Prognostic significance of caspase-3 expression in primary resected esophageal squamous cell carcinoma

EJSO 2003; 29: 44±48 doi:10.1053/ejso.2002.1338

Prognostic significance of caspase-3 expression in primary resected esophageal squamous cell carcinoma Jiun-Yi Hsia, Chih-Yi Chen, Jung-Ta Chen*, Chung-Ping Hsu, Sen-Ei Shai, Shyh-Sheng Yang, Cheng-Yen Chuang, Pei-Yen Wang and Jen Miaw Division of Thoracic Surgery, Department of Surgery, *Department of Pathology, Taichung Veterans General Hospital, Taichung, Taiwan

Aims: The present study retrospectively examines the expression of caspase-3 in primary resected esophageal squamous cell carcinoma (ESCC) and the correlation between the outcome of patients and the expression of proteins. Methods: Immunohistochemistry and Western blot analyses were used to analyse the expression of caspase-3 in 40 archival specimens of patients with primary resected ESCC. Results: According to our cut-off point of the staining for caspase-3, 24 (60%) cases were positive and 16 (40%) negative. Caspase-3 expression correlated with a significant favorable prognosis in primary resected ESCC (P ˆ 0.02). A multivariate analysis of clinical and biological factors indicated that stage, tumor differentiation, and caspase-3 expression were independent prognostic factors. Conclusions: Caspase-3 expression might be a good and new prognostic indicator for primary resected ESCC. # 2002 Elsevier Science Ltd. All rights reserved. Key words: caspase-3; esophageal squamous cell carcinoma.

INTRODUCTION Squamous cell carcinoma of esophagus accounts for almost all esophageal malignancy in Taiwan. The prognosis of patients with ESCCs has only slightly improved during recent years. Even in resectable stage, the results of surgical excision have been unsatisfactory, with 5-year survival rates about 20%.1 The situation makes the availability of prognostic factors for outcome highly desirable to guide the use of (neo) adjuvant treatment modalities. Deranged regulation of apoptotic cell death has evolved as an important keyplayer not only for the pathogenesis of cancer but also for its biological behavior.2,3 A recently discovered family of proteases, called caspases, are the key effectors of the cellular death.4,5 Among the caspases, caspase-3 (also known as CPP32, YAMA, or apopain) is probably the one that so

Correspondence to: Jiun-Yi Hsia, MD, FCCP, Division of Thoracic Surgery, Department of Surgery, Taichung Veterans General Hospital, 160, Sec 3, Taichung-Kang Rd, Taichung, Taiwan. Tel: 886 4 23592525 ext. 5045; Fax: 886 4 23599715; E-mail: [email protected] 0748±7983/03/$30.00

far best correlates with apoptosis.5,6 Furthermore, caspase-3 expression could be detected in several human malignancies such as neuroblastomas,7 gliomas,8 and lung cancers.9 Koomagi et al.9 found that caspase-3 was expressed at 72% of non-small cell lung cancers with a favorable prognosis. Until now, no data have been available concerning the prognostic role of caspase-3 expression in primary resected esophageal cancer. In order to properly evaluate this, the present study examined the expression pattern and the prognostic significance of the caspase-3 in primary resected ESCCs.

MATERIALS AND METHODS Patients Tumor samples were obtained from 40 consecutive patients who underwent surgical intervention for ESCC at the division of Thoracic Surgery of Taichung Veterans General Hospital between June 1994 and July 1996. No pre-operative radio- or chemotherapy had been #

2002 Elsevier Science Ltd. All rights reserved.

PROGNOSTIC SIGNIFICANCE OF CASPASE-3 EXPRESSION performed. All patients have been followed up for overall survival.

Immunohistochemistry Paraffin-embedded tumor specimens that had been fixed in neutral-buffered formalin were sectioned (4 mm) and placed on poly-L-lysine-coated glass slides. After microwave pretreatment in 10 mM citric acid monohydrate (pH 6.0) three times for 5 min at 750 W, slides were incubated overnight at 4 C with monoclonal antibodies against caspase-3 (clone E-8; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After a second incubation with biotin-conjugated antimouse antibody, slides were incubated with avidin±biotin-peroxidase reagent (LSAB kit, Dakopatts, Denmark). Reaction products were visualised by immersing slides in diaminobenidine tetrachloride and finally counterstained with hematoxylin. Negative controls were performed by replacing the primary antibodies with dilution buffer. Positive staining of normal squamous cell epithelium provided a positive internal control for immunostaining. Using light microscopy, the immunohistochemical expression of caspase-3 was examined by two observers without knowledge of the clinical outcome. The immunohistochemical staining was analyzed according to a scoring method that previously validated by Koomagi et al.9 The tumor samples were graded as negative and either weakly (, 25% positive cells), moderately (25±50% positive cells) or strongly positive (>50% positive cells). Finally, we grouped the tumors into two groups: the tumors without or with weak staining were classified as negative, the moderately and strongly stained tumors as positive.

45

Statistical analysis Survival rates were calculated by the Kaplan±Meier method for analysis of censored data. The significance of differences in survival was analyses by means of the log-rank test: Ps , 0.05 were considered significant. In multivariate analysis, independent prognostic factors were determined by the Cox proportional hazards model. The level of significance was set at P , 0.05.

RESULTS A total of 40 samples of primary resected ESCCs were processed for caspase-3 expression by immunohistochemistry. Table 1 lists the clinicopathological features and caspase-3 expression in primary resected ESCCs. All patients were men and the median age was 61 (range 40±79).

Expression of caspase-3 in primary resected ESCCs When present, the staining for caspase-3 was strong in 17 cases, moderate in 7 cases, weak in 7 cases, and negative in 9 cases. According to our cut-off point of the staining for caspase-3, 24 (60%) cases were positive (Fig. 1) and 16 (40%) negative. No significant relationship between caspase-3 and the clinical parameters of age, stage, lymph node metastasis, and histological type was found. Table 1 Clinicopathological features and caspase-3 expression in ESCCs Variable

Caspase-3

Western blot analysis Protein was isolated from esophageal cancer tissues and normal esophageal tissues by using the RIPA buffer (Boehringer Mannheim, Germany). After electrophoresis on a 12% polyacrylamide gel in the presence of SDS and transfer to a PVDF membrane (Amersham Pharmacia Biotech AB, Uppsala, Sweden) through electroblotting, the transferred protein was detected with 0.2% Ponceau S in 3% TCA. Blocking the membrane in 5% non-fat dried milk/PBST for 60 min. The immunodetection of caspase-3 was performed using mouse anti-caspase-3 monoclonal antibody diluted in 1% non-fat dried milk/ PBST (1:200) for 90 min. After washing, the membrane was incubated with goat anti-mouse IgG (BD Biosciences, Palo Alto, CA, USA) labeled horseradish peroxidase as second antibody diluted in 1% non-fat dried milk/PBST (1:2000) for 60 min. After washing, the membrane was incubated in the chemiluminescence reagent (NEN, Boston, MA, USA) for one min, excess reagent was removed and the membrane placed in a plastic sheet protector and exposed to X-film for 10 s.

Positive

Negative

Sex Male Female

40 0

24

16

Age 60 >60

18 22

10 14

8 8

Stage (UICC, 1987) I & IIA IIB & III

15 25

12 12

3 13

Histological differentiation Moderate Poor

30 10

18 6

12 4

Lymph node metastasis Positive Negative

22 18

11 13

11 5

Surgical intervention Thoracotomy with esophagectomy Transhiatal esophagectomy

33 7

46

J.-Y. HSIA ET AL.

Figure 1 Immunohistochemical staining of caspase-3 (200) in primary resected ESCC (A: strongly stained, B: moderately stained, C: weakly stained).

Immunoblot analysis of caspase-3 expression in primary resected ESCCs The specificity of the caspase-3 protein was accessed by immunoblot analysis. As shown in Fig. 2, the anticaspase-3 antibody reacted with a single 32 kDa band in solid tumors and normal esophageal tissue, corresponding to the expected molecular weight.

Correlation between expression of caspase-3 in primary resected ESCCs and survival The overall survival of all 40 patients had been followed up every 3 months for the first 2 years after the end of treatment; afterward every 6 months. At the end of the follow-up period ( July 31, 2001), 7 of the 40 patients (17.5%) were still alive. Analysis of the immunohistochemical expression of caspase-3 with respect to survival is shown in Fig. 3. Analysis based on the log rank test revealed that patients with caspase-3 positive tumors had a significantly more favorable overall survival than patients with caspase-3 negative tumors (P ˆ 0.0201; Fig. 3). A multivariate analysis of the clinical and immunohistochemical data indicated that stage, tumor differentiation, and caspase-3 expression were independent prognostic factors (Table 2).

