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Annals of Surgical Oncology 9:364-370 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Intraoperative Molecular Detection of Circulating Tumor Cells by Reverse Transcription-Polymerase Chain Reaction in Patients With Biliary-Pancreatic Cancer Is Associated With Hematogenous Metastasis

Keiichiro Uchikura, MD, Sonshin Takao, MD, Akihiro Nakajo, MD, Futoshi Miyazono, MD, Saburou Nakashima, MD, Kouki Tokuda, MD, Masataka Matsumoto, MD, Hiroyuki Shinchi, MD, Shoji Natsugoe, MD and Takashi Aikou, MD

From the First Department of Surgery, Kagoshima University School of Medicine, Kagoshima, Japan.

Correspondence: Address correspondence and reprints to: Sonshin Takao, MD, First Department of Surgery, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan; Fax: 81-99-265-7426; E-mail: sonshin{at}m2.kufm.kagoshima-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Circulating tumor cells in the blood were frequently detected by reverse transcription-polymerase chain reaction during operation in patients with biliary-pancreatic cancer. We investigated the relationship between circulating tumor cells during operation and hematogenous metastases.

Methods: Blood samples from 67 patients with biliary-pancreatic cancer were obtained from the portal vein, peripheral artery, and superior vena cava during operation. After total RNA was extracted from each blood sample, carcinoembryonic antigen (CEA)–specific reverse transcription-polymerase chain reaction was performed.

Results: Intraoperative CEA-messenger RNA (mRNA) expression was detected in the blood of 32 (47.8%) of 67 patients with biliary-pancreatic cancer, although it was not detected in the blood obtained from 20 healthy volunteers or 15 patients with benign disease of the biliary pancreas. The incidence (37.5%) of hematogenous metastases after surgery in the CEA-mRNA–positive group (n = 32) was significantly higher than that (11.4%) in the negative group (n = 35; P = .01). In stage I, II, and III patients, survival of the CEA-mRNA–positive group was significantly worse compared with that of negative group (P = .03).

Conclusions: Intraoperative molecular detection of circulating tumor cells in patients with biliary-pancreatic cancer relates to a high risk of hematogenous metastasis and is associated with unfavorable prognosis even after curative resection.

Key Words: CEA-mRNA • RT-PCR • Circulating tumor cells • Hematogenous metastasis • Liver metastasis • Biliary-pancreatic cancer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In patients with biliary-pancreatic cancer, liver metastases occur at a high frequency after surgery, and the prognosis remains very poor despite the curative operation.1–4 However, whether the operation itself triggers the hematogenous spread of tumor cells resulting in liver metastases is unclear. Reverse transcriptase-polymerase chain reaction (RT-PCR) is a powerful and highly sensitive molecular biological technique that can be used to detect micrometastases that are undetectable by histological examination in bone marrow5,6 and lymph nodes.7,8 In recent years, the dissemination of prostatic epithelial cells in patients with prostatic carcinoma9 and the dissemination of tumor cells in patients with breast cancer were detected during surgery by RT-PCR.10 By use of this technique, we have reported the intraoperative molecular detection of circulating tumor cells in patients with biliary-pancreatic cancer11 and gastric cancer.12 Whether these circulating tumor cells provoke a poor prognosis caused by the hematogenous dissemination is vital to the field of surgical oncology. The purpose of this study was to investigate the relationship between circulating tumor cells during surgery and hematogenous metastases in patients with biliary-pancreatic cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
From August 1996 to December 1999, 67 Japanese patients with biliary-pancreatic cancer underwent surgical resection of the primary tumor, and carcinoembryonic antigen (CEA)-messenger RNA (mRNA) was assayed in the blood at the First Department of Surgery, Kagoshima University School of Medicine. The patients’ ages ranged from 39 to 85 years, with a median of 65 years. According to tumor-node-metastasis criteria for cancer staging, 34 patients with pancreatic cancer (stage I, n = 9; stage II, n = 1; stage III, n = 9; stage IV, n = 15), 24 patients with biliary duct cancer (stage I, n = 5; stage II, n = 9; stage III, n = 1; stage IV, n = 9), and 9 patients with carcinoma of the papilla of Vater (stage I, n = 2; stage II, n = 4; stage III, n = 1; stage IV, n = 2) were enrolled. Ultrasonography and computed tomography were performed for all patients to examine hematogenous and distant metastases before operation. No patient had what would be considered hematogenous metastasis before operation. Consequently, curative tumor resection was performed for all patients, including those with locally resectable stage IV disease without distant metastasis. Fifteen patients with benign disease (intraductal papillary tumor, n = 4; Mirizzi syndrome, n = 1; gallbladder polyp, n = 1; gallbladder stone, n = 2; insulinoma, n = 1; solid and cystic tumor, n = 1; cyst adenoma, n = 2; anomalous arrangement of pancreaticobiliary ductal system, n = 1; intrahepatic stone, n = 1; abdominal aortic aneurysm, n = 1) were also studied.

