Molecular detection of circulating cancer cells in the peripheral blood of patients with colorectal cancer by using membrane array with a multiple mRNA marker panel.

The objective of this study was mainly to evaluate the simultaneous detection of expression levels of a multiple mRNA marker panel in the peripheral blood of colorectal cancer (CRC) patients for use in complementary CRC diagnosis. Twenty-seven tumor tissue specimens and 80 peripheral blood specimens were collected from CRC patients. Firstly, the levels of multiple molecular markers in the tumor tissue and blood specimens were evaluated by using real-time quantitative PCR (RT-QPCR) and membrane array. The result of linear regression showed a high degree of correlation (r=0.954, P<0.0001) between the data of these two methods. CK-19 was the marker with the highest detection rate (87.5%) in the peripheral blood, followed by CEA (82.6%), REG4 (80.8%), and then uPA (80.0%) and TLAM1 (80.0%). The levels of the six markers in the peripheral blood were extensively explored. In the 80 patients, the frequency of CK-19, CK-20, CEA, REG4, uPA, and TIAM1 mRNA overexpression was 82.5% (66/80), 78.8% (63/80), 82.5% (66/80), 80.0% (64/80), 78.8% (63/80), and 80.0% (64/80), respectively. Then, a panel combining these 6 mRNA markers was evaluated for its utility in the clinical diagnosis of CRC. The sensitivity, specificity, and accuracy of membrane array-based diagnostic method were 88.8%, 87.8%, and 88.2%, respectively; much higher than those of examinations with single markers. Finally, lymph node metastasis (P=0.024) and TNM stage (P=0.009) were found to be significantly correlated with overexpression of the multiple mRNA marker panel. The detection rates of stage-I and -II CRC by using the multi-marker membrane array were 54.5% (6/11) and 92.0% (23/25), respectively. In conclusion, the results of the present study have shown that this innovative membrane array technique with a multiple mRNA marker panel can significantly improve the diagnosis rate of early colorectal cancer.

[1]  L. Frati,et al.  Detection of CK19, CK20 and EGFR mRNAs in peripheral blood of carcinoma patients: correlation with clinical stage of disease. , 2003, Oncology reports.

[2]  Yiwang,et al.  Clinical features, diagnosis, treatment and prognosis of multiple primary colorectal carcinoma , 2004 .

[3]  M. Loda,et al.  Evaluation of Microsatellite Instability and Updated Version Cited Articles Citing Articles E-mail Alerts Evaluation of Microsatellite Instability and Immunohistochemistry for the Prediction of Germ-line Msh2 and Mlh1 Mutations in Hereditary Nonpolyposis Colon Cancer Families , 2022 .

[4]  L. Boix,et al.  Detection of colonic cells in peripheral blood of colorectal cancer patients by means of reverse transcriptase and polymerase chain reaction. , 1998, British Journal of Cancer.

[5]  M. Berger,et al.  Molecular detection of clinical colorectal cancer metastasis: how should multiple markers be put to use? , 2005, International Journal of Colorectal Disease.

[6]  G. Tsavellas,et al.  Flow cytometry correlates with RT-PCR for detection of spiked but not circulating colorectal cancer cells , 2004, Clinical & Experimental Metastasis.

[7]  L. Frati,et al.  UDP-glucuronosyltransferases 1A expression in human urinary bladder and colon cancer by immunohistochemistry. , 2005, Oncology reports.

[8]  W. Kruis,et al.  Chronic inflammatory bowel disease and cancer. , 2000, Hepato-gastroenterology.

[9]  D. Parkin,et al.  Global cancer statistics in the year 2000. , 2001, The Lancet. Oncology.

[10]  C.-H. Wu,et al.  Detection of circulating cancer cells with K-ras oncogene using membrane array. , 2005, Cancer letters.

[11]  Jiang Cao,et al.  [Significance of CK20 mRNA expression in peripheral blood of colorectal cancer patients by real-time fluorescent quantitative RT-PCR]. , 2004, Zhejiang da xue xue bao. Yi xue ban = Journal of Zhejiang University. Medical sciences.

[12]  R K Jain,et al.  Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[13]  P. Dettmar,et al.  Tumour-associated urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in normal and neoplastic tissues of patients with squamous cell cancer of the oral cavity - clinical relevance and prognostic value. , 2005, Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-Maxillo-Facial Surgery.

[14]  C. Glover,et al.  Clearance of Circulating Tumor Cells After Excision of Primary Colorectal Cancer , 2002, Annals of surgery.

[15]  I. Fleming,et al.  AJCC/TNM cancer staging, present and future , 2001, Journal of surgical oncology.

[16]  Y. Takeshima,et al.  Expression and localization of Reg IV in human neoplastic and non‐neoplastic tissues: Reg IV expression is associated with intestinal and neuroendocrine differentiation in gastric adenocarcinoma , 2005, The Journal of pathology.

[17]  Ya-Li Zhang,et al.  Early diagnosis for colorectal cancer in China. , 2002, World journal of gastroenterology.

