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

Fiorella Guadagni; Judith Kantor; Simona Aloe; Maria Daniela Carone; Antonella Spila; Roberta D’Alessandro; Maria Rosaria Abbolito; Maurizio Cosimelli; Franco Graziano; Fabio Carboni; Sandro Carlini; Pasquale Perri; Francesco Sciarretta; John W. Greiner; Syed V. S. Kashmiri; Seth M. Steinberg; Mario Roselli; Jeffrey Schlom

DOI: Published March 2001

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Fig. 1.
Optimization of the sensitivity and specificity of the RT-PCR assay for CEA mRNA. A, improvement in RT-PCR sensitivity using different amounts of total RNA. Agarose gel electrophoresis of CEA RT-PCR amplification products from peripheral blood cells of three different metastatic colorectal carcinoma patients when cDNA was synthesized using either 5 μg (Lanes 1–3) or 1 μg (Lanes 4–6) of total RNA. Lane 7, cDNA negative control. Arrow, 131-bp CEA band. M, 100-bp DNA ladder (also seen in B–D). B, evaluation of two different blood samples obtained at days 7 and 1 prior to surgery, using two different blood volumes from the same colorectal cancer patient. Lane 1, MC38(CEA) cell line. Lane 2, MC38 cell line. Lanes 3 and 4, 15 and 7 ml of blood, respectively, from a colorectal cancer patient 7 days prior to surgery. Lanes 5 and 6, 15 and 7 ml of blood, respectively, from the same patient 1 day prior to surgery. Lane 7, cDNA negative control. C, evaluation of the specificity of the RT-PCR reaction. Lane 1, MC38(CEA) cells expressing CEA. Lanes 2 and 3, MC38 and MC38(NCA), respectively. Lanes 4–11 (and Lanes 1–11 in D), blood samples from 19 different, apparently healthy donors.
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Fig. 2.
Relationship between CEA RT-PCR results and CEA serum levels and CEA expression in tumor tissue. A, modest overall association between serum CEA levels and RT-PCR analysis for CEA in the blood of colorectal cancer patients. [P2 = 0.11, comparing four groups as shown; P2 = 0.02 comparing (+) versus (−)]. B, strong relationship between CEA expression in tumor tissue by immunohistochemistry and RT-PCR analysis for CEA in the blood of colorectal cancer patients (P2 = 0.0041). NEG, negative.

Tables

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Table 1

Summary of CEA RT-PCR results in blood samples from apparently healthy volunteers and patients with benign or malignant colorectal disease

	No. of cases	No. of positive cases^a	No. of borderline positive (+/−) cases^a	No. of negative cases
Series I
Donors
1 μg of RNA	20	0	0	20
2 μg of RNA	20	0	0	20
5 μg of RNA	20	0	0	20
Patients
1 μg of RNA	20	3 (15)	0	17
2 μg of RNA	20	7 (35)	0	13
5 μg of RNA	20	15 (75)	0	5
Series II^b
Donors^c	40	0	2 (5.0)	38 (95.0)
Patients
Benign	18	0	0	18 (100.0)
Malignant	51	34 (66.7)	1 (2.0)	16 (31.4)

a Numbers in parentheses represent percentages.
b 5 μg of RNA were used for all series II samples.
c Donors are apparently healthy individuals, as described in “Materials and Methods.”

Table 2

RT-PCR results and serum tumor marker levels in nonmetastatic colorectal cancer patients

Serum CEA levels were determined by an enzyme immunoassay test kit, using a value of 5 ng/ml as positive. CA19.9 was determined using the CA19.9 kit with the suggested value 37 units/ml as positive. Serum CA72-4 antigen levels were determined using the CA72-4 kit with the suggested value 6 units/ml as positive.
Patient	Site of primary	Stage	Site of metastasis	Serum levels			CEA RT-PCR
Patient	Site of primary	Stage	Site of metastasis	CEA (ng/ml)	CA19.9 (units/ml)	CA72-4 (units/ml)	CEA RT-PCR
1	Colon	A		2.9 (−)	8.0 (−)	1.7 (−)	−
2	Rectum	A		1.4 (−)	39.9 (+)	10.0 (+)	+++
3	Rectum	A		0.8 (−)	3.7 (−)	0.1 (−)	−
4	Colon	A		0.6 (−)	25.6 (−)	1.9 (−)	++
5	Colon	A		0.6 (−)	12.2 (−)	2.1 (−)	−
6	Colon	A		0.5 (−)	15.3 (−)	1.4 (−)	−
7	Rectum	B1		3.0 (−)	6.2 (−)	1.8 (−)	++
8	Rectum	B1		2.8 (−)	30.9 (−)	0.7 (−)	++
9	Rectum	B1		2.7 (−)	4.8 (−)	2.0 (−)	−
10	Rectum	B1		2.1 (−)	7.0 (−)	1.9 (−)	−
11	Sigma	B1		11.5 (+)	8.3 (−)	0.9 (−)	+
12	Rectum	B1		0.9 (−)	15.8 (−)	2.2 (−)	−
13	Colon/Rectum	B1^a		1.7 (−)	8.7 (−)	2.4 (−)	++
14	Colon	B2		77.3 (+)	40.0 (+)	22.9 (+)	++
15	Sigma	B2		3.1 (−)	9.3 (−)	4.5 (−)	+++
16	Rectum	B2		1.8 (−)	7.4 (−)	12.5 (+)	+
17	Rectum	B2		1.7 (−)	7.3 (−)	2.1 (−)	+
18	Rectum	B2		1.4 (−)	4.0 (−)	0.8 (−)	−
19	Rectum	B2		1.1 (−)	8.1 (−)	1.8 (−)	+
20	Colon	B2		0.4 (−)	18.3 (−)	3.4 (−)	−
21	Colon	B2		0.2 (−)	2.2 (−)	3.0 (−)	−
22	Rectum	C1		6.2 (+)	12.7 (−)	1.8 (−)	+
23	Rectum	C1		1.0 (−)	12.1 (−)	2.3 (−)	+
24	Colon	C1		0.5 (−)	8.9 (−)	2.0 (−)	+
25	Sigma	C2		16.1 (+)	0.1 (−)	1.6 (−)	−
26	Colon	C2		5.6 (+)	0.4 (−)	0.4 (−)	++
27	Colon	C2		1.2 (−)	87.1 (+)	18.5 (+)	++
28	Rectum	C2		0.8 (−)	6.2 (−)	2.3 (−)	+
29	Rectum	C2		0.6 (−)	11.8 (−)	2.8 (−)	+++
30	Colon	D	Liver	433.5 (+)	1364.2 (+)	18.9 (+)	+
31	Colon	D	Liver	311.6 (+)	6490.1 (+)	31.3 (+)	+++
32	Colon	D	Liver	257.4 (+)	44.7 (+)	65.3 (+)	+
33	Sigma	D	Liver	139.0 (+)	963.0 (+)	151.9 (+)	−
34	Colon	D	Multiple	126.2 (+)	53.4 (+)	2.6 (−)	+
35	Cecum	D	Liver	20.5 (+)	75.8 (+)	0.7 (−)	+
36	Rectum	D	Peritoneum	20.0 (+)	238.6 (+)	15.5 (+)	+
37	Colon	D	Liver	13.5 (+)	84.6 (+)	22.5 (+)	+++
38	Rectum	D	Liver	10.2 (+)	2.0 (−)	0.1 (−)	+++
39	Colon	D	Peritoneum	8.7 (+)	1.7 (−)	3.8 (−)	+
40	Rectum	D	Lung-Liver	5.0 (+)	43.4 (+)	2.0 (−)	+
41	Rectum	D	Liver	4.7 (−)	25.7 (−)	1.6 (−)	+
42	Colon	D	Liver	4.2 (−)	34.9 (−)	0.1 (−)	+
43	Rectum	D	Liver	2.1 (−)	8.3 (−)	2.8 (−)	−
44	Colon	D	Multiple	1.5 (−)	99.5 (+)	22.7 (+)	−
45	Colon	D	Liver	1.5 (−)	148.3 (+)	10.2 (+)	−
46	Sigma	D	Liver	1.0 (−)	0.4 (−)	0.3 (−)	++
47	Sigma	REC^b	Locoreg.	278.1 (+)	25.5 (−)	1.9 (−)	+
48	Rectum	REC	Locoreg.	47.4 (+)	124.2 (+)	3.9 (−)	+
49	Rectum	REC	Locoreg.	3.9 (−)	9.8 (−)	2.0 (−)	+
50	Rectum	REC	Locoreg.	3.1 (−)	13.4 (−)	1.9 (−)	−
51	Colon	REC	Locoreg.	0.9 (−)	127.3 (+)	104.4 (+)	+

