Clinical Cancer Research Vol. 9, 2678-2682, July 2003
© 2003 American Association for Cancer Research
Molecular Oncology, Markers, Clinical Correlates |
Persistence of Epidermal Growth Factor Receptor and Interleukin 10 in Blood of Colorectal Cancer Patients after Surgery Identifies Patients with High Risk to Relapse1
Luciano Giacomelli,
Walter Gianni,
Cristiano Belfiore,
Orietta Gandini,
Lazzaro Repetto,
Angelo Filippini,
Luigi Frati,
Anna Maria Aglianò and
Paola Gazzaniga2
Dipartimento di Scienze Chirurgiche, Policlinico Umberto I [L. G., C. B., A. F.], Rome; INRCA, Unità Operativa Oncologica, Sede di Roma [W. G., L. R.], Rome; and Dipartimento di Medicina Sperimentale e Patologia, Università degli Studi di Roma, "La Sapienza," 324 00161 Rome [O. G., L. R., L. F., A. A., P. G.], Italy
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ABSTRACT
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Purpose: Despite the great number of studies performed to detect circulatingmarkers of disease progression in colorectal cancer, few have shown a clinical use; among those, epidermal growth factor receptor (EGFR) and, more recently, interleukin (IL)-10. In this article, we sought to investigate how primary surgery could affect expression levels of EGFR, IL-6, and IL-10 in blood from colorectal cancer patients.
Experimental Design: We investigated by reverse transcriptase-PCR assay the expression at mRNA level of EGFR, IL-6, and IL-10 in blood samples taken from 56 colorectal cancer patients. Each gene expression was evaluated 1 day before and 20 days after primary surgery. Persistence of each gene in blood after surgery was then correlated to the relapse free time in a follow-up of 3 years.
Results: In blood samples taken before surgery, EGFR, IL-6, and IL-10 were found expressed in 62, 100, and 100% of patients, respectively. EGFR expression, but not IL-6 and IL-10, correlates with stage of disease. In the group of 41 patients who underwent follow-up studies, EGFR was found persistently high in 67%; 94% of them had relapse. Persistence of IL-10 after surgery also identifies relapses in 89% of cases. IL-6 persistence was not found to significantly correlate to progression of disease.
Conclusions: Persistence of both EGFR and IL-10 in blood of colorectal cancer patients after surgery identifies patients with high propensity to relapse. These findings may suggest a clinical use of preoperative EGFR/IL-10 reverse transcriptase-PCR assay in the prediction of tumor recurrence.
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INTRODUCTION
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Patients who undergo potentially curative surgery for colorectal cancer have a risk of recurrence mainly because of tumor dissemination via blood or lymphatic circulation. In recent years, many studies have been focused on the use of circulating molecular markers in the clinical approach to colorectal cancer patients, as well as in patients with other solid tumors (1)
. Studies performed in the last years have investigated the role of marker genes in blood of gastrointestinal cancer patients; among those, CEA3
(2)
, cytokeratins 19 and 20 (3)
, guanylyl cyclase C (3)
, and, more recently, carcinoembryonic gene member 2 (4)
. Through the increasing number of reports, the clinical use of these markers was found limited, mainly because of their detection in variable percentage of blood samples from healthy donors.
The recent observation that EGFR is absent in hematopoietic cells and overexpressed in 50–70% of blood drawings from colorectal cancer patients led to the hypothesis that it might represent a promising candidate in the early identification of circulating tumoral cells (5)
. EGFR transcripts were found in a high percentage of patients with metastatic bladder tumors (6)
; furthermore, in bladder cancer, its persistence after primary surgery seems to identify patients with high risk to relapse (7)
.
The prognostic significance of IL-6 and IL-10 in colon cancer patients was also recently investigated (8)
. Both were found elevated in blood of patients with different cancer types, and several reports have focused on the role of serum levels of these cytokines in monitoring tumor recurrence after surgery. Application of more than one molecular marker would be auspicable to enhance the sensitivity and specificity of RT-PCR assays in the diagnosis of circulating tumoral cells.
In this study, we analyzed by RT-PCR assay a combination of three markers, specifically EGFR, IL-6, and IL-10, in blood drawings performed before and after surgery from 56 patients affected by colorectal cancer. We found that persistence of EGFR and IL-10 transcripts in blood after surgery identifies patients with high risk to relapse.
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PATIENTS AND METHODS
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Patients.
Sixty patients were accrued, all with histologically proven colorectal cancer, who came to our observation between March 1999 and March 2001. We had 21 stage I, 9 stage II, 19 stage III, and 7 stage IV. (Table 1)
. Informed consent was obtained from all patients. A main inclusion criterion was the absence of any concomitant pathological conditions that could influence the expression levels of ILs 6 and 10:acute or chronic inflammatory disease, autoimmune disorders, and allergic conditions. Four patients were initially excluded from the study for the following reasons: 1 recent acute myocardial infarction; 1 multiple sclerosis; 1 anamnesis positive for allergy; and 1 high fever at the moment of blood drawing.
Because of the lack of clinical data from 15 patients after surgery, follow-up data were available only in 41 patients, who enter now the fourth year of follow-up. Follow-up was performed regularly at 3–6-month intervals by standard procedures. CEA values of the first year of follow-up are shown in Table 2
.
Sixteen of 41 patients who entered in follow-up studies relapsed; among them, 10 developed distant metastases, another 5 had local recurrence, and 1 died because of the disease. The median time of recurrence was 12 months (range, 3–30 months). Blood samples from 30 healthy donors were used as controls. Healthy donors were chosen in a group of young, healthy, nonsmoking subjects with anamnesis negative for allergic conditions and the absence of inflammatory disease at the moment of blood drawing. Subjects were ages between 25 and 40 years (median age, 32 years). There were 15 males and 15 females.
Blood Collection.
In all patients two blood drawings were performed, the first the day before surgery (day -1) and the second 20 days after surgery (+20). In the 16 patients who relapsed, a blood drawing was performed also at time of relapse. RNA extraction was performed using Trizol LS according to manufacturer’s guidelines. The quantity and quality of RNAs were determined by absorbance at 260 and 280 nm.
Reverse Transcriptase-PCR.
Total RNA (1 µg) from blood samples was reverse transcribed in a final volume of 20 µl with 100 pmol of random examer and 50 units of murine leukemia virus reverse transcriptase (Perkin-Perkin-Elmer Corp., Norwalk, CT), according to the manufacturer’s guidelines.
Aliquots from blood samples corresponding to 100 ng of RNA were then amplified in PCR buffer containing 25 pmol each primer and 1.25 units of Taq Polymerase in a final volume of 50 µl.
Aliquots of the same cDNA were amplified with ß-actin, EGFR, IL-6, and IL-10 primers. Amplification for ß-actin and EGFR was performed for 35 cycles; a cycle profile consisted in denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Amplification for IL-6 was performed for 30 cycles; each cycle profile consisted in denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s. Amplification for IL-10 was performed for 30 cycles, with an annealing temperature of 55°C. A final extension step at 72°C for 7 min completed the reaction. A sample without RNA was included in each experiment of reverse transcriptase-PCR as negative control; RNA extracted from HeLa cell line was used as positive control for the expression of ß-actin and EGFR, whereas RNA from PBLs was used as control for IL-6 and IL-10 amplifications.
The expected size of the amplified products were 168 bp for ß-actin, 441 bp for EGFR, 652 bp for IL-6, and 356 bp for IL-10.
Amplification products were then size-fractionated by agarose gel electrophoresis and quantified by densitometric scanner. To evaluate differences in each gene expression before and after surgery, we related the values obtained by densitometric scanning of EGFR, IL-6, and IL-10 signals to the values obtained for ß-actin in the corresponding sample.
Statistical Analysis.
Statistical analysis was performed with BMDP statistical package (BMDP Statistical Software, Inc., Los Angeles, CA). 
2 test was used to correlate EGFR, IL-6, and IL-10 mRNA expression with clinical stage of tumors and relapses. P < 0.05 was considered statistically significant.
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RESULTS
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Reverse transcriptase-PCR performed on blood samples from healthy donors showed the presence of EGFR, IL-6, and IL-10 transcripts in 0 of 30, 30 of 30, and 30 of 30, respectively. Expression levels of IL-6 and IL-10 were lower than those observed in cancerous patients (Fig. 1)
.
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Fig. 1. EGFR, IL-6, and IL-10 amplification products in blood from 5 healthy donors. Pos: positive controls: HeLa cell line for EGFR; PBL for IL-6 and IL-10 amplifications.
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We then evaluated basal levels of EGFR, IL-6, IL-10 mRNA expression in blood samples taken from all 56 patients before surgery. EGFR was expressed in 36 of 56 patients (62%), whereas IL-6 and IL-10 were both expressed at high levels in all samples (100%). When we correlated each gene expression with stage of disease, we found that EGFR is expressed in 9% of stage I/II and in 100% of stage III/IV. The correlation between EGFR expression and stage was found statistically significant (P = 0.0001; Table 3
). Both IL-6 and IL-10 were found expressed in 100% of low stage, as well as in 100% of high stage.
In the group of 41 patients who underwent follow-up studies, we then performed a correlation between the persistence of each gene expression after surgery and the emergency of relapse. EGFR was found persistently highly expressed in 67% of patients; among them, 94% were in progression of disease, whereas 6% were disease free after 36 months (P = 0.0001). Persistence of IL-6 mRNA expression after surgery was found in 49% of patients, 52% who progressed, and 48% in stability of disease. This difference was not found statistically significant. IL-10 mRNA levels remained extremely high in 44% of patients; among them, 89% relapsed, whereas 11% are at present in stability of disease (P < 0.05; Table 4A
).
In the group of 16 patients who relapsed, the persistence of both IL-10 and EGFR after surgery was found in 94% of patients, whereas in the group of disease free, such persistence was not found in any patient (P = 0.0001). On the contrary, in all disease-free patients we observed a clear decrease of IL-10 and EGFR levels after surgery (Table 4B
, Fig. 2
). Persistence of IL-10 and EGFR in relapsed patients is shown in Fig. 3
. We did not find always a clear correlation between CEA levels and relapses (Table 2)
. In fact, reduction of CEA levels 3 months after surgery does not correlate in our casuistry to the appearance of relapses (45% of free patients versus 54% of relapsed).
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Fig. 2. EGFR and IL-10 amplification products in blood of three patients with disease-free colorectal cancer. For each patient: left band, amplification product in presurgical blood drawing; right band, amplification product in postsurgical blood drawing. Pos: positive controls: HeLa cell line for EGFR; PBL for IL-10 amplifications.
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Fig. 3. ß-Actin, EGFR, and IL-10 amplification products in blood of four patients with relapsed colorectal cancer. For each patient: left band, amplification product in presurgical blood drawing; right band, amplification product in postsurgical blood drawing. Pos: positive controls: HeLa cell line for ß-actin and EGFR; PBL for IL-10 amplifications.
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DISCUSSION
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The role of EGFR-based reverse transcriptase-PCR assay to detect circulating cells was already established (5
, 6)
. Here, for the first time in colorectal cancer, we found a correlation between persistence of high EGFR levels after surgery and poor prognosis of patients, maybe reflecting the inefficacy of curative surgery in this group of patients. Similar results have been described by our group in bladder cancer, where progression of disease was observed in patients who had persistently high EGFR expression levels in blood drawings performed after surgery (7)
. Some studies have suggested that many cancer types are able to secrete their own IL-10; to stress this hypothesis, this cytokine has been detected in sera from patients with solid and hematopoietic malignancies (9, 10)
. In our hands, high IL-10 mRNA expression after surgery identifies patients with propensity to relapse; these results are in agreement with what published by Galizia et al. (8)
, despite the different methods used. Patients who are disease free after 36 months had a decrease or disappearance of both EGFR and IL-10 transcripts after surgical removal of the tumoral mass. In these patients, we hypothesize that decreasing levels of both IL-10 and EGFR after surgery may reflect the efficiency of the curative surgery. By the contrary, data concerning IL-6 persistence in blood of patients after surgery are not so encouraging. Although IL-6 is also produced by the tumor itself and its expression levels seem higher in cancerous patients compared with healthy donors, its persistence after surgery was not found to correlate to relapse-free time. Thus, its prognostic significance seems extremely limited in our casuistry, as well as in other studies (8)
. In our series of patients, the use of CEA in the first months of follow-up seems quite controversial because its reduction 3 months after surgery was found in 54% of patients who had relapses in the following months. CEA seems to be more useful after 6 or 12 months of follow-up, when its elevation is often correlated to the appearance of relapses. This additionally confirms the use of EGFR/IL-10 assays because elevated levels immediately after surgery seem to have prognostic significance.
Additional studies with larger number of patients are still required to validate the clinical use of IL-10/EGFR-based reverse transcriptase-PCR assays in the follow-up of colorectal cancer patients after surgery.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Partially supported by Associazione Ricerche Geriatriche ed Oncologiche. 
2 To whom requests for reprints should be addressed, at Dipartimento di Medicina Sperimentale e Patologia, Università degli Studi di Roma "La Sapienza," Viale Regina Elena, 324 00161 Rome, Italy. Phone: 39-6-49973011; Fax: 39-6-4454820; E-mail: paola.gazzaniga{at}uniroma1.it
3 The abbreviations used are: CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; PBL, peripheral blood lymphocyte.
Received 7/17/02;
revised 2/11/03;
accepted 2/13/03.
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ABSTRACT
INTRODUCTION
