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, 16 (47), 5965-74

Multiplex RT-PCR-based Detections of CEA, CK20 and EGFR in Colorectal Cancer Patients

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Multiplex RT-PCR-based Detections of CEA, CK20 and EGFR in Colorectal Cancer Patients

Aikaterini Tsouma et al. World J Gastroenterol.

Abstract

Aim: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting circulating tumor cells in the peripheral blood of colorectal cancer (CRC) patients.

Methods: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo).

Results: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P < 0.001) and preoperative serum levels of CEA (P = 0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented significant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts.

Conclusion: These data suggest that multiplex RT-PCR assay can provide useful information concerning disease stage and overall survival of CRC patients.

Figures

Figure 1
Figure 1
Detection of carcinoembryonic antigen, cytokeratin 20 and epidermal growth factor receptor mRNAs in colorectal cancer tissue samples, HT-29 colorectal cancer cells and colorectal cancer patient peripheral blood samples. A: Detection of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA in pairs of colorectal cancer (CRC) and normal adjacent tissue samples. Lane 0 = molecular weight marker (100 bp); lane 1 = normal tissue; lane 2 = CRC tissue; B: Assessment of the sensitivity of the reverse transcription polymerase chain reaction (RT-PCR) detections for CEA, CK20 and EGFR mRNA. Lane 0 = molecular weight marker (100 bp); lane 1 = 105 HT-29 cells in 3 mL of normal blood; lane 2 = 104 HT-29 cells in 3 mL of normal blood; lane 3 = 103 HT-29 cells in 3 mL of normal blood; lane 4 = 102 HT-29 cells in 3 mL of normal blood; lane 5 = 10 HT-29 cells in 3 mL of normal blood; lane 6 = 1 HT-29 cell in 3 mL of normal blood; lane 7 = normal blood; C: Assessment of the sensitivity of the multiplex RT-PCR detections for CEA (131 bp), CK20 (371 bp) and EGFR (474 bp) mRNA. Lane 0 = molecular weight marker (100 bp); lane 1 = 105 HT-29 cells in 3 mL of normal blood; lane 2 = 104 HT-29 cells in 3 mL of normal blood; lane 3 = 103 HT-29 cells in 3 mL of normal blood; lane 4 = 102 HT-29 cells in 3 mL of normal blood; lane 5 = 10 HT-29 cells in 3 mL of normal blood; lane 6 = 1 HT-29 cell in 3 mL of normal blood; lane 7 = normal blood; D: An example of multiplex RT-PCR-based detection pattern of tumor marker transcripts in the peripheral blood samples of CRC patients. Left: lane 0 = molecular weight marker (100 bp); lane 1 = patient sample positive for CEA (131 bp) and CK20 (371 bp) mRNA; lane 2 = patient sample positive for CEA (131 bp) mRNA; lane 3 = patient sample positive for CEA (131 bp), CK20 (371 bp) and EGFR (474 bp) mRNA; lane 4 = patient sample negative for all markers. Right: lane 0 = molecular weight marker (100 bp); lanes 1-4 = amplification of β-actin (172 bp) cDNA of the patient samples presented in the left side of this panel.
Figure 2
Figure 2
Kaplan-Meier analysis comparing the overall survival of patient groups as developed according to transcript detection patterns for carcinoembryonic antigen, cytokeratin 20 and epidermal growth factor receptor. Group 1: Carcinoembryonic antigen (CEA) positive; Group 2: Positive for 2 markers (CEA and cytokeratin 20); Group 3: Positive for all markers; Group 4: Combination of groups 1 and 2; Group 5: Combination of groups 2 and 3. Log Rank test P values were as follows: Groups 1 vs 3: P = 0.002; Groups 2 vs 3: P = 0.007; Groups 4 vs 3: P = 0.001; Groups 1 vs 5: P = 0.041; Groups 1 vs 2: P = 0.116.

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