a Synchronous adenocarcinomas of the right colon and rectum.
b REC, recurrent; Locoreg, locoregional.

Table 3

Detection of CEA-specific mRNA by nested RT-PCR in peripheral blood cells vs. serum marker levels of colorectal cancer patients

Stage	No. of patients	No. of patients positive for^a
Stage	No. of patients	CEA RT-PCR	Serum CEA	Any of the three serum markers^b	Any of the three serum markers and RT-PCR	CEA RT-PCR and negative by all three serum markers	Any of the three serum markers and/or RT-PCR
A	6	2 (33.3)	0 (0.0)	1 (16.7)	1 (16.7)	1 (16.7)	2 (33.3)
B	15	9 (60.0)	2 (13.3)	3 (20.0)	3 (20.0)	6 (40.0)	9 (60.0)
C	8	7 (87.5)	3 (37.5)	4 (50.0)	3 (37.5)	4 (50.0)	8 (100.0)
D	17	13 (76.5)	11 (64.7)	13 (76.5)	10 (58.8)	3 (17.6)	16 (94.1)
Locoregional recurrence	5	4 (80.0)	2 (40.0)	3 (60.0)	3 (60.0)	1 (20.0)	4 (80.0)
Total	51	35 (68.3)	18 (35.3)	24 (47.1)	20 (39.2)	15 (29.4)	39 (76.5)

a Values in parentheses are percentages.
b CEA, CA19.9, CA72-4.

Table 4

Correlation between RT-PCR results and tissue CEA expression in colorectal cancer patients

Patients diagnosed with	Patient	Site of primary tumor	Stage	Site of metastasis	Serum CEA (ng/ml)	CEA RT-PCR	Tissue CEA expression^a (%)
Primary nonmetastatic cancer	2	Rectum	A		1.4 (−)	+++	85 (++)
	7	Rectum	B1		3.0 (−)	++	40 (+++)
	8	Rectum	B1		2.8 (−)	++	>90 (++)
	9	Rectum	B1		2.7 (−)	−	10 (+)
	12	Rectum	B1		0.9 (−)	−	25 (+)
	13	Colon & rectum	B1^b		1.7 (−)	++	70 (+++)
	14	Sigma	B2		2.3 (−)	+++	>90 (+++)
	17	Rectum	B2		1.7 (−)	+	50 (++)
	19	Rectum	B2		1.1 (−)	+	>90 (+++)
	20	Colon	B2		0.4 (−)	−	65 (++)
	21	Colon	B2		0.2 (−)	−	50 (++)
	29	Rectum	C2		0.6 (−)	+++	50 (+++)
Metastatic disease	30	Colon	D	Liver	433.5 (+)	+	10 (+/−)
	31	Colon	D	Liver	311.6 (+)	+++	>90 (+++)
	32	Colon	D	Liver	257.4 (+)	+	20 (+)
	38	Rectum	D	Liver	10.2 (+)	+++	85 (++)
	39	Sigma	D	Peritoneum	8.7 (+)	+	50 (++)
	44	Colon	D	Multiple	1.5 (−)	−	Neg^c
	45	Colon	D	Liver	1.5 (−)	−	Neg
	46	Sigma	D	Liver	1.0 (−)	++	15 (+++)
	49	Rectum	REC^d	Locoregional	3.9 (−)	+	10 (+/−)

