Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

Table 1 Primer sequences used for reverse transcription polymerase chain reaction amplification of target transcripts

Tumor marker	Sense	Antisense	PCR product size (bp)	Ref.
CEA	5'-GGGCCACTGTCGGCATCATGATTG-3'	5'-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC-3'	131	[38]
CK20	5'-CAGACACACGGTGAACTATGG-3'	5'-GATCAGCTTCCACTGTTAGACG-3'	371	[39]
EGFR	5'-TCTCAGCAACATGTCGATGG-3'	5'-TCGCACTTCTTACACTTGCG-3'	474	[40]

CEA: Carcinoembryonic antigen; CK20: Cytokeratin 20; EGFR: Epidermal growth factor receptor; PCR: Polymerase chain reaction.

Table 2 Clinical and pathological characteristics of the colorectal cancer patients enrolled in the study

	No. of cases
Total cases	88
Sex	56
Male	32
Female
Stage (Astler-Coller)
A	4
B	26
C	27
D	31
Lymph node metastasis
No	34
Yes	54
Surgical intervention
Resection	83
No intervention	5
Distant metastasis
No	57
Yes (liver/lungs/both)	24/2/5
Age (yr), median (min-max)	69 (43-88)
Tumor size (cm), median (min-max)	4.0 (1.0-11.0)

Table 3 Correlation of the number of multiplex reverse transcription polymerase chain reaction positive detections in each blood sample with disease stage in colorectal cancer patients n (%)

CRC patients (n = 88)	No. of positive marker detections in each blood sample
	0	1	2	3
Stage A (n = 4)	1 (25)	2 (50)	1 (25)	0 (0)
Stage B (n = 26)	3 (11.5)	5 (19.2)	16 (61.5)	2 (7.7)
B1 (n = 9)	1 (11.1)	1 (11.1)	7 (77.7)	0 (0)
B2 (n = 17)	2 (11.8)	4 (23.5)	9 (52.9)	2 (11.8)
Stage C (n = 27)	0 (0)	6 (22.2)	17 (63)	4 (14.8)
C1 (n = 5)	0 (0)	1 (20)	3 (60)	1 (20)
C2 (n = 22)	0 (0)	5 (22.7)	14 (63.6)	3 (13.6)
Stage D (n = 31)	0 (0)	2 (6.5)	18 (58.1)	11 (35.5)

Disease staging according to the modified Astler-Coller classification. A positive correlation was revealed using Spearman’s test (Spearman’s ρ = 0.396, P < 0.001). CRC: Colorectal cancer.

Table 4 Analysis of the clinical and pathological characteristics in relation to the detection pattern of circulating tumor cells using the carcinoembryonic antigen, cytokeratin 20 and epidermal growth factor receptor multiplex assay n (%)

Parameter	No. of positive markers in each blood sample				P-value
	0	1	2	3
Age (yr)
< 69	1 (2.4)	7 (17.1)	26 (63.4)	7 (17.1)
≥ 69	3 (6.4)	8 (17)	26 (55.3)	10 (21.3)	0.963
Sex
Male	3 (5.4)	8 (14.3)	32 (57.1)	13 (23.2)
Female	1 (3.1)	7 (21.9)	20 (62.5)	4 (12.5)	0.294
Stage (Astler-Coller)
A + B	4 (13.3)	7 (23.3)	17 (56.7)	2 (6.7)
C	0 (0)	6 (22.2)	17 (63)	4 (14.8)
D	0 (0)	2 (6.5)	18 (58.1)	11 (35.5)	< 0.001
Lymph node involvement1
≤ 3	4 (7.1)	11 (19.6)	35 (62.5)	6 (10.7)
> 3	0 (0)	4 (16)	14 (56)	7 (28)	0.055
Tumor size (cm)2
< 3	2 (10.5)	2 (10.5)	15 (79)	0 (0)
3-5	1 (2.5)	8 (20)	21 (52.5)	10 (25)
> 5	1 (4.8)	5 (23.8)	11 (52.4)	4 (19)	0.774
Differentiation3
Well + moderate	4 (6.8)	10 (17)	36 (61)	9 (15.2)
Poor + no	0 (0)	4 (22.2)	10 (55.6)	4 (22.2)	0.560
Serum CEA (ng/mL)4
< 5	3 (10)	9 (30)	12 (40)	6 (20)
5-10	0 (0)	1 (7.1)	11 (78.6)	2 (14.3)
> 10	0 (0)	4 (13.3)	17 (56.7)	9 (30)	0.029

P-values were calculated by Spearman’s test.

¹P-value calculation using data from 81 patients (7 missing values);

²P-value calculation using data from 80 patients (8 missing values);

³P-value calculation using data from 77 patients (11 missing values);

⁴P-value calculation using data from 74 patients (14 missing values). CEA: Carcinoembryonic antigen.