[18]  C. Moskaluk,et al.  CK20 and CK7 protein expression in colorectal cancer: demonstration of the utility of a population-based tissue microarray. , 2005, Human pathology.

[19]  M. Fiegl,et al.  Mammaglobin Gene Expression: A Superior Marker of Breast Cancer Cells in Peripheral Blood in Comparison to Epidermal-Growth-Factor Receptor and Cytokeratin-19 , 2000, Laboratory Investigation.

[20]  M. Monden,et al.  Application of RT-PCR to clinical diagnosis of micrometastasis of colorectal cancer: A translational research study. , 2004, International journal of oncology.

[21]  W Blumenfeld,et al.  Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. , 1993, The American journal of pathology.

[22]  A. Dobrovic,et al.  Molecular detection of blood‐borne epithelial cells in colorectal cancer patients and in patients with benign bowel disease , 2000, International journal of cancer.

[23]  Taylor Murray,et al.  Cancer statistics, 2000 , 2000, CA: a cancer journal for clinicians.

[24]  A. Öberg,et al.  Detection of occult tumour cells in lymph nodes of colorectal cancer patients using real‐time quantitative RT‐PCR for CEA and CK20 mRNAS , 2004, International journal of cancer.

[25]  E. Thiel,et al.  Quantitative real‐time RT‐PCR for detection of disseminated tumor cells in peripheral blood of patients with colorectal cancer using different mRNA markers , 2004, International journal of cancer.

[26]  T. Ishikawa,et al.  Real-time PCR (TaqMan PCR) quantification of carcinoembryonic antigen (CEA) mRNA in the peripheral blood of colorectal cancer patients. , 2003, Anticancer research.

[27]  J. Nährig,et al.  Quantification of CK20 gene and protein expression in colorectal cancer by RT‐PCR and immunohistochemistry reveals inter‐ and intratumour heterogeneity , 2002, The Journal of pathology.

[28]  R. Coombes,et al.  The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19. , 1997, European journal of cancer.

[29]  E. Paskett,et al.  Predictors of stage of adoption for colorectal cancer screening. , 2000, Preventive medicine.

[30]  M. Szyf,et al.  Methylation and inhibition of expression of uPA by the RAS oncogene: divergence of growth control and invasion in breast cancer cells. , 2004, Carcinogenesis.

[31]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[32]  Y. Kodera,et al.  Quantitative detection of CEA expressing free tumor cells in the peripheral blood of colorectal cancer patients during surgery with real-time RT-PCR on a LightCycler. , 2002, Cancer letters.

[33]  F. Hsieh,et al.  Clinicopathologic Correlation of Up-regulated Genes Identified Using cDNA Microarray and Real-time Reverse Transcription-PCR in Human Colorectal Cancer , 2005, Cancer Epidemiology Biomarkers & Prevention.

[34]  E. Kim,et al.  Cloning and characterization of T-cell lymphoma invasion and metastasis 2 (TIAM2), a novel guanine nucleotide exchange factor related to TIAM1. , 1999, Genomics.

[35]  K. Miller,et al.  Quantitative evaluation of telomerase subunits in urine as biomarkers for noninvasive detection of bladder cancer , 2005, International journal of cancer.

[36]  Shiu-Ru Lin,et al.  Fatty acid metabolism pathway play an important role in carcinogenesis of human colorectal cancers by Microarray-Bioinformatics analysis. , 2006, Cancer letters.

[37]  De-Hua Wu,et al.  Tiam1 gene expression and its significance in colorectal carcinoma. , 2005, World journal of gastroenterology.

[38]  C. Boring,et al.  Cancer statistics for african americans , 1992, CA: a cancer journal for clinicians.

[39]  A. Neugut,et al.  Benefits of Colonoscopic Surveillance After Curative Resection of Colorectal Cancer , 1994, Annals of surgery.

[40]  C. Glover,et al.  Increased detection of circulating tumor cells in the blood of colorectal carcinoma patients using two reverse transcription-PCR assays and multiple blood samples. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[41]  A. Paller,et al.  Gangliosides inhibit urokinase-type plasminogen activator (uPA)-dependent squamous carcinoma cell migration by preventing uPA receptor/alphabeta integrin/epidermal growth factor receptor interactions. , 2005, The Journal of investigative dermatology.

[42]  M. Federico,et al.  Detection of circulating tumor cells by reverse transcriptase polymerase chain reaction of maspin in patients with breast cancer undergoing conventional-dose chemotherapy. , 2000, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[43]  D. Beck,et al.  Colon surveillance after colorectal cancer surgery , 1996, Diseases of the colon and rectum.

[44]  R. Paridaens,et al.  A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood , 2001 .

[45]  F Graziano,et al.  Detection of blood-borne cells in colorectal cancer patients by nested reverse transcription-polymerase chain reaction for carcinoembryonic antigen messenger RNA: longitudinal analyses and demonstration of its potential importance as an adjunct to multiple serum markers. , 2001, Cancer research.