a CEA tissue expression was evaluated by immunohistochemical analysis as described in “Material and Methods.”
b Synchronous adenocarcinomas of the right colon and the rectum.
c Neg, CEA was evaluated only on biopsies obtained from liver metastases (taken for diagnostic purposes).
d REC, recurrent.

Table 5

Summary of the CEA RT-PCR results on blood samples from stage A and B2 colorectal cancer patients during postsurgical follow-up

Patient	Stage	Days post surgery	CEA RT-PCR	Serum levels^a
Patient	Stage	Days post surgery	CEA RT-PCR	CEA	CA19.9	CA72-4
2	A	−2	+++	1.4 (−)	39.9 (+)	10.0 (+)
		30	−	1.3 (−)	27.3 (−)	2.1 (−)
		206	−	1.6 (−)	28.1 (−)	1.8 (−)
		367	−	1.2 (−)	29.5 (−)	2.2 (−)
		444	−	1.8 (−)	22.7 (−)	2.5 (−)
		675	−	2.2 (−)	28.5 (−)	1.7 (−)
4	A	−37	++	0.6 (−)	25.6 (−)	1.9 (−)
		0	++	2.8 (−)	29.8 (−)	0.7 (−)
		3	+	2.1 (−)	25.6 (−)	1.2 (−)
		47	+	2.2 (−)	26.4 (−)	1.3 (−)
		122	+	2.2 (−)	24.9 (−)	1.3 (−)
5	A	−3	−	0.6 (−)	12.2 (−)	2.1 (−)
		2	−	0.8 (−)	11.3 (−)	1.8 (−)
		4	−	1.2 (−)	6.9 (−)	1.7 (−)
		8	−	0.9 (−)	12.4 (−)	2.3 (−)
		120	−	1.4 (−)	7.9 (−)	2.1 (−)
		240	−	1.1 (−)	9.8 (−)	1.9 (−)
1	A	0	−	2.9 (−)	8.0 (−)	1.7 (−)
		3	−	2.3 (−)	8.1 (−)	1.5 (−)
		31	−	1.8 (−)	9.5 (−)	1.8 (−)
		90	−	1.9 (−)	6.8 (−)	1.6 (−)
		202	−	2.0 (−)	8.1 (−)	1.3 (−)
19	B2	−2	+	1.1 (−)	8.1 (−)	1.8 (−)
		5	+	2.1 (−)	9.9 (−)	2.3 (−)
		39	−	2.4 (−)	6.2 (−)	3.7 (−)
		126	−	3.1 (−)	4.9 (−)	4.2 (−)
17	B2	−1	+	1.7 (−)	7.3 (−)	2.1 (−)
		47	+	0.6 (−)	5.5 (−)	1.6 (−)
		75	−	2.7 (−)	3.5 (−)	1.7 (−)
		103	−	1.0 (−)	5.5 (−)	2.0 (−)
15	B2	−1	+++	3.1 (−)	9.3 (−)	4.5 (−)
		41	+++	2.3 (−)	4.7 (−)	1.9 (−)
		62	+	1.8 (−)	6.7 (−)	2.0 (−)
		90	−	2.6 (−)	8.9 (−)	1.5 (−)
		118	−	1.9 (−)	9.7 (−)	1.7 (−)
		146	−	2.5 (−)	13.6 (−)	2.0 (−)
		309	−	2.0 (−)	12.0 (−)	1.9 (−)
20	B2	0	−	0.4 (−)	18.3 (−)	3.4 (−)
		30	−	1.4 (−)	25.3 (−)	5.2 (−)
		90	−	1.8 (−)	225.4 (+)	7.4 (+)
		120	−	3.0 (−)	525.3 (+)	13.1 (+)
		236	−	2.8 (−)	1368.4 (+)	21.0 (+)
		280	−	10.3 (+)	2466.6 (+)	57.4 (+)
		292	−	15.1 (+)	4273.5 (+)	60.6 (+)
21	B2	−2	−	0.2 (−)	2.2 (−)	3.0 (−)
		108	+++	2.6 (−)	0.1 (−)	1.5 (−)
		677	+++	2.5 (−)	4.1 (−)	7.8 (+)

