Immunohistochemical expression of C-reactive protein in squamous cell carcinoma of the esophagus - significance as a tumor marker

Cancer Letters 192 (2003) 89–95 www.elsevier.com/locate/canlet

Immunohistochemical expression of C-reactive protein in squamous cell carcinoma of the esophagus - significance as a tumor marker Tadahiro Nozoea,*, Daisuke Korenagaa, Motonori Futatsugib, Hiroshi Saekib, Yoshihiko Maeharab, Keizo Sugimachib a b

Department of General Surgery, Fukuoka Dental College Hospital, 2-15-1, Tamura, Sawara Ward, Fukuoka, 814-0193, Japan Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi Ward, Fukuoka, 812-8582, Japan Received 20 August 2002; received in revised form 17 October 2002; accepted 28 October 2002

Abstract C-reactive protein (CRP) is a prototype acute phase protein which has been known to be synthesized in hepatocytes. Although serum elevation of CRP has been reported to be an indicator of the unfavorable outcome of the patients in some malignant tumors, the expression of the protein in carcinoma cells has not been investigated. The aim of the current study was to assess the immunohistochemical expression of CRP in squamous cell carcinoma (SCC) of the esophagus and to find its biological significance. Immunohistochemical examination for CRP expression was performed for 37 advanced esophageal SCCs with the depth of T2, T3 or T4, which had been surgically resected without preoperative therapy for the patients. Eighteen carcinomas (48.6%) demonstrated immunohistochemical CRP expression. Univariate analysis showed that the prognosis of the patients with esophageal SCCs expressing CRP was significantly worse than that in patients with tumors without CRP expression (P ¼ 0:017). Moreover, CRP expression was found to be an independent prognosticator in patients with esophageal SCCs in the multivariate analysis (P ¼ 0:036). To the best of authors’ knowledge, this is the first report that demonstrated the possible carcinoma-related expression of CRP in SCCs of the esophagus and its biological significance as the prognosticator of the patients. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Esophagus; Squamous cell carcinoma; C-reactive protein; Immunohistochemistry; Tumor marker

1. Introduction C-reactive protein (CRP) was initially identified as a substance which reacted to the C polysaccaride of Streptococcus pneumoniae in the serum of a patient * Corresponding author. Fax: þ 81-92-801-0735. E-mail address: [email protected] (T. Nozoe).

with pneumonia. CRP is a prototype acute phase protein and it has been well known to be synthesized in hepatocytes in response to the inflammatory changes [1]. CRP is an acute phase protein which increased in the serum responsively to the inflammatory diseases and its expression has been known to be induced by pro-inflammatory cytokine, interleukin-6

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 8 3 5 ( 0 2 ) 0 0 6 3 0 - 4

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[2,3], which acts as the growth factor in the malignant tumors. The significance of serum elevation of CRP as an indicator of the malignant potential of the tumor and/or therefore the unfavorable outcome of the patients has been investigated in human gastrointestinal carcinomas [4 –11], renal cell carcinoma [12], ovarian carcinoma [13], or myeloma [14]. Our motivation of adopting CRP as a marker of malignant potential in colorectal carcinoma [7] and esophageal carcinoma [11] was that the malignant tumor must be one of the inflammatory diseases which might possibly cause the serum elevation of CRP. Previous investigations including our studies with regard to the clinicopathological significance of serum elevation of CRP indicated that CRP might be one of the tumor markers. However the origin of the serum elevation of CRP in patients with malignant tumors has not been focused and it also remains controversial whether the serum elevation of the protein is solely derived from the production of CRP by the hepatocytes as the response to the inflammatory events or from the production and accumulation of carcinoma cells. In the current study, we investigated immunohistochemically whether CRP is expressed in the carcinoma cells of esophageal squamous cell carcinoma and elucidated its biological significance as a marker of malignant potential.

2. 2. Materials and methods 2.1. Patients and samples The tissue samples were collected from the 37 patients with squamous cell carcinoma of the esophagus with the depth of T2, T3 or T4 determined according to TNM classification of malignant tumors decided by International Union Against Cancer [15]. All of the patients who had these tumors had been treated with esophageal resection and reconstruction from September 1989 to December 1998 in the Department of Surgery and Science, Kyushu University. None of the patients was treated with such preoperative therapies as radiation and chemotherapy. The study population was consisted of 32 male and

five female, and the age ranged from 48 to 89 years with a mean of 66. Follow-up for the patients was continued until death and only patients who died of esophageal SCCs were included in the tumor-related deaths. The interval of the follow-up after surgical treatment ranged from 32 days to 9 years and 7 months with a mean of 3 years. The clinicopathological investigation was made on the basis of guidelines for clinical and pathologic studies on carcinoma of the esophagus established by the Japanese Society for Esophageal Diseases [16], and the clinical stages were determined according to TNM classification of International Union Against Cancer [15]. 2.2. Immunohistochemical expression of CRP In order to detect CRP expression, an avidinbiotin-peroxidase complex method using a sheep antihuman polyclonal CRP antibody (Biogenesis Ltd, England, UK) was performed. The antibody is specific for CRP and is minimal cross-reactive with other related proteins, according to the manufacturer. 4 mmthick sections sliced from paraffin-embedded specimen were prepared on the slideglasses pre-coated with silane. After removing paraffin in xylene and washing in graded series of ethanol, the sections were placed in Tris-buffered saline (TBS) for 10 min. Then the sections were incubated with a blocking solution for 5 min to block an endogenous peroxidase activity, and were placed in TBS. Next the sections were incubated with TBS including 1% concentration of bovine serum albumin for 20 min to block nonspecific binding of the immunoreagents. The nonspecific binding of the secondary antibody was blocked with normal rabbit serum for 15 min. Sections were incubated with a 1:100 diluted sheep anti-human polyclonal CRP antibody for 30 min and rinsed with TBS. Avidin-biotin-peroxidase complex method was performed using VECTASTAIN ABC systems (Vector laboratories, Inc., Burlingame, CA). Then the localization of CRP was visualized with diaminobenzidine tetrahydrochloride. The specimens with CRP positive cells more than 10% of examined cancer cells were considered to have a positive immunostaining of CRP.

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2.3. Serum value of CRP

2.4. Statistical analysis

Serum CRP was measured by latex photometric immunoassay [17] using CRP-L kit (Yatoron Co., Ltd, Tokyo, Japan). Serum levels of CRP ranging from 0 to 5 mg/l are normal in this assay and therefore, the serum CRP concentration of more than 5 mg/l was considered as a positive elevation.

The chi-square test and Student’s t-test were used to compare the clinicopathological data. The cumulative survival rates were calculated by the Kaplan – Meier method and the survival curves were tested by the Mantel – Cox method. Multivariate analysis for survival was calculated according to Cox’s pro-

Fig. 1. Representative findings of immunohistochemical expression of CRP in SCC cells. (A) Moderately differentiated SCC ( £ 200). (B) Poorly differentiated SCC ( £ 200).

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Table 1 Correlation of CRP expression with clinicopathological factors in esophageal SCCa Variable

Sex

CRP expression

Male Female

Age (years) Location of tumor

Histology

Tumor size (cm) Depth of tumor

Lymph nodes metastasis Lymphatic invasion Venous invasion TNM stage

Curability

Upper Middle Lower Well Moderate Poor T2 T3 T4 Positive Negative Positive Negative Positive Negative IA IIB III Curative resection Noncurative resection

P value

Positive (n ¼ 18)

Negative (n ¼ 19)

16 (88.9) 2 (11.1) 65.3 ^ 9.7 2(11.1) 11 (61.1) 5 (27.8) 2 (11.1) 13 (72.2) 3 (16.7) 5.4 ^ 2.3 3 (16.7) 11 (61.1) 4 (22.2) 11(61.1) 7 (38.9) 11(61.1) 7 (38.9) 9 (50.0) 9 (50.0) 6 (33.3) 2(11.1) 10 (55.6) 12 (66.7) 6 (33.3)

16 (84.2) 3(15.8) 67.3 ^ 10.0 0 13 (68.4) 6 (31.6) 3 (15.8) 9 (47.4) 7 (36.8) 5.8 ^ 2.3 6 (31.6) 10 (52.6) 3 (15.8) 8 (42.1) 11 (57.9) 7 (36.8) 12 (63.2) 7(36.8) 12 (63.2) 8 (42.1) 3(15.8) 8 (42.1) 12 (63.2) 7 (36.8)

0.677 0.545 0.328

0.286

0.628 0.559

0.248 0.140 0.419 0.711

0.823

a All data are expressed as mean ^ SEM. CRP, C-recative protein; SCC, squamous cell carcinoma; Well ¼ well differentiated squamous cell carcinoma; Moderate ¼ moderately differentiated squamous cell carcinoma; Poor ¼ poorly differentiated squamous cell carcinoma. Values in the parentheses are the percentages.

portional hazards model with the forward stepwise manner A P value of less than 0.05 was regarded as significant.

3. Results There were 18 patients (48.6%) with esophageal

SCCs showing CRP expression in carcinoma cells (Fig. 1) and the other 19 SCCs did not express CRP. The clinicopathological characteristics of the patients were shown in Table 1. No significant difference was observed with regard to sex, age, and location, histological differentiation, size and depth of the tumors. Regarding such pathological factors as lymph node metastasis, lymphatic invasion and

Table 2 Relationship between immunohistochemical expression and serum elevation of CRPa Immunohistochemical expression of CRP

Serum elevation of CRP

a

Positive Negative Total

Positive

Negative

5 (62.5) 3 (37.5) 8 (100)

2 (15.4) 11 (84.6) 13 (100)

P ¼ 0.026. CRP, C-reactive protein. Values in the parentheses are the percentages.

