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Prediction of Distant Metastasis by Using Reverse Transcriptase Polymerase Chain Reaction for Epithelial and Variant CD44 Mrna I

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Prediction of Distant Metastasis by Using Reverse Transcriptase Polymerase Chain Reaction for Epithelial and Variant CD44 Mrna I 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 Shozo Yokoyama, MD; Hiroki Yamaue, MD Background: Reverse transcriptase–polymerase chain Main Outcome Measure: The clinical significance of reaction (RT-PCR) has been used to identify small num- RT-PCR for epithelial and variant CD44 mRNA in pe- bers of tumor cells. Molecular detection is thought to pro- ripheral blood. vide useful information for the clinical management of postoperative adjuvant therapy regimens. Results: During 3 years of follow-up, 2 patients whose peripheral blood had carcinoembryonic antigen and Objective: To use RT-PCR to identify messenger RNA CD44 variant mRNA also had distant metastases (lung (mRNA) coding for carcinoembryonic antigen, epithelial or spleen). Expression of epithelial and variant CD44 and variant CD44, and matrix metalloproteinase 7 in the mRNA in peripheral blood was more highly correlated portal venous and peripheral blood of patients with colo- with the clinical cancer stage than with expression rectal carcinoma to predict live or distant metastasis. of carcinoembryonic antigen and matrix metallopro- teinase 7. Design: Prospective consecutive series. Conclusions: Molecular detection of epithelial and Setting: University hospital. variant CD44 mRNA in the peripheral blood may help determine distant metastases in patients with colorectal Patients and Methods: Portal venous and peripheral carcinoma. Molecular detection in the peripheral blood blood samples were obtained from 22 patients with colo- at surgical treatment suggests that systemic hemato- rectal cancer during surgical manipulation. Using comple- genic tumor cell dissemination is an early event of dis- mentary DNA primers specific for carcinoembryonic tant metastasis. antigen, CD44, and matrix metalloproteinase 7, RT-PCR was performed to detect tumor cells. Arch Surg. 2002;137:1069-1073 F PATIENTS with colorec- (CEA) messenger RNA (mRNA), matrix tal cancer who have cura- metalloproteinese 7 (MMP-7) mRNA, and tive surgical treatment, epithelial and variant CD44 mRNA in cir- 44% develop metastatic culating-blood specimens from patients disease.1 The sites of me- with colorectal carcinoma. The presence Otastases are the lymph nodes, peritoneum, of cancer cells in the blood does not prove liver, lungs, and bone marrow. Many meth- that there has been metastasis to distant ods have been established to increase the organs. It seems to be necessary for can- sensitivity of detection of these early me- cer cells to acquire certain properties for tastases. Cytologic and immunochemical metastasis. Thus, one should consider methods have been used to detect colorec- which tumor marker genes are related to tal cancer cells in peripheral and mesen- the formation of metastatic foci in dis- teric venous blood–draining tumors.2,3 tant organs, including the liver, lungs, and However, the prognostic and clinical value spleen. In this study, we selected CD44 of this detection is not clear. Technical ad- variants and the MMP-7 gene as markers. vances have also made it possible to detect The CD44 variant exons and MMP-7 are micrometastases at the molecular level in frequently overexpressed in human colo- circulating blood, and recent studies have rectal carcinoma, and many studies have From the Second Department of reported and discussed the clinical signifi- suggested that CD44 variants and MMP-7 4,5 Surgery, Wakayama Medical cance of such detection. are associated with metastases and inva- University School of Medicine, In the present study, we detected the sion,6-14 with CD44 variants related to the Wakayama, Japan. expression of carcinoembryonic antigen properties of adhesion and MMP-7 re- (REPRINTED) ARCH SURG/ VOL 137, SEP 2002 WWW.ARCHSURG.COM 1069 ©2002 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 lated to tumor invasion. On the basis of our results, we sion step for 10 minutes. Then, 1 µL of the reaction mixture discuss the clinical value of detecting epithelial and vari- was transferred into a second tube with 50 µL of a solution con- ant CD44 and MMP-7 mRNA in the circulating blood. taining buffer, primers (CD44 and MMP-7 primers were the same as for the first PCR; 0.4µM each), 2.5 U of Taq polymer- ase, and 200µM of deoxynucleoside triphosphate. Thirty am- PATIENTS AND METHODS plification cycles were performed at 95°C (1 minute), 67°Cfor CEA, 55°C for CD44, or 55°C for MMP-7 (1 minute), and 72°C PATIENTS (1 minute), with a final extension step for 10 minutes. We re- peated the sensitivity tests and determined the adequate PCR Twenty-two patients with colorectal cancer were enrolled in cycle. The most appropriate cycles are 20 for the first PCR and this study. The locations of the tumor were the ascending co- 30 for the second PCR. The PCR products were then electro- lon (n=4), the