a Serum CEA levels were determined by an enzyme immunoassay test kit, using a value of 5 ng/ml as positive. CA19.9 was determined using the CA19.9 kit with the suggested value 37 units/ml as positive. Serum CA72-4 antigen levels were determined using the CA72-4 kit with the suggested value 6 units/ml as positive.

Table 6

Summary of the CEA RT-PCR results on blood samples from colorectal cancer patients undergoing chemotherapy

Patient	Stage	Site of metastasis	Days post surgery	CEA RT-PCR	Serum levels^a			Postsurgical treatment
Patient	Stage	Site of metastasis	Days post surgery	CEA RT-PCR	CEA	CA19.9	CA72-4	Postsurgical treatment
26	C2		−5	++	5.6 (+)	0.4 (−)	0.4 (−)	CHT-RT^b
			90	+	9.0 (+)	18.4 (−)	1.2 (−)
			210	+	12.5 (+)	29.3 (−)	1.5 (−)
		Lung	420	+++	793.8 (+)	2319.0 (+)	0.6 (−)	Lung metastasis
		Lung	483	+++	894.0 (+)	1090.0 (+)	1.8 (−)
38	D	Multiple	−2	+++	10.2 (+)	2.0 (−)	0.1 (−)	CHT
			36	++	9.4 (+)	0.1 (−)	0.1 (−)	Stable disease
			53	++	16.2 (+)	0.1 (−)	1.8 (−)	Stable disease
			235	++	51.3 (+)	0.1 (−)	2.6 (−)	Stable disease
			319	+++	82.0 (+)	0.8 (−)	2.4 (−)	PD
			530	+++	573.1 (+)	5.3 (−)	3.3 (−)	PD
31	D	Liver	−1	+++	311.6 (+)	6490.1 (+)	1.3 (−)	CHT
			33	−	224.0 (+)	182.9 (+)	2.4 (−)
			51	−	60.5 (+)	462.0 (+)	2.6 (−)	Responsive
			182	++	226.5 (+)	487.1 (+)	42.6 (+)	PD
37	D	Liver	−1	+++	13.5 (+)	84.6 (+)	22.5 (+)	CHT
			33	−	4.3 (−)	45.3 (+)	8.4 (+)	Responsive
			51	−	4.1 (−)	39.5 (+)	6.9 (+)	Responsive
			182	++	9.5 (+)	76.4 (+)	18.9 (+)	PD
46	D	Liver	−11	+++	1.0 (−)	0.4 (−)	0.3 (−)	CHT
			41	++	1.4 (−)	0.1 (−)	1.4 (−)	Responsive
			56	++	2.2 (−)	0.1 (−)	0.4 (−)	CR
			219	+	1.1 (−)	0.8 (−)	2.1 (−)	CR

a Serum CEA levels were determined by an enzyme immunoassay test kit, using a value of 5 ng/ml as positive. CA19.9 was determined using the CA19.9 kit with the suggested value 37 units/ml as positive. Serum CA72-4 antigen levels were determined using the CA72-4 kit with the suggested value 6 units/ml as positive.
b CHT-RT, chemotherapy + radiotherapy; PD, progressive disease; CR, complete remission.