperative ITC detection and recurrence. Patel et al. (27) showed that the clearance of ITC in blood within 24 hours of colorectal cancer excision was greatest in tumors with the best prognosis. In another report, postoperative detection of ITC at 24 hours after curative resection had no prognostic significance in colorectal cancer (28). Patients with high blood loads of ITC at 2 weeks postoperatively have poor prognosis about disease recurrence and tumor-related mortality in colorectal cancer (29). In head and neck cancer patients, detection of ITC in blood at 3 months postoperatively was not useful in predicting recurrence (30). The relationship between the presence of postoperative ITC and clinical outcome thus remains controversial. In these reports, the clinical significance of ITC in blood obtained once or twice within 3 months after surgery was discussed. We speculated that longitudinal analysis was necessary to detect ITC in blood based on relapse circumstances. Therefore, we investigated ITC in blood together with serum CEA and SCC during regular follow-up examinations at our outpatient clinic.

An important issue is the origin of ITC in blood. Fidler (31) reported that most circulating tumor cells are cleared from circulation within 24 hours of surgery. O'Sullivan et al. (32) suggested that preoperative detection of micrometastasis may reflect either transient shedding of cells, metastatic potential, or residual disease, but postoperative micrometastases are likely to indicate minimal residual disease. In the present study, we found that ITC released from metastatic tumor were detected in blood during the follow-up period in relation to recurrent growth.

In the present study, 26 of 34 (76.5%) patients with relapse had positive CEA mRNA findings in blood during the follow-up period before recurrence was confirmed by imaging. The incidence of recurrence was significantly higher in patients positive for CEA mRNA than in those negative for CEA mRNA. Interestingly, the mean period from surgery to first detection of CEA mRNA by RT-PCR was ~6 months before discovery on imaging. This suggests that ITC positivity in blood can be observed, even in occult recurrence that cannot be detected on imaging.

Serum CEA has been well documented as a tumor marker for cancers of the gastrointestinal tract (4) and serum SCC has a high sensitivity and specificity for ESCC (2, 3). In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of CEA mRNA were higher than those of serum CEA and SCC. In patients with relapse, CEA mRNA expression was found earlier than positive serum CEA and SCC values, and the incidence of discovery at the same time or before recurrence was seen on imaging was higher for CEA mRNA (76.4%) than for serum CEA (35.3%) or SCC (35.4%). Moreover, according to multivariate regression analysis, CEA mRNA positivity during follow-up was an independent factor for recurrence. At present, serum SCC, CEA, and CYFRA 21-1 are used as tumor markers in ESCC. Recurrent disease is detected by the elevation of such markers in some patients. However, it is difficult to detect early recurrence with these markers before imaging diagnosis. In this series, high CEA mRNA level was detected in about half of patients with recurrence before imaging diagnosis. Therefore, examination of CEA mRNA in blood may be a new marker for the prediction of ESCC recurrence.

Mataki et al. (33) reported the longitudinal analysis of CEA mRNA expression and two tumor markers, serum CEA and serum carbohydrate antigen 19-9, and found that CEA mRNA was expressed in two recurrent cases without tumor marker elevation among 53 biliary-pancreatic cancer patients. Similarly, Guadagni et al. (34) reported that CEA mRNA expression was detected in two relapse cases in spite of normal tumor marker values in 51 colorectal cancer patients. In our case, however, 8 of 69 (11.6%) patients that showed negative CEA mRNA expression had tumor relapse and 6 patients had lymph node recurrence. CEA mRNA negativity might be due to deficient blood flow around the lymph node, as compared with blood-borne recurrence. Among these six patients, elevation of serum CEA was found in four and elevation of SCC was found in two. Therefore, serum tumor markers in combination with CEA mRNA are useful for more precisely predicting recurrent disease. On the other hand, because patients positive for CEA mRNA and without overt recurrence on imaging seem to have a higher risk of recurrence, meticulous follow-up is essential. We should consider the adjuvant therapy for patients with positive CEA mRNA expression because of high risk of recurrence.

In conclusion, although large randomized prospective studies are required to define the role of CEA mRNA detection in blood, the studies reported here show that such methods are promising for the early detection of ITC in patients with ESCC. If occult recurrent disease is detected before imaging diagnosis during follow-up, prognosis can be improved by early treatment.


   Footnotes
 
Grant support: Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan.

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.

Received 3/15/06; revised 7/20/06; accepted 8/10/06.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
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