transverse colon (n=4), the descending colon phoresed on 2% agarose gels containing 1 µg/mL of ethidium (n=2), the sigmoid colon (n=6), and the rectum (n=6). All pa- bromide and visualized with ultraviolet light. The PCR prod- tients received surgical treatment in which the tumors were re- ucts were identified by direct sequencing. sected completely. The clinical stages according to TNM stag- ing were stage I (n=4), stage II (n=8), stage III (n=8), and stage STATISTICAL ANALYSIS IV (n=2). None of the patients had received any previous treat- ment, including anticancer chemotherapy or radiation therapy. The significance of differences was determined by the Fisher Ͻ Thirteen patients had well-differentiated adenocarcinoma, 6 pa- exact test. P .05 was considered statistically significant. tients had moderately differentiated adenocarcinoma, and 3 pa- tients showed other histologic types (2 with mucinous adeno- carcinoma and 1 with poorly differentiated adenocarcinoma). RESULTS Informed consent was obtained from all patients in accor- dance with the guidelines of the Ethics Committee on Human To determine the sensitivity of reverse transcriptase– Research, Wakayama Medical University, Wakayama, Japan. PCR (RT-PCR) for CEA, CD44, and MMP-7, triplicate reconstitution experiments were performed, in which EXTRACTION OF RNA serial 10-fold dilutions of HT29 cells, a human colon cancer cell line, were mixed with 1ϫ107 mononucle- Blood samples from the portal and peripheral veins (10 mL) ated cells from a healthy volunteer. In the preliminary and tumor tissue (100 mg) were obtained from each patient ϫ 7 during surgical treatment. The mononucleated cell fraction was study, even 1 tumor cell in 1 10 normal cells could be isolated by centrifugation on a Ficoll-Hypaque gradient at 400g detected by RT-PCR. for 30 minutes. Total RNA was isolated from mononuclear cells None of the healthy volunteers (n=5) had CEA, epi- and tumor tissues by guanidinium thiocyanate extraction, us- thelial or variant CD44, or MMP-7 mRNA expressed in ing the method described by Chomczynski and Sacchi.15 their peripheral blood; 3 patients with colorectal ad- enoma showed expression of CEA, epithelial and vari- REVERSE TRANSCRIPTASE–POLYMERASE ant CD44, and MMP-7 mRNA in adenoma tissues but had CHAIN REACTION (RT-PCR) no expression in portal venous and peripheral blood samples. All 22 patients with colorectal cancer showed The oligonucleotide primers specific for CEA, CD44, and MMP-7 expression of CEA, epithelial and variant CD44, and mRNA were synthesized according to the sequences described by Gerhard et al16 and Ichikawa et al.17 To detect CEA expres- MMP-7 mRNA in all tumor tissues (Table 1). Tumor sion, the primers used for the first polymerase chain reaction (PCR) cells were detected in portal venous and peripheral blood were sense, 5Ј-TCTGGAACTTCTCCTGGTCTCTCAGCTGG- samples of patients with stage I, II, III, and IV disease by 3Ј, and antisense, 5Ј-GGGCCACTGCTGGCATCATGATTGG- using the RT-PCR assay for CEA, whereas the expres- 3Ј. The heminested sense primer for CEA expression was sion of epithelial and variant CD44 was detected in por- 5Ј-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC 3Ј, and tal venous and peripheral blood samples of patients with the antisense primer was the same as that used for the first PCR. stage II, III, and IV disease. The rates of expression of Ј The CD44 primers were sense, 5 -TCCCAGACGAAGACAGTC- epithelial and variant CD44 were correlated with TNM CCTGGAT-3Ј, and antisense, 5Ј-CACTGGGGTGGAATGT- Ј Ј stage in portal venous and peripheral blood. In all tu- GTCTTGGTC-3 . The MMP-7 primers were sense, 5 - mor tissues, including liver metastatic lesions, MMP-7 TCTTTGGCCTACCTATAACTGG-3Ј, and antisense, 5Ј- CTAGACTGCTACCATCCGTC-3Ј. Efficient amplification of mRNA was expressed. However, there was no expres- the RNA samples was confirmed by an internal control by am- sion of MMP-7 mRNA in the portal venous or periph- plification of ␤-actin mRNA. The primers used for ␤-actin were eral blood of any patients, including patients with liver sense, 5Ј-ATCTGGCACCACACCTTCTACAATGAGCTGCG- metastases. The expression of these tumor marker genes 3Ј, and antisense, 5Ј-CGTCATACTCCTGCTTGCTGATCCA- in 2 representative cases is shown in Figure 1. The pat- CATCTGC-3Ј. tern of expression of CEA mRNA in patients with stage Complementary DNA was synthesized from 2 µg of total I and II cancers was similar to that in patients with stage RNA with a Ready-to-Go T-Primed First-Strand Kit (Amer- III and IV cancers (Table 2). The CD44 variant mRNA sham Biosciences, Piscataway, NJ) following the manufactur- were detected in 1 (8%) of the portal venous and periph- ers’ instructions. For the first PCR, 50 µL of a solution con- eral blood samples from patients with localized disease taining buffer (Takara Bioch Inc, Shiga, Japan), primers (0.4 µmol/L each), 1 µL of complementary DNA, 2.5 U of Taq poly- (stage I and II) and were detected in 9 portal venous blood merase, and 200µM of deoxynucleoside triphosphate was added samples (90%) and 6 peripheral blood samples (60%) of to each tube.
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