Blood Samples
Before operation, 5 mL of blood was collected in ethylenediaminetetraacetic acid (EDTA) from a peripheral vein after discarding the first 10 mL of blood to prevent contamination with epithelial cells. Each 5 mL of blood was collected in EDTA from three sites—a peripheral artery, the superior vena cava (SVC), and the portal vein—at the beginning of the operation and at tumor resection. Blood samples from the portal vein and the SVC were obtained from a catheter replaced in the portal vein via the umbilical vein and from a catheter for monitoring the central venous pressure, respectively. Control blood samples from 15 healthy volunteers were obtained from a peripheral vein after the first 10 mL of blood was discarded. The fraction of nucleated cells was obtained by centrifugation with Mono-poly resolving medium (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan). Blood samples were diluted by adding 5 mL of .05 M phosphate-buffered saline. Diluted samples were layered on 5 mL of Mono-poly resolving medium. Each sample was centrifuged at 420 x g at room temperature for 20 minutes. The cell layer was collected into 40 mL of phosphate-buffered saline. The fraction of nucleated cells was collected after centrifugation at 1350 x g at 4°C for 15 minutes. The fraction of nucleated cells was suspended in 1 mL of Isogen (Nippon Gene, Toyama, Japan) and stored at -80°C until use.

RNA Extraction
Total RNA was extracted by using Isogen. The method of RNA extraction conformed to the manufacturer’s instructions. Total RNA was dissolved in 10 µL of diethylpyrocarbonate-treated water. The volume and quality of total RNA obtained were checked by absorption measurements at optical density 260 and 280 nm with a BioSpec-1600 ultraviolet-visible spectrophotometer (Shimazu Co., Kyoto, Japan).13

Reverse Transcriptase-Polymerase Chain Reaction
Before the synthesis of complementary DNA (cDNA), deoxyribonuclease I (Stratagene, La Jolla, CA) was added to the total RNA. One-half unit of deoxyribonuclease I and 1 µL of 10x PCR buffer (Takara Shuzo Co., Ltd., Otsu, Japan) were added to 5 µg of total RNA to yield a total volume of 9.5 µL. The reaction solution was incubated for 60 minutes at 37°C. Then 1.5 µL of 20 mM EDTA was added, and 11 mL of reaction solution was incubated for 20 minutes at 70°C. After 50 ng of random hexamer was added, the 5 µg of total RNA in a volume of 12 µL was incubated for 10 minutes at 72°C. One microliter of 10x PCR buffer, 2 µL of 25 mM MgCl2, 1 µL of .1 M dithiothreitol, 1 µL of 10 mM deoxynucleotide triphosphate (dNTP) mix, and 2 µL of diethylpyrocarbonate-treated water were added to the reaction solution. Nineteen microliters of reaction mix was incubated for 10 minutes at 25°C, and then 100 U of Super Script II RT was added. The reaction solution was incubated for 15 minutes at 25°C, 90 minutes at 42°C, and 15 minutes at 70°C and was quickly chilled on ice. One unit of Escherichia coli ribonuclease H (all from Life Technologies, Rockville, MD) was added to the reaction solution. A 20-µL sample of reaction mixture was incubated for 40 minutes at 37°C. Five microliters of Tris-EDTA was added to the cDNA, which was stored at - 20°C until use.

To monitor the synthesis of cDNA, glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) RT-PCR was performed by using the published GAPDH primer. The GAPDH forward primer was 5'-TCCCATCACCATCTTCCA-3', and the GAPDH reverse primer was 5'-CATCACGCCACAGTTTCC-3'. Fifty microliters of the PCR mixture contained .5 µg of cDNA, 5 µL of 10x PCR buffer, .2 µM of dNTP mixture, .2 µM of forward primer, .2 µM of reverse primer, and 1.25 U of Taq DNA polymerase (all from Takara Shuzo Co., Ltd.). Denaturation for 4 minutes at 95°C, amplification with denaturation for 1 minute at 95°C, annealing for 1 minute at 57°C, and extension for 45 seconds at 72°C for 40 cycles were performed, followed by a final extension for 9 minutes at 72°C. The GAPDH RT-PCR product was separated by 2% agarose gel electrophoresis in Tris-acetate-EDTA buffer and visualized by ethidium-bromide staining. The RT-PCR product was detected as a 390-base pair (bp) fragment.

To detect the more specific CEA-mRNA, we performed nested RT-PCR. We used three types of CEA primers designed by Gerhard et al.14 The CEA-A primer was 5' -TCTGGAACTTCTCCTGGTCTCTCAGCTGG-3', the CEA-B primer was 5' -TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC-3', and the CEA-C primer was 5' -GGGCCACTGTCGGCATCATGATTGG-3'. In the first RT-PCR, 20 µL of reaction mix contained .5 µL of cDNA, 2 µL of 10x PCR buffer, .2 mM of dNTP mixture, .2 µM of CEA primers A and B, and .5 U of Taq DNA polymerase (all from Takara Shuzo Co., Ltd.). Denaturation for 4 minutes at 95°C, amplification with denaturation for 30 seconds at 95°C, annealing for 1 minute at 69°C, and extension for 30 seconds at 72°C for 30 cycles were performed, followed by a final extension for 6 minutes at 72°C. In nested RT-PCR, 40 µL of reaction mix contained 1 µL of the first PCR product, 4 µL of 10x PCR buffer, .2 mM of dNTP mixture, .2 µM of CEA primers B and C, and 1 U of Taq DNA polymerase (all Takara Shuzo Co., Ltd.). Denaturation for 4 minutes at 95°C, amplification with denaturation for 30 seconds at 95°C, annealing for 1 minute at 64°C, and extension for 30 seconds at 72°C for 32 cycles were performed, followed by a final extension for 6 minutes at 72°C. The CEA RT-PCR product was separated by 2% agarose gel electrophoresis in Tris-acetate-EDTA buffer and visualized by ethidium-bromide staining. The RT-PCR product was detected as a 131-bp fragment.

Setting for CEA-Specific RT-PCR
We previously reported our setting for CEA-specific RT-PCR.11 Briefly, MCF-7 cells, a breast carcinoma cell line, were used to test the sensitivity of CEA-specific nested RT-PCR. Diluted numbers of MCF-7 cells (105, 104, 103, 102, 10, and 1) were added to separate aliquots of the 5-mL blood samples obtained from a healthy volunteer. In this series, 10 cells of MCF-7 in 1 x 106 normal lymphocytes were detected as the level of the lower limit of CEA-mRNA positivity (Fig. 1).



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  		FIG. 1. Products of reverse transcriptase-polymerase chain reaction for carcinoembryonic antigen (CEA)-messenger RNA (mRNA) separated by electrophoresis and stained with ethidium bromide in 8 samples containing 106, 105, 104, 103, 102, 10, 1, and no cancer cells of the MCF-7 cell line in 5-mL aliquots of blood obtained from healthy volunteers. CEA-mRNA of 10 or more cancer cells in 5 mL of blood could be detected. Glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) served as an internal control. M, molecular weight markers; P, positive control of RNA extracted from MCF-7 cells; N, negative control.

 
Clinical Follow-Up
Postsurgical follow-up data were obtained from all patients after discharge. Follow-up examinations included physical examination, routine blood tests, serum tumor marker tests, and x-ray examinations every 1 to 3 months, as well as computed tomography, magnetic resonance imaging, and ultrasonography every 3 to 6 months. All patients were observed for >2 years after surgery.

Statistical Analysis
Statistical analyses were performed with the {chi} 2 test. Differences with P values <.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In healthy volunteers and patients with benign disease, CEA-mRNA was not detected before or during operation. In the patients with biliary-pancreatic cancer, however, 32 (47.8%) of 67 patients tested positive for CEA-mRNA during operation, although no patient was positive for CEA-mRNA before operation (Fig. 2). Of these 32 patients, 6 were positive for CEA-mRNA at the beginning of the operation, whereas 28 patients were positive for CEA-mRNA in the blood obtained from the portal vein, SVC, or peripheral artery at the time of tumor resection. The detection rate of circulating tumor cells at the time of tumor resection was significantly more than that at the beginning of the operation. However, detection rates among the portal vein, SVC, and peripheral artery were not significantly different (Table 1). Surgical procedures, tumor staging, lymph node metastasis, lymphatic invasion, and venous invasion were not significantly different between CEA-mRNA–positive and –negative groups (Table 2).



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  		FIG. 2. Products of reverse transcriptase-polymerase chain reaction (RT-PCR) for carcinoembryonic antigen (CEA)-messenger RNA separated by electrophoresis and stained with ethidium bromide in 7 samples obtained from a patient with pancreatic cancer. Lane 1, peripheral vein sample before operation; lane 2, portal vein sample at the beginning of the operation; lane 3, portal vein sample during tumor resection; lane 4, peripheral artery sample at the beginning of the operation; lane 5, peripheral artery sample during tumor resection; lane 6, superior vena cava (SVC) sample at the beginning of the operation; lane 7, SVC sample during tumor resection; M, molecular weight markers; P, positive control of RNA extracted from MCF-7 cells; N, negative control. The samples of lanes 3, 5, and 7 show positive CEA expression by RT-PCR. Glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) was an internal control.

 

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  		TABLE 1. Intraoperative detection of CEA-mRNA in the blood obtained from three different vessels in 67 patients who underwent curative resection for biliary-pancreatic cancer
 

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  		TABLE 2. Relationships between intraoperative detection of CEA-mRNA in the blood and stage, operation procedures, and clinicopathological findings
 
Twelve (37.5%) of 32 patients with positive CEA-mRNA during operation had recurrences of hematogenous metastases after curative surgery. This recurrence rate was significantly higher than that (11.4%) of 35 patients with negative CEA-mRNA (P = .01), although neither local nor peritoneal recurrence rates were significantly different between CEA-mRNA–positive and –negative groups (Table 3). Of 12 patients with hematogenous recurrence in the CEA-mRNA–positive group, the primary tumor sites were 7 of the pancreas, 4 of the bile duct, and 1 of the papilla of Vater. According to cancer staging, there was one stage II patient, and there were four stage III and seven stage IV patients. Organs that had hematogenous metastases were 10 of the liver and 1 each of the bone and the lung.


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  		TABLE 3. Comparison of recurrence rates between CEA-mRNA–positive and –negative groups in 67 patients who underwent curative resection for biliary-pancreatic cancer according to recurrence type
 
Overall survival curves between CEA-mRNA–positive and – negative groups were not significantly different (Fig. 3A). In stage I to III patients, however, the actual survival curve of the CEA-mRNA–negative group was significantly better than that of the positive group (P = .03; Fig. 3B). In these patients with stage I to III disease, the hematogenous recurrence rate (29.4%) in the CEA-mRNA–positive group (n = 17) was significantly higher than that (4.2%) in the CEA-mRNA–negative group (n = 24; P = .024). Neither the local nor peritoneal recurrence rate, however, was significantly different between the CEA-mRNA–positive and –negative groups (Table 4).



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  		FIG. 3. The comparison of actual survival curves between carcinoembryonic antigen (CEA)-messenger RNA (mRNA)–positive and –negative groups. (A) Overall survival curves between CEA-mRNA–positive (n = 32) and –negative (n = 35) groups were not significantly different. (B) In patients with stage I to III disease, the actual survival curve of the CEA-mRNA– positive group (n = 17) was significantly worse than that of the negative group (n = 24; P = .03). N.S., not significant.

 

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  		TABLE 4. Comparison of recurrence rates between CEA-mRNA–positive and –negative groups in 41 patients with stage I to III disease who underwent curative resection for biliary-pancreatic cancer according to recurrence type
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Liver metastasis is the main mode of recurrence after curative resection in patients with biliary-pancreatic cancer. Consequently, the prognosis of the patients who undergo curative resection for biliary-pancreatic cancer is still poor.2,4 Whether or not the operation itself triggers the hematogenous spread of tumor cells resulting in liver metastases is an important issue for surgical oncology. Evidence from animal studies has shown that malignant cells are shed into the bloodstream during surgical manipulation of a primary tumor,15,16 leading to an increased incidence of distant metastases.17,18 We recently reported that tumor resection or manipulation in biliary-pancreatic surgery was strongly related to the increase of circulating tumor cells in the blood11 and that the presence of circulating tumor cells had a high risk of hematogenous metastasis in patients with gastric cancer.12

In recent years, circulating tumor cells in the blood during surgery have been detected by the PCR method in patients with prostatic carcinoma, 9 breast cancer,10,19 gastrointestinal cancer, 20 pancreatic cancer,21 and hepatocellular carcinoma.22 Nomoto et al.21 reported that stress (such as anesthesia or laparotomy) and manipulation increase the dissemination of tumor cells with K-ras gene point mutations in the blood during operation. Gion et al.23 also reported the same results by using quantitative albumin-specific RT-PCR in patients with hepatocellular carcinoma. In this study, we chose CEA-mRNA for the detection of the circulating tumor cells because CEA-mRNA is present in all epithelial cells, including carcinoma cells.14 A CEA-mRNA–positive result means that viable tumor cells released or detached from the primary tumor beds were detected in the circulatory system.11,12

Our results indicate that surgical maneuvers are a possible cause of hematogenous metastasis because of promoting the detachment of tumor cells and increasing the incidence of circulating tumor cells in the blood during operation. Whether these findings affect the incidence of hematogenous metastasis is important for predicting outcome. Micrometastases in the lymph nodes and the bone marrow or disseminated carcinoma cells in the peritoneal cavity with use of the PCR method are significantly associated with survival of patients with gastrointestinal cancer.24–28 In this study, CEA-mRNA in the blood was detected after general anesthesia or just after laparotomy in a few patients, although it was not detected on the day before operation. Then, the detection rate of CEA-mRNA in the blood rapidly increased during surgical procedures for tumor resection. Thus, the molecular detection of circulating tumor cells during surgery seems to be strongly related to surgical maneuvers. Detection of CEA-mRNA, however, did not relate to the sort of surgical procedure, tumor staging, lymph node metastasis, lymphatic invasion, or even venous invasion. The presence of circulating cancer cells during surgery might play an important role in hematogenous metastasis and might be an independent factor for predicting outcome. Thus, surgery for biliary-pancreatic cancer not only offers a possibility for curative treatment, but unfortunately may also induce the detachment of cancer cells from the primary tumor beds.

The clinical significance of detection of circulating tumor cells in the blood during operation depends on whether or not circulating tumor cells relate to hematogenous metastasis and survival. We recently reported that the presence of circulating tumor cells caused by surgical maneuvers had a high risk of hematogenous metastasis even after curative resection in patients with gastric cancer.12 In this study, the CEA-mRNA–positive group had a higher recurrence rate and showed a worse prognosis in stage I to III patients compared with the negative group. The reason is that the incidence of hematogenous metastasis in the positive group was higher than that in the negative group, although the incidence of other recurrence types between the two groups was similar. We previously reported that the poor prognoses were associated with liver metastases, but not local recurrence, in the patients with biliary cancer who underwent curative resection.2 Thus, hematogenous metastasis was strongly associated with the prognosis of patients with biliary-pancreatic cancer who underwent curative resection.

However, 13 patients (40.6%) in the CEA-mRNA–positive group have no recurrence yet. This result suggests that hematogenous metastasis may depend not only on the hematogenous spread of tumor cells, but also on the biological behavior of circulating tumor cells, including adhesive, angiogenic, and invasive functions.29–31

In conclusion, our results suggest that the molecular detection of circulating tumor cells during operation is associated with poor prognosis, depending on hematogenous metastasis. Therefore, new strategies implemented during the operation should be considered to prevent hematogenous metastases and to improve the survival of patients with biliary-pancreatic cancer.


   Acknowledgments
 
Supported in part by grants-in-aid for scientific and cancer research from the Ministry of Science and Culture, Japan.

Received for publication May 22, 2001. Accepted for publication January 25, 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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