DISCUSSION Squamous cell carcinoma of esophagus is one of the most malignant tumors, and patients with this disease have a dismal prognosis. The prognosis of patients with this disease has only slightly improved during recent years. Recently, intensive molecular and biological studies have demonstrated the significance of oncogenes, tumor suppressor genes, and growth factors in carcinogenesis and malignant transformation of cells, but there is still no

Figure 2 Western blot analysis identified the 32 kDa caspase-3 expression in esophageal carcinomas (Lanes 1±3) and normal esophageal tissue (Lane 4).

significant prognostic indicator that could be used for esophageal carcinoma. Multiple factors are responsible for modulation of tumor growth and prognosis of patients with malignant tumors. Recently, much attention has been paid to the maintenance of tumor volume by cellular proliferation and apoptosis. An imbalance between apoptosis and proliferation is believed to underlie tumor development and prognosis. Theoretical considerations and in vitro studies suggest that caspase-3 plays a major role in apoptosis.4,8,10,11 Kondo et al.8 demonstrated that the retroviral transfer of caspase-3 induces apoptosis in malignant glioma cell in vitro. Additionally, treatment of mouse tumors with a retrovirus that contained caspase-3 significantly inhibited tumor growth through an induction of apoptosis. In this study, caspase-3 positive staining was a significant prognostic factor in predicting survival in primary resected esophageal squamous cell carcinoma: there was a 25% 5-year survival in caspase-3 positive patients compared with a 6% 5-year survival in caspase-3 negative patients (P ˆ 0.02). To date, no data are available with regard to the prognostic significance of caspase-3 expression in primary resected esophageal carcinoma patients. But consistent with the previous studies on neuroblastomas7 and non-small cell lung

PROGNOSTIC SIGNIFICANCE OF CASPASE-3 EXPRESSION

47

100% 90% 80%

Survival

70% 60% 50% 40%

Casp 3-pos. ( n=24 ) =>25.00 %

30% 20%

Casp 3-neg. ( n=16 ) => 6.25 %

10%

P=0.0201

0% 0

6

12

18

24

30

36

42

48

54

60

66

72

Months

Figure 3 Overall survival in 40 primary resected ESCC patients in relation to expression of caspase-3.

Table 2 Multivariate analysis of prognostic factors for survival using Cox's proportional hazard model Parameters Age (60 vs >60) Stage (I, IIA vs IIB, III) Lymph node metastasis (positive vs negative) Tumor differentiation (moderate vs poor) Caspase-3 expression (positive vs negative)

Coefficient

P

Risk ratio

0.73 2.02 ÿ0.57 ÿ1.13 ÿ1.12

0.097 0.0227* 0.493 0.0095* 0.0042*

2.08 7.57 0.56 0.32 0.33

95% CIa 0.88±4.93 1.33±43.19 0.11±2.91 0.14±0.76 0.15±0.70

a

95% CI, 95% confidence interval. * P , 0.05.

cancers,9 the moderate to strong caspase-3 staining in these tumors is to be associated with a favorable prognosis. These results suggest that a reduced expression of caspase-3 in tumors promotes the growth of tumor cells and results in a shorter overall survival. Kurabayashi et al.12 demonstrated no significant relationship of caspase-3 expression and survival in esophageal squamous cell carcinoma, but all their patients underwent chemotherapy preoperatively. We consider this is why their results are different from our present and previous studies.13 To date, pathological staging remains the most reliable determinant of prognosis in ESCC, and the main factor in the choice of curative treatment. However, it is of great interest to verify new biological markers to define the risk of relapse or to decide on the use of (neo) adjuvant treatment. In conclusion, our investigation demonstrates that caspase-3 staining is associated with a favorable prognosis and the expression of caspase-3 might be developed as a good, new prognostic factor in primary resected squamous cell carcinomas.

ACKNOWLEDGEMENTS This research was supported by a grant TCVGH914701A from the Research Committee of Taichung Veterans General Hospital, Taiwan.

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