Association between Bcl-2 expression and tumor recurrence in cervical cancer: A matched case-control study

Gynecologic Oncology 102 (2006) 263 – 269 www.elsevier.com/locate/ygyno

Association between Bcl-2 expression and tumor recurrence in cervical cancer: A matched case-control study Sumonmal Manusirivithaya a,⁎, Sumalee Siriaunkgul b , Surapan Khunamornpong b , Manit Sripramote a , Pichet Sampatanukul c , Siriwan Tangjitgamol a , Jatupol Srisomboon d a

Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Bangkok Metropolitan Administration Medical College and Vajira Hospital, 681 Samsen Road, Dusit District, Bangkok 10330, Thailand b Department of Pathology, Faculty of Medicine, Chiangmai University, Chiangmai, Thailand c Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand d Department of Obstetrics and Gynecology, Faculty of Medicine, Chiangmai University, Chiangmai, Thailand Received 28 August 2005 Available online 17 February 2006

Abstract Objectives. To evaluate the association between bcl-2 expression, some pathological factors (lymph-vascular space invasion (LVSI), tumor grade, depth of stromal invasion), and tumor recurrence in cervical cancer. Methods. A matched case-control study was performed in cervical cancer patients who underwent radical hysterectomy and were found to have negative lymph node between January 1992 and June 1998 in Bangkok Metropolitan Administration Medical College and Vajira Hospital and Maharaj Nakorn Chiangmai Hospital, Thailand. Cases were 32 patients who had tumor recurrence within 5 years after surgery. Controls were 63 patients who were disease free for at least 5 years after surgery. Cases and controls were matched for age, stage, histology, and tumor size. All except one case, who had only one matching control, had two controls. Paraffin-embedded cervical cancer tissues of cases and controls were immunohistochemical stained for bcl-2. Results. Bcl-2 positive was demonstrated in 43.7% of tumor from recurrent cases, which was not different from 50.8% from non-recurrent controls (P = 0.378). Tumor grade and LVSI had significant association with tumor recurrence. From multivariable analysis, the patients with grade 2–3 tumors had the odds ratio of 6.9 for recurrence compared to patients with grade 1 tumors. The patients whose tumors had LVSI had the recurrent odds ratio of 5.4 compared to those without LVSI. Conclusions. There was no association between bcl-2 expression and tumor recurrence in cervical cancer, while tumor grade and LVSI had significant association with tumor recurrence. © 2005 Elsevier Inc. All rights reserved. Keywords: bcl-2; Cervical cancer; Radical hysterectomy; Recurrence; Prognosis

Introduction Cervical cancer is the most common cancer, which causes a significant health problem in Thailand. Approximately 5500 Thai women develop invasive cervical cancer per year [1]. Among these, 15–25% of them are in early stage of disease (stage Ib or IIa) [2]. These patients are usually treated by radical hysterectomy and pelvic lymphadenectomy. The 5⁎ Corresponding author. Fax: +66 2 2437907. E-mail address: [email protected] (S. Manusirivithaya). 0090-8258/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2005.12.037

year survival rate of this particular group of patients ranges from 80 to 90% [3,4]. Many factors are found to be of prognostic significance in association with recurrence and survival in cervical cancer. The most significant of which is lymph node metastasis [5–7]. Recurrent rates of the patients with metastases to lymph node are much higher than those with negative node, 33–42% compared to 7–11% [5–7]. The recurrences in cervical patients who have no lymph node metastasis indicate that other tumorrelated variables may contribute to the risk of recurrence as well. These factors are close surgical margin [8], parametrial

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invasion [9], large tumor size [10], deep stromal invasion [11,12], lymph-vascular space invasion (LVSI) [11], adenocarcinoma cell type [10,12], and high histologic grade [11,13]. However, neither of these single risk factors (with the possible exception of parametrial invasion and positive surgical margin) signifies a sufficient adverse prognosis justifying a routine adjuvant therapy [14]. To date, there is no consensus on the criteria to select the patients with negative node and free surgical margin but with other risk factors for adjuvