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Original Article |

Prediction of Distant Metastasis by Using Reverse Transcriptase–Polymerase Chain Reaction for Epithelial and Variant CD44 mRNA in the Peripheral Blood of Patients With Colorectal Cancer FREE

Shozo Yokoyama, MD; Hiroki Yamaue, MD
[+] Author Affiliations

From the Second Department of Surgery, Wakayama Medical University School of Medicine, Wakayama, Japan.


Arch Surg. 2002;137(9):1069-1073. doi:10.1001/archsurg.137.9.1069.
Text Size: A A A
Published online

Background  Reverse transcriptase–polymerase chain reaction (RT-PCR) has been used to identify small numbers of tumor cells. Molecular detection is thought to provide useful information for the clinical management of postoperative adjuvant therapy regimens.

Objective  To use RT-PCR to identify messenger RNA (mRNA) coding for carcinoembryonic antigen, epithelial and variant CD44, and matrix metalloproteinase 7 in the portal venous and peripheral blood of patients with colorectal carcinoma to predict live or distant metastasis.

Design  Prospective consecutive series.

Setting  University hospital.

Patients and Methods  Portal venous and peripheral blood samples were obtained from 22 patients with colorectal cancer during surgical manipulation. Using complementary DNA primers specific for carcinoembryonic antigen, CD44, and matrix metalloproteinase 7, RT-PCR was performed to detect tumor cells.

Main Outcome Measure  The clinical significance of RT-PCR for epithelial and variant CD44 mRNA in peripheral blood.

Results  During 3 years of follow-up, 2 patients whose peripheral blood had carcinoembryonic antigen and CD44 variant mRNA also had distant metastases (lung or spleen). Expression of epithelial and variant CD44 mRNA in peripheral blood was more highly correlated with the clinical cancer stage than with expression of carcinoembryonic antigen and matrix metalloproteinase 7.

Conclusions  Molecular detection of epithelial and variant CD44 mRNA in the peripheral blood may help determine distant metastases in patients with colorectal carcinoma. Molecular detection in the peripheral blood at surgical treatment suggests that systemic hematogenic tumor cell dissemination is an early event of distant metastasis.

Figures in this Article

OF PATIENTS with colorectal cancer who have curative surgical treatment, 44% develop metastatic disease.1 The sites of metastases are the lymph nodes, peritoneum, liver, lungs, and bone marrow. Many methods have been established to increase the sensitivity of detection of these early metastases. Cytologic and immunochemical methods have been used to detect colorectal cancer cells in peripheral and mesenteric venous blood–draining tumors.2,3 However, the prognostic and clinical value of this detection is not clear. Technical advances have also made it possible to detect micrometastases at the molecular level in circulating blood, and recent studies have reported and discussed the clinical significance of such detection.4,5

In the present study, we detected the expression of carcinoembryonic antigen (CEA) messenger RNA (mRNA), matrix metalloproteinese 7 (MMP-7) mRNA, and epithelial and variant CD44 mRNA in circulating-blood specimens from patients with colorectal carcinoma. The presence of cancer cells in the blood does not prove that there has been metastasis to distant organs. It seems to be necessary for cancer cells to acquire certain properties for metastasis. Thus, one should consider which tumor marker genes are related to the formation of metastatic foci in distant organs, including the liver, lungs, and spleen. In this study, we selected CD44 variants and the MMP-7 gene as markers. The CD44 variant exons and MMP-7 are frequently overexpressed in human colorectal carcinoma, and many studies have suggested that CD44 variants and MMP-7 are associated with metastases and invasion,614 with CD44 variants related to the properties of adhesion and MMP-7 related to tumor invasion. On the basis of our results, we discuss the clinical value of detecting epithelial and variant CD44 and MMP-7 mRNA in the circulating blood.

PATIENTS

Twenty-two patients with colorectal cancer were enrolled in this study. The locations of the tumor were the ascending colon (n = 4), the transverse colon (n = 4), the descending colon (n = 2), the sigmoid colon (n = 6), and the rectum (n = 6). All patients received surgical treatment in which the tumors were resected completely. The clinical stages according to TNM staging were stage I (n = 4), stage II (n = 8), stage III (n = 8), and stage IV (n = 2). None of the patients had received any previous treatment, including anticancer chemotherapy or radiation therapy. Thirteen patients had well-differentiated adenocarcinoma, 6 patients had moderately differentiated adenocarcinoma, and 3 patients showed other histologic types (2 with mucinous adenocarcinoma and 1 with poorly differentiated adenocarcinoma). Informed consent was obtained from all patients in accordance with the guidelines of the Ethics Committee on Human Research, Wakayama Medical University, Wakayama, Japan.

EXTRACTION OF RNA

Blood samples from the portal and peripheral veins (10 mL) and tumor tissue (100 mg) were obtained from each patient during surgical treatment. The mononucleated cell fraction was isolated by centrifugation on a Ficoll-Hypaque gradient at 400g for 30 minutes. Total RNA was isolated from mononuclear cells and tumor tissues by guanidinium thiocyanate extraction, using the method described by Chomczynski and Sacchi.15

REVERSE TRANSCRIPTASE–POLYMERASE CHAIN REACTION (RT-PCR)

The oligonucleotide primers specific for CEA, CD44, and MMP-7 mRNA were synthesized according to the sequences described by Gerhard et al16 and Ichikawa et al.17 To detect CEA expression, the primers used for the first polymerase chain reaction (PCR) were sense, 5′-TCTGGAACTTCTCCTGGTCTCTCAGCTGG-3′, and antisense, 5′-GGGCCACTGCTGGCATCATGATTGG-3′. The heminested sense primer for CEA expression was 5′-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC 3′, and the antisense primer was the same as that used for the first PCR. The CD44 primers were sense, 5′-TCCCAGACGAAGACAGTCCCTGGAT-3′, and antisense, 5′-CACTGGGGTGGAATGTGTCTTGGTC-3′. The MMP-7 primers were sense, 5′-TCTTTGGCCTACCTATAACTGG-3′, and antisense, 5′-CTAGACTGCTACCATCCGTC-3′. Efficient amplification of the RNA samples was confirmed by an internal control by amplification of β-actin mRNA. The primers used for β-actin were sense, 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′, and antisense, 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′.

Complementary DNA was synthesized from 2 µg of total RNA with a Ready-to-Go T-Primed First-Strand Kit (Amersham Biosciences, Piscataway, NJ) following the manufacturers' instructions. For the first PCR, 50 µL of a solution containing buffer (Takara Bioch Inc, Shiga, Japan), primers (0.4 µmol/L each), 1 µL of complementary DNA, 2.5 U of Taq polymerase, and 200µM of deoxynucleoside triphosphate was added to each tube. Twenty amplification cycles were performed at 95°C (1 minute), 55°C for CEA, 67°C for CD44, or 63°C for MMP-7 (1 minute), and 72°C (1 minute), with a final extension step for 10 minutes. Then, 1 µL of the reaction mixture was transferred into a second tube with 50 µL of a solution containing buffer, primers (CD44 and MMP-7 primers were the same as for the first PCR; 0.4µM each), 2.5 U of Taq polymerase, and 200µM of deoxynucleoside triphosphate. Thirty amplification cycles were performed at 95°C (1 minute), 67°C for CEA, 55°C for CD44, or 55°C for MMP-7 (1 minute), and 72°C (1 minute), with a final extension step for 10 minutes. We repeated the sensitivity tests and determined the adequate PCR cycle. The most appropriate cycles are 20 for the first PCR and 30 for the second PCR. The PCR products were then electrophoresed on 2% agarose gels containing 1 µg/mL of ethidium bromide and visualized with ultraviolet light. The PCR products were identified by direct sequencing.

STATISTICAL ANALYSIS

The significance of differences was determined by the Fisher exact test. P<.05 was considered statistically significant.

To determine the sensitivity of reverse transcriptase–PCR (RT-PCR) for CEA, CD44, and MMP-7, triplicate reconstitution experiments were performed, in which serial 10-fold dilutions of HT29 cells, a human colon cancer cell line, were mixed with 1 × 107 mononucleated cells from a healthy volunteer. In the preliminary study, even 1 tumor cell in 1 × 107 normal cells could be detected by RT-PCR.

None of the healthy volunteers (n = 5) had CEA, epithelial or variant CD44, or MMP-7 mRNA expressed in their peripheral blood; 3 patients with colorectal adenoma showed expression of CEA, epithelial and variant CD44, and MMP-7 mRNA in adenoma tissues but had no expression in portal venous and peripheral blood samples. All 22 patients with colorectal cancer showed expression of CEA, epithelial and variant CD44, and MMP-7 mRNA in all tumor tissues (Table 1). Tumor cells were detected in portal venous and peripheral blood samples of patients with stage I, II, III, and IV disease by using the RT-PCR assay for CEA, whereas the expression of epithelial and variant CD44 was detected in portal venous and peripheral blood samples of patients with stage II, III, and IV disease. The rates of expression of epithelial and variant CD44 were correlated with TNM stage in portal venous and peripheral blood. In all tumor tissues, including liver metastatic lesions, MMP-7 mRNA was expressed. However, there was no expression of MMP-7 mRNA in the portal venous or peripheral blood of any patients, including patients with liver metastases. The expression of these tumor marker genes in 2 representative cases is shown in Figure 1. The pattern of expression of CEA mRNA in patients with stage I and II cancers was similar to that in patients with stage III and IV cancers (Table 2). The CD44 variant mRNA were detected in 1 (8%) of the portal venous and peripheral blood samples from patients with localized disease (stage I and II) and were detected in 9 portal venous blood samples (90%) and 6 peripheral blood samples (60%) of patients with stage III and IV disease, including patients with regional lymph node involvement or metastatic lesions (portal venous blood, P<.001; peripheral blood, P = .02). Two patients with liver metastases had positive expression of epithelial and variant CD44 in all samples, including tumor tissues, portal venous blood, peripheral blood, and liver metastatic foci. Furthermore, patients with expression of epithelial and variant CD44 mRNA had cancer recurrences by 3 years after surgical treatment. Patient 20 had lung metastasis after 1 year, and patient 21 had spleen metastasis after 6 months (Figure 2). Of 6 patients with stage III or IV disease who had CEA and CD44 variant mRNA expressed in their peripheral blood, 3 had metastases to distant organs. Of 4 patients with stage III or IV disease but without DEA and CD44 variant mRNA, none had metastases to distant organs (χ2 = 2.86; P = .09).

Table Graphic Jump LocationTable 1. Clinicopathological Characteristics of Patients With Colon Cancer and Detection of CEA, CD44 Variants, and MMP-7 mRNA by Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)*
Place holder to copy figure label and caption
Figure 1.

Reverse transcriptase–polymerase chain reaction assays for carcinoembryonic antigen (CEA), CD44, and matrix metalloproteinase (MMP-7) messenger RNA (mRNA) in tumor tissue, portal venous blood, and peripheral blood of 2 representative patients with colorectal cancer. Patient 10 with stage II cancer (A) showed expression of CEA mRNA in all samples and of epithelial and variant CD44 and MMP-7 mRNA in tumor tissues, but not in portal venous or peripheral blood. Patient 20 with stage III cancer (B) showed expression of CEA mRNA in all samples and also expression of epithelial and variant CD44 mRNA in all samples. However, MMP-7 mRNA was expressed in tumor tissues but not in portal venous or peripheral blood. CD44E indicates epithelial CD44; CD44H, hematopoietic CD44; M, molecular markers; T, tumor tissue; D, drainage (portal) venous blood; P, peripheral blood; and bp, base pair.

Graphic Jump Location
Table Graphic Jump LocationTable 2. TNM Stage and Reverse Transcriptase–Polymerase Chain Reaction Detection of CEA and CD44 Variant mRNA*
Place holder to copy figure label and caption
Figure 2.

A, Plain computed-tomographic (CT) scan of the upper abdomen demonstrates spleen metastasis 6 months after surgical treatment in patient 21. B, Plain CT scan of the chest demonstrates lung metastasis 1 year after surgical treatment in patient 20 (arrows indicate the lung metastasis). Epithelial and variant CD44 messenger RNA were expressed in the peripheral blood of 2 patients during the surgical treatment.

Graphic Jump Location

For patients with colorectal carcinoma, distant metastasis is one of the most common recurrence patterns after surgical treatment.1821 In particular, colorectal cancer patients with stage III disease often have distant metastasis and poor survival.8,18 The presence of tumor cells in peripheral blood is by itself not sufficient to prove metastasis to distant organs. Rather, it seems to be necessary for cancer cells to acquire some additional properties for metastasis. For improving survival of patients with stage III disease, early detection of the cancer cells with the properties necessary for metastasis in portal venous or peripheral blood is required.

Using RT-PCR, the dissemination of tumor cells in the blood has been shown in patients with breast cancer,18,22 gastrointestinal cancer,18 malignant melanoma,2325 and lung cancer.26 Colorectal cancer cells are detected by using RT-PCR for CEA,1820,27 cytokeratin 19,28 cytokeratin 20,29 K-ras, 30 and p5328 mutations, and CEA mRNA is the most commonly used marker for detection of cancer cells in the bloodstream of patients with colorectal cancer. Positive expression of CEA mRNA in the bloodstream indicates the existence of viable cancer cells, which have been released or detached from the primary tumor beds.1820 We examined CEA mRNA for detection of circulating cancer cells; however, there was no significant difference between patients with stage I and II vs stage III and IV cancers. Indeed, the portal vein is the main drainage vein for colorectal cancer, and CEA mRNA was expressed in portal venous blood, but the presence of cancer cells in the bloodstream was not sufficient to cause metastasis. Therefore, in addition to examining CEA mRNA in the portal venous and peripheral blood samples of patients with colorectal cancer, we also analyzed the expression of CD44 and MMP-7 mRNA, which are likely candidates for conferring properties to promote metastasis.

The CD44 variants containing the products of variant exons 8 through 10 (CD44 v8-10) play an important role in the adhesion of tumor cells to the capillaries of distant organs in the metastatic process.31 Moreover, the level of expression of CD44 v8-10 is significantly higher in cancers associated with liver metastasis than in those without liver metastasis. In addition, expression of CD44 v8-10 in liver metastases is stronger than that in primary colorectal cancers.6 Therefore, expression of CD44 v8-10 has emerged as an independent prognostic indicator.7 It has been reported that CD44 variant exon 6 (CD44 v6) expression in tumors is associated with tumor-related death in patients with colorectal cancer. Expression of CD44 v6 has prognostic value independent of the Dukes classification and reflects the propensity for metastasis after apparently curative surgical treatment.8 Epithelial and variant CD44 were detected in 8% of portal venous and peripheral blood samples of patients with stage I and II disease, whereas they were detected in 90% of portal venous and 60% of peripheral blood samples of patients with stage III and IV disease. Two patients with liver metastases had epithelial and variant CD44 expressed in all samples, including tumor tissue, portal venous blood, peripheral blood, and liver metastatic lesions. The presence of CEA and CD44 variant mRNA may indicate hematogenous metastasis. Based on 3 years of follow-up data, 2 patients whose peripheral blood had expression of CEA and CD44 variant mRNA had distant metastases (lung or spleen). In the present study, the pattern of expression of CEA mRNA in stage I and II cancers was similar to that in stage III and IV cancers. The CD44 variant mRNA were detected in 8% of peripheral blood samples of patients with stage I and II disease and in 60% of peripheral blood samples of patients with stage III and IV disease. Therefore, RT-PCR for epithelial and variant CD44 mRNA may be a more sensitive tool than RT-PCR for CEA for delineating a high-risk group for hematogenous metastasis. In our results, expression of epithelial and variant CD44 mRNA in portal venous blood could not predict liver metastasis. On the other hand, expression of epithelial and variant CD44 mRNA in peripheral blood could predict distant metastasis (lung or spleen). Therefore, the detection of epithelial and variant CD44 expression in peripheral blood might provide the tool for diagnosis of distant metastasis and therapy for prevention of recurrence of stage III colorectal cancer.

Matrilysin, or MMP-7, is a matrix metalloproteinase that may be involved in the metastasis of colorectal cancer14 because of its ability to degrade the extracellular matrix, especially type IV collagen, a major component of the basement membrane.32 We chose MMP-7 mRNA because it is known to be epithelium-specific, unlike the other MMPs. Most MMPs are secreted not only from epithelial cells, but also from mesenchymal cells. In colonic tissue, MMP-7 is detected consistently in tumors but not in normal surrounding mucosa or mononucleated cells.11 Therefore, the detection of MMP-7 mRNA in lymph nodes and blood is definite proof of the presence of colorectal cancer cells. Our study demonstrates that MMP-7 mRNA is expressed in all tumor tissues, but not in all portal venous and peripheral blood samples, even in patients with stage III and IV disease, which means that adherence and interaction with mesenchymal cells seems to be necessary for expression of MMP-7 mRNA in cancer cells.32 These results suggest that MMP-7 gene expression may be associated with cell invasion. Therefore, expression of MMP-7 mRNA in portal venous and peripheral blood is not a useful tool for predicting liver and distant metastases.

In conclusion, expression of epithelial and variant CD44 mRNA of circulating cancer cells in peripheral blood may provide the ability to predict the hematogenous dissemination of cancer cells in patients with colorectal cancer. The present study suggests that the detection of cancer cells by RT-PCR assays for epithelial and variant CD44 mRNA may be more useful than CEA mRNA for predicting metastases in patients with stage III disease. These differences seem most evident in patients with stage III disease. Currently, chemotherapy is the standard of care for these patients.33 Further data on genetic changes in response to chemotherapy would be enlightening. Further investigations are required to determine the usefulness of RT-PCR assay for epithelial and variant CD44 mRNA.

This study was supported by the Mitsui Life Social Welfare Foundation, Tokyo, Japan.

Corresponding author and reprints: Hiroki Yamaue, MD, Second Department of Surgery, Wakayama Medical University, School of Medicine 811-1 Kimiidera, Wakayama 641-8510, Japan (e-mail: yamaue-h@wakayama-med.ac.jp).

Goldberg  RMFleming  TRTangen  CM  et al.  Surgery for recurrent colon cancer: strategies for identifying resectable recurrence and success rates after resection. Ann Intern Med. 1998;12927- 35
Link to Article
Fisher  ERTurnbull  RB  Jr The cytologic demonstration and significance of tumor cells in the mesenteric venous blood in patients with colorectal carcinoma. Surg Gynecol Obstet. 1955;100102- 108
Griffiths  JDMcKinna  JARowbotham  HD  et al.  Carcinoma of the colon and rectum: circulating malignant cells and 5-year survival. Cancer. 1973;31226- 236
Link to Article
Koch  MWeitz  JKienle  P  et al.  Comparative analysis of tumor cell dissemination in mesenteric, central, and peripheral venous blood in patients with colorectal cancer. Arch Surg. 2001;13685- 89
Link to Article
Yamaguchi  KTakagi  YAoki  SFutamura  MSaji  S Significant detection of circulating cancer cells in the blood by reverse transcriptase–polymerase chain reaction during colorectal cancer resection. Ann Surg. 2000;23258- 65
Link to Article
Takeuchi  KYamaguchi  AUrano  T  et al.  Expression of CD44 variant exons 8-l0 in colorectal cancer and its relationship to metastasis. Jpn J Cancer Res. 1995;86292- 297
Link to Article
Yamaguchi  AUrano  TGoi  T  et al.  Expression of a CD44 variant containing exons 8 to 10 is a useful independent factor for the prediction of prognosis in colorectal cancer patients. J Clin Oncol. 1996;141122- 1127
Mulder  JWRKruyt  PMSewnath  M  et al.  Colorectal cancer prognosis and expression of exon-v6–containing CD44 proteins. Lancet. 1994;3441470- 1472
Link to Article
Gotley  DCFawcett  JWalsh  MD  et al.  Alternatively spliced variants of the cell adhesion molecule CD44 and tumor progression in colorectal cancer. Br J Cancer. 1996;74342- 351
Link to Article
Yamamoto  HItoh  FHinoda  Y  et al.  Suppression of matrilysin inhibits colon cancer cell invasion in vitro. Int J Cancer. 1995;61218- 222
Link to Article
McDonnell  SNavre  MCoffey  RJ  et al.  Expression and localization of the matrix metalloproteinase pump-1 (MMP-7) in human gastric and colon carcinomas. Mol Carcinog. 1991;4527- 533
Link to Article
Yoshimoto  MItoh  FYamamoto  H  et al.  Expression of MMP-7 (PUMP-1) mRNA in human colorectal cancers. Int J Cancer. 1993;54614- 618
Link to Article
Mori  MBarnard  GMimori  K  et al.  Overexpression of matrix metalloproteinase 7 mRNA in human colon carcinomas. Cancer. 1995;751516- 1519
Link to Article
Ishikawa  TIchikawa  YMitsuhashi  M  et al.  Matrilysin is associated with progression of colorectal tumor. Cancer Lett. 1996;1075- 10
Link to Article
Chomczynski  PSacchi  N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162156- 159
Link to Article
Gerhard  MJuhl  HKalthoff  HSchreiber  HWWagener  CNeumaier  M Specific detection of carcinoembryonic antigen–expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol. 1994;12725- 729
Ichikawa  YIshikawa  TMomiyama  N  et al.  Detection of regional lymph node metastases in colon cancer by using RT-PCR for matrix metalloproteinase 7, matrilysin. Clin Exp Metastasis. 1998;163- 8
Link to Article
Mori  MMimori  KUeo  H  et al.  Clinical significance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcription–polymerase chain reaction in patients with gastrointestinal or breast carcinomas. J Clin Oncol. 1998;16128- 132
Jonas  SWindeatt  SO-Boateng  A  et al.  Identification of carcinoembryonic antigen–producing cells circulating in the blood of patients with colorectal carcinoma by reverse transcriptase polymerase chain reaction. Gut. 1996;39717- 721
Link to Article
Castells  ABessa  LGargallo  L  et al.  Detection of colonic cells in peripheral blood of colorectal cancer patients by means of reverse transcriptase and polymerase chain reaction. Br J Cancer. 1998;781368- 1372
Link to Article
Wharton  RQJonas  SKGlover  C  et al.  Increased detection of circulating tumor cells in the blood of colorectal carcinoma patients using 2 reverse transcription-PCR assays and multiple blood samples. Clin Cancer Res. 1999;54158- 4163
Datta  YHAdams  PTDrobyski  WR  et al.  Sensitive detection of occult breast cancer by the reverse–transcriptase polymerase chain reaction. J Clin Oncol. 1994;12475- 482
Mellado  BColomer  DCastel  T  et al.  Detection of circulating neoplastic cells by reverse transcriptase–polymerase chain reaction in malignant melanoma: association with clinical stage and prognosis. J Clin Oncol. 1996;142091- 2097
Jung  FABuzaid  ACRoss  MI  et al.  Evaluation of tyrosinase mRNA as a tumor marker in the blood of melanoma patients. J Clin Oncol. 1997;152826- 2831
Glaser  RRass  KSeiter  SHauschild  AChristophers  ETilgen  W Detection of circulating melanoma cells by specific amplification of tyrosinase complementary DNA is not a reliable tumor marker in melanoma patients: a clinical, 2-center study. J Clin Oncol. 1997;152818- 2825
Castaldo  GTomaiuolo  RSanduzzi  A  et al.  Lung cancer metastatic cells detected in blood by reverse transcriptase–polymerase chain reaction and dot-blot analysis. J Clin Oncol. 1997;153388- 3393
Mori  MMimori  KUeo  H  et al.  Molecular detection of circulating solid carcinoma cells in the peripheral blood: the concept of early systemic disease. Int J Cancer. 1996;68739- 743
Link to Article
Nakamori  SKameyama  MFurukawa  H  et al.  Genetic detection of colorectal cancer cells in circulation and lymph nodes. Dis Colon Rectum. 1997;40(suppl 10)S29- S36
Link to Article
Funaki  NOTanaka  JOhshio  G  et al.  Cytokeratin 20 mRNA in peripheral venous blood of colorectal carcinoma patients. Br J Cancer. 1998;771327- 1332
Link to Article
Fujita  SSugano  KFukayama  N  et al.  Detection of K-ras point mutations in mesenteric venous blood from colorectal cancer patients by enriched polymerase chain reaction and single-strand confirmation polymorphism analysis. Jpn J Clin Oncol. 1996;26417- 421
Link to Article
Seki  KYamaguchi  AGoi  T  et al.  Inhibition of liver metastasis formation by anti-CD44 variant exon 9 monoclonal antibody. Int J Oncol. 1997;111257- 1261
Wilson  CLMatrisian  LM Matrilysin: an epithelial matrix metalloproteinase with potentially novel functions. Int J Biochem Cell Biol. 1996;28123- 136
Link to Article
Ragnhammar  PHafstrom  LNygren  PGlimelius  B A systematic overview of chemotherapy effects in colorectal cancer. Acta Oncol. 2001;40282- 308
Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.

Reverse transcriptase–polymerase chain reaction assays for carcinoembryonic antigen (CEA), CD44, and matrix metalloproteinase (MMP-7) messenger RNA (mRNA) in tumor tissue, portal venous blood, and peripheral blood of 2 representative patients with colorectal cancer. Patient 10 with stage II cancer (A) showed expression of CEA mRNA in all samples and of epithelial and variant CD44 and MMP-7 mRNA in tumor tissues, but not in portal venous or peripheral blood. Patient 20 with stage III cancer (B) showed expression of CEA mRNA in all samples and also expression of epithelial and variant CD44 mRNA in all samples. However, MMP-7 mRNA was expressed in tumor tissues but not in portal venous or peripheral blood. CD44E indicates epithelial CD44; CD44H, hematopoietic CD44; M, molecular markers; T, tumor tissue; D, drainage (portal) venous blood; P, peripheral blood; and bp, base pair.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

A, Plain computed-tomographic (CT) scan of the upper abdomen demonstrates spleen metastasis 6 months after surgical treatment in patient 21. B, Plain CT scan of the chest demonstrates lung metastasis 1 year after surgical treatment in patient 20 (arrows indicate the lung metastasis). Epithelial and variant CD44 messenger RNA were expressed in the peripheral blood of 2 patients during the surgical treatment.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Clinicopathological Characteristics of Patients With Colon Cancer and Detection of CEA, CD44 Variants, and MMP-7 mRNA by Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)*
Table Graphic Jump LocationTable 2. TNM Stage and Reverse Transcriptase–Polymerase Chain Reaction Detection of CEA and CD44 Variant mRNA*

References

Goldberg  RMFleming  TRTangen  CM  et al.  Surgery for recurrent colon cancer: strategies for identifying resectable recurrence and success rates after resection. Ann Intern Med. 1998;12927- 35
Link to Article
Fisher  ERTurnbull  RB  Jr The cytologic demonstration and significance of tumor cells in the mesenteric venous blood in patients with colorectal carcinoma. Surg Gynecol Obstet. 1955;100102- 108
Griffiths  JDMcKinna  JARowbotham  HD  et al.  Carcinoma of the colon and rectum: circulating malignant cells and 5-year survival. Cancer. 1973;31226- 236
Link to Article
Koch  MWeitz  JKienle  P  et al.  Comparative analysis of tumor cell dissemination in mesenteric, central, and peripheral venous blood in patients with colorectal cancer. Arch Surg. 2001;13685- 89
Link to Article
Yamaguchi  KTakagi  YAoki  SFutamura  MSaji  S Significant detection of circulating cancer cells in the blood by reverse transcriptase–polymerase chain reaction during colorectal cancer resection. Ann Surg. 2000;23258- 65
Link to Article
Takeuchi  KYamaguchi  AUrano  T  et al.  Expression of CD44 variant exons 8-l0 in colorectal cancer and its relationship to metastasis. Jpn J Cancer Res. 1995;86292- 297
Link to Article
Yamaguchi  AUrano  TGoi  T  et al.  Expression of a CD44 variant containing exons 8 to 10 is a useful independent factor for the prediction of prognosis in colorectal cancer patients. J Clin Oncol. 1996;141122- 1127
Mulder  JWRKruyt  PMSewnath  M  et al.  Colorectal cancer prognosis and expression of exon-v6–containing CD44 proteins. Lancet. 1994;3441470- 1472
Link to Article
Gotley  DCFawcett  JWalsh  MD  et al.  Alternatively spliced variants of the cell adhesion molecule CD44 and tumor progression in colorectal cancer. Br J Cancer. 1996;74342- 351
Link to Article
Yamamoto  HItoh  FHinoda  Y  et al.  Suppression of matrilysin inhibits colon cancer cell invasion in vitro. Int J Cancer. 1995;61218- 222
Link to Article
McDonnell  SNavre  MCoffey  RJ  et al.  Expression and localization of the matrix metalloproteinase pump-1 (MMP-7) in human gastric and colon carcinomas. Mol Carcinog. 1991;4527- 533
Link to Article
Yoshimoto  MItoh  FYamamoto  H  et al.  Expression of MMP-7 (PUMP-1) mRNA in human colorectal cancers. Int J Cancer. 1993;54614- 618
Link to Article
Mori  MBarnard  GMimori  K  et al.  Overexpression of matrix metalloproteinase 7 mRNA in human colon carcinomas. Cancer. 1995;751516- 1519
Link to Article
Ishikawa  TIchikawa  YMitsuhashi  M  et al.  Matrilysin is associated with progression of colorectal tumor. Cancer Lett. 1996;1075- 10
Link to Article
Chomczynski  PSacchi  N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162156- 159
Link to Article
Gerhard  MJuhl  HKalthoff  HSchreiber  HWWagener  CNeumaier  M Specific detection of carcinoembryonic antigen–expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol. 1994;12725- 729
Ichikawa  YIshikawa  TMomiyama  N  et al.  Detection of regional lymph node metastases in colon cancer by using RT-PCR for matrix metalloproteinase 7, matrilysin. Clin Exp Metastasis. 1998;163- 8
Link to Article
Mori  MMimori  KUeo  H  et al.  Clinical significance of molecular detection of carcinoma cells in lymph nodes and peripheral blood by reverse transcription–polymerase chain reaction in patients with gastrointestinal or breast carcinomas. J Clin Oncol. 1998;16128- 132
Jonas  SWindeatt  SO-Boateng  A  et al.  Identification of carcinoembryonic antigen–producing cells circulating in the blood of patients with colorectal carcinoma by reverse transcriptase polymerase chain reaction. Gut. 1996;39717- 721
Link to Article
Castells  ABessa  LGargallo  L  et al.  Detection of colonic cells in peripheral blood of colorectal cancer patients by means of reverse transcriptase and polymerase chain reaction. Br J Cancer. 1998;781368- 1372
Link to Article
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