Total

7 14 21

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Fig. 2. Survival curves. Survival rate of patients with esophageal squamous cell carcinomas expressing CRP (thick line) was significantly worse that that of patients with carcinomas without CRP expression (thin line) (P ¼ 0:017)

venous invasion, although the incidence was comparatively higher in carcinomas expressing CRP than that in carcinomas without CRP expression, there was not a significant difference. No significant difference was observed with regard to TNM stage and the proportion of curative resection. Value of preoperative serum CRP was measured in 21 patients. Preoperative elevation of serum CRP was found in five (62.5%) out of eight patients with SCCs associated with immunohistochemical expression of CRP and two (15.4%) out of 13 patients with SCCs without CRP expression. There existed a significant difference between these proportions (P ¼ 0:026; Table 2). 1-, 3-, and 5-year survival rates in patients with esophageal SCCs expressing CRP were 81.6%, 29.7% and 22.2%, respectively, which were significantly worse than those in patients with tumors without CRP

expression (87.5%, 67.7% and 60.2%, respectively, P ¼ 0:017; Fig. 2). Additionally, a multivariate analysis demonstrated that TNM stage of the tumors (P ¼ 0:017) and CRP expression (P ¼ 0:018) were found to be an independent prognosticator in patients with esophageal SCCs (Table 3).

4. Discussion During the series of our investigations with regard to the correlation of serum CRP with the biological behavior of esophageal carcinoma [11,18] and colorectal carcinoma [7,19], we have considered that the elevation of the enzyme could be derived from the inflammatory response of the host to the existence of

Table 3 Factors independently associated with prognosis of patients with esophageal SCCa Variable

Regression coefficient

Standard error

Odds ratio (95% Confidence interval)

P value

TNM stage CRP expression

1.167 1.185

0.489 0.501

3.215 (1.233–8.403) 3.268 (1.225–8.696)

0.017 0.018

a

SCC, squamous cell carcinoma; CRP, C-recative protein.

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malignant tumor which accounts for one of the inflammatory diseases. It is well known that CRP is synthesized mainly in hepatocytes [1,20]. However there have been some investigations presenting that an excessive elevation of CRP is occasionally found in such patients with liver failure who are requiring liver transplantation [21,22]. Moreover although there has been some investigations showing interleukin-6 (IL-6) expression in malignant tumor cells [23 – 26] including esophageal squamous cell carcinoma (SCC) [23], the possible carcinoma-related expression of CRP, which is generally known to be regulated by IL-6, has not been focused. Therefore we thought that there might be the other pathway of serum elevation of CRP in patients with malignant tumors and have been considering the possibility that carcinoma cells itself might produce CRP other than the established theory that hepatocytes synthesize CRP responsively to the carcinoma. The validity to perform an immunohistochemical investigation for CRP expression in esophageal SCCs in the current study is that an immunohistochemical examination would instantly demonstrate whether carcinoma cells have positive response to CRP and that if carcinoma cells responses positively, the localization of the CRP expression would be also revealed. Moreover to the best of our knowledge, the investigation on the immunohistochemical expression of CRP in the human malignant tumors has not been reported before. The proportion of CRP expression (48.6%, 18 out of 37) was roughly concomitant with the proportion of serum elevation of CRP in patients with esophageal carcinoma with the tumor depth of T2, T3 or T4 in our recent investigation [11]. Moreover a significant correlation between immunohistochemical expression of CRP of the tumor and preoperative elevation of serum CRP could be found in the current study. While this report is the first one referring to the significance of immunohistochemical expression of CRP in carcinoma cells, the appropriateness of the boarder line to determine the positive response for CRP set in the current study should be discussed more fully in the further investigation. Although the mechanism for the production of CRP in carcinoma cells is yet to be elucidated, our results demonstrated that esophageal SCC cells might produce CRP and

furthermore CRP expression in carcinoma cells might also be a marker for predicting the outcome of patients with esophageal SCCs. In conclusion, CRP was found to be expressed in esophageal SCC cells and the serum elevation of the protein may be partially derived from the instant produce of the substance by the carcinoma cells as well as by the synthesis in the hepatocytes as a bioresponse to carcinoma. To the best of our knowledge, this is the first report with regard to the possible carcinoma-related expression of CRP and its biological significance.

Acknowledgements This work was supported in part by a research grant from the Ministry of Education, Science, Sports and Culture in Japan (Frontier Research Grant and Grant for Science Research 13671308).

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