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Annals of Surgical Oncology 8:407-412 (2001)
© 2001 Society of Surgical Oncology

ORIGINAL ARTICLES

Clinical Significance of K-Ras Mutations in Intraoperative Tumor Drainage Blood From Patients With Colorectal Carcinoma

Tsuyoshi Etoh, MD, Hiroaki Ueo, MD, Hiroshi Inoue, MD, Koichi Sato, MD, Tohru Utsunomiya, MD, Graham F. Barnard, MD, Seigo Kitano, MD and Masaki Mori, MD

From the Department of Surgery (TE, HI, KS, TU, MM), Medical Institute of Bioregulation, Kyushu University, Beppu, Japan; Department of Surgery (HU,), Oita Prefectual Hospital, Oita, Japan; Division of Digestive Disease and Nutrition (GFB), University of Massachusetts Medical School, Worcester, Massachusetts; and Department of Surgery I (TE, SK), Faculty of Medicine, Oita Medical University, Oita, Japan.

Correspondence: Address correspondence and reprint requests to: Masaki Mori, MD, Department of Surgery, Medical Institute of Bioregulation, Kyushu University 4546, Tsurumihara, Beppu 874-0838, Japan; Fax: 81-977-27-1651; E-mail: mmori{at}tsurumi.beppu.kyushu-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Recurrent and metastatic carcinoma of the colorectum remains a major problem. This may be ascribed to the presence of micrometastasis at diagnosis. The purpose of this study was to analyze prospectively the clinical value of detecting K-ras mutations in the perioperative circulating blood from patients with colorectal carcinoma.

Methods: Twenty-four patients whose tumor carried mutations in codon 12 of the K-ras gene were studied for the presence of cancer cells in perioperative blood samples, in particular, tumor drainage samples. A detection assay using CD45 immunomagnetic separation plus nested mutant allele specific amplification (MASA) was performed.

Results: K-ras mutations in CD45 negative cells in tumor drainage blood were detected in 7 (29.2%) of 24 patients. There was no significant relationship between the presence of a K-ras mutation and clinicopathological features. Four (57.1%) of the seven patients with a positive K-ras mutation in drainage blood had early recurrent disease. Of the 17 patients with no K-ras mutation, none developed metastatic disease. The recurrence rate of the K-ras mutation positive group was higher than that of the K-ras mutation negative group (P < .01). There was a significant difference, regarding prognosis, between K-ras mutation positive and negative groups (P < .01).

Conclusions: This preliminary study demonstrates that the detection of circulating cancer cells in the tumor drainage blood by our new assay system may provide a predictor of recurrence and metastasis of colorectal cancer.

Key Words: K-ras mutation • MACS system • Prognosis • Drainage blood • Colorectal carcinoma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Approximately 30%–50% of patients with colorectal cancer who initially present with resectable tumors subsequently develop metastatic disease.1,2 Early dissemination of cancer cells is thought to be one of the leading causes of relapse at distant sites and of death from cancer.3–5 To detect disseminated cancer cells, several approaches have been used. Using conventional cytological and immunohistochemical examination of blood samples from patients with colorectal carcinoma, the sensitivity and specificity for cancer cell detection seems to be low.6,7 Polymerase chain reaction (PCR)-based protocol has improved the sensitivity of detection, allowing the identification of approximately 1 to 10 malignant cells in a background of 107 normal peripheral blood mononuclear cells.8 Transcripts of cytokeratin (CK) or carcinoembryonic antigen (CEA) have been used as effective target genes for disseminated colorectal cancer cells.4,9 However, these genes are not specific to cancer cells and can also be detected in noncancerous epithelial cells. Therefore, false positive results have been troublesome in practical use.10,11

Recently, we have focused on the detection of K-ras gene mutations that might be more specific to cancer cells.12 Several studies using genetic analysis of adenocarcinomas have shown frequent DNA mutations of the K-ras gene in codons 12, 13, or 61. In particular, a K-ras gene mutation in codon 12 is found in 50%–60% of colorectal adenocarcinomas. These mutations have been detected in metastatic tissues of colorectal cancer such as lymph nodes,13,14 peripheral blood including plasma or serum,15,16 ascites,17 and feces.18 To increase the detection rate of K-ras mutations in a background of normal blood cells, we have established a detection assay system using CD45 immunomagnetic separation plus nested mutant allele specific amplification (MASA)-PCR.12 In this study, we tried to separate and enrich circulating cancer cells in the blood samples by this assay system and to estimate its clinical utility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
A total of 24 subjects with colorectal carcinoma were recruited for this study. The patients’ primary tumor had been confirmed (by means of MASA-PCR) to have a K-ras mutation in codon 12. The patients’ median age was 60 years (range, 45–88), with 12 men and 12 women. They had undergone surgery at the Department of Surgery, Medical Institute of Bioregulation, Kyushu University (Beppu, Japan) between 1998 and 2000. All patients had surgery with a no-touch isolation technique according to the guidelines of Turnbull et al.19 Twenty-one patients had undergone curative surgery; three patients had undergone noncurative surgery due to multiple liver metastasis (n = 1) and para-aortic lymph node metastases (n = 2). None of these patients received preoperative treatment, such as radiation and/or chemotherapy. The histopathological type and staging of colorectal carcinoma were classified based on the TNM (tumor, node, metastasis) classification.

Blood Samples
In this study, we focused on the drainage vein, which was defined as the closest mesenteric vein to the primary tumor. Each heparin-treated blood sample (10 ml) was obtained from the peripheral blood before laparotomy, within 1 hour after operation, and from the drainage vein just before tumor resection during the operation. Each collected sample was diluted with 10 ml of PBS (phosphate-buffered saline) and laid on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). Peripheral blood mononuclear cells (PBMC) in the buffy coat layer, separated by centrifugation at 2500 for 25 minutes at room temperature, were incubated with CD45 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, the cells were separated using the magnetic-activated cell separation (MACS) system (Miltenyi Biotec). As a result, almost all of the CD45 negative fractioned cells were composed of non-leukocytes such as epithelial cells including cancer cells.

DNA Extraction and Detection of K-Ras Mutations
DNA was extracted with a QIAamp Blood kit (Qiagen GmbH, Hilden, Germany) by the MACS system from CD45 negative cells obtained from blood samples. Furthermore, DNA was extracted from the primary tumor of these patients; genetic alternations in codon 12 of the K-ras gene were examined by the modified nested MASA-PCR as described previously.12

Clinical Follow-Up
Data concerning the patient outcome, including overall survival and development of metastases, were available for all 24 patients. The observation period after the operation ranged from 4 to 26 months (median follow-up period, 15.4 months). All patients were seen every 3 to 4 months postoperatively. A clinical history, physical examination, blood cell count, routine blood work, and serum tumor marker tests were performed during each visit. An ultrasound examination and a chest X-ray were performed every 6 months. If indicated, computed tomography or magnetic resonance imaging was also performed.

Statistical Analysis
Associations among the variables were tested by the Fisher’s exact probability test. The Kaplan-Meier survival curves were constructed and analyzed by the log-rank test. All statistical differences were deemed significant at P < .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of K-Ras Mutations in the Perioperative Blood Samples
The results from all 24 patients of the determination of K-ras mutations in the perioperative blood samples are summarized in Table 1. There was no detectable mutation in the CD45 negative cells from the peripheral blood samples before operation. Regarding the drainage blood samples during the operation, seven cases (29.2%) had codon 12 K-ras mutations in the CD45 negative cells. Of the seven patients with a K-ras mutation in the operative drainage blood sample, two were stage II, two were stage III, and three were stage IV. Only two cases (8.3%) had codon 12 K-ras mutations in the CD45 negative cells from the peripheral blood samples after operation. These two cases showed the same mutations in both samples obtained during and after the operation. Representative cases are shown in Fig. 1. In all cases that had K-ras mutations in the blood samples, the same mutation was seen in the primary tumors (data not shown).


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  		TABLE 1. Summary of clinical findings in 24 patients with a codon 12 K-ras mutation in colorectal carcinoma
 


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  		FIG. 1. Representative cases with the presence of a K-ras codon 12 mutation in the perioperative blood samples. DNA from the blood samples was obtained by the immunomagnetic separation technique and was amplified by the nested MASA-PCR method. Case 4 had amplification of an AGT (Ser) mutation in both venous drainage and peripheral blood. Case 10 had amplification of a GAT (Asp) mutation in only venous drainage blood. Case 21 had no K-ras mutation in either venous drainage or peripheral blood. (M, marker; PC, positive control.)

 
Relationship Between the Presence of K-Ras Mutations in the Drainage Blood Samples and Clinicopathological Variables
We compared the K-ras mutation positive (n = 7) and negative (n = 17) cases in the drainage blood samples relating to clinicopathological variables. There were no significant differences in variables such as age, sex, tumor localization, tumor size, depth of invasion, node status, distant metastasis at surgery, TNM stage, and resectability between the two groups (Table 2).


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  		TABLE 2. Association between codon 12 K-ras gene mutation in tumor drainage blood and clinicopathological features in colorectal carcinoma
 
Relationship Between the Presence of a K-Ras Mutation and Clinical Outcome
The clinical outcome for all 24 patients is summarized in Table 1. Four (57.1%) of the seven patients with a positive K-ras mutation result in the blood samples had a tumor recurrence after the operation. Of these four patients, one had multiple lung metastases and another one had multiple liver metastases; both died from these diseases within 15 and 9 months after the operation, respectively. The other two had peritoneal dissemination after a noncurative operation due to para-aortic lymph node metastasis; they died within 8 and 4 months, respectively. On the other hand, none of the 17 patients without a K-ras mutation in the blood samples recurred postoperatively. The recurrence rate for the patients with K-ras mutation in the blood samples was higher than those without K-ras mutation (P < .01). There was a significant difference, regarding prognosis, between the two groups (P < .01) (Fig. 2).



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  		FIG. 2. Survival curves of 24 patients with colorectal cancer according to the presence of a K-ras codon 12 mutation in the venous drainage blood samples. Patients with a K-ras mutation exhibit a significantly poorer prognosis than those with no K-ras mutation (P < .01).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several reports have demonstrated the detection of gene alternations such as K-ras mutation or p53 mutation,13,16,20 transcripts such as CEA or CK mRNA (messenger RNA)21,22 using blood samples from patients with colorectal carcinoma. In this study, we selected K-ras mutation as a target for circulating cancer cells due to higher specificity for cancer cells, compared with CEA or CK mRNA. Regarding the DNA-based PCR method, MASA is a tumor-specific DNA amplification technique because of the use of mutant specific primers. This method’s usefulness is restricted to tumors with specific genetic mutations. The upper limit of its detection is reported to be 1/500 to 1/105; most investigators report it to be 1/104.23,24 For example, Hayashi et al.25 reported that MASA could detect a few tumor cells with K-ras or p53 mutations in a background of thousands of normal cells. However, this upper limit of sensitivity seems less able to detect circulating tumor cells in blood samples. Indeed, to detect one tumor cell in 1 ml of blood, a sensitivity of 1/106 is required. To overcome this problem, we used a CD45 immunomagnetic separation system to enrich tumor cells from blood samples and then performed the nested MASA-PCR. As a result, the combination of these methods enabled us to detect one tumor cell in one million normal cells.

To avoid dissemination of cancer cells into the drainage blood during operation, a no-touch isolation technique with initial vascular ligation for the resection of colorectal carcinoma was performed. Because it is thought that cancer cells released from tumor tissues are disseminated into portal blood, we focused not only on peripheral blood, but also on the tumor drainage blood from the colorectal carcinoma. Relating to mesenteric venous blood, Fujita et al.26 have reported the circulating cancer cells detection rate of 4%. Hayashi et al.25 have reported that no-touch isolation surgery is able to reduce the frequency (to 0%) of circulating cancer cells spread into portal blood. On the other hand, Sales et al.27 have reported that they looked for cell dissemination in mesenteric blood during no-touch isolation surgery and showed such spillage in 12.5% of patients with colorectal cancer. These reports indicate that the clinical value of the determination of K-ras mutation in blood samples has not been determined. In our study, the frequency (29.2%) of circulating cancer cells in the drainage blood was higher than that of previous reports even though only a single operative blood sample was obtained. One of the probable reasons is that our immunomagnetic separation technique increases the sensitivity of cancer cell detection compared to conventional methods.

Despite the detection of micro-shedding cancer cells into blood, using molecular analysis such as PCR-based methods, the exact relationship between these cells and the development of recurrent disease in colorectal cancer is not yet understood.28 It is not clear whether the detected cancer cells may or may not be responsible for the development of recurrent disease. Several studies demonstrated that tumor cells in the mesenteric venous blood are significantly associated with tumor stage23,29 and synchronous liver metastasis.30 On the other hand, our study demonstrated that the presence of a K-ras mutation in the drainage blood was not significantly associated with clinicopathological features including depth of invasion, node status, distant metastasis at surgery, and TNM stage. In this study, there was an interesting finding regarding recurrence and prognosis of patients with a K-ras mutation in the drainage blood. Two patients with TNM stage II had a K-ras mutation in the drainage blood, suggesting that the cancer cells are able to disseminate into the blood even at an early stage. Furthermore, patients with a K-ras mutation in the drainage blood showed an early relapse of colorectal carcinoma compared with those without a K-ras mutation. During the observation period (which ranged from 4 to 26 months), there was a significant relationship between the presence of K-ras mutation and survival after the operation. These results suggest that the detection of the cells in tumor drainage blood is a sensitive prognostic marker for relapse of the disease.

In conclusion, our immunomagnetic separation techniques plus nested MASA-PCR increased the sensitivity of cancer cell detection in blood samples. The detection of micro-shedding of cancer cells in tumor drainage blood during the operation may be valuable as a predictor of prognosis in colorectal carcinoma.


   Acknowledgments
 
The authors thank Ms. J. Miyake, Mrs. Ogata, and Mr. K Sato for excellent technical assistance.

Received for publication October 30, 2000. Accepted for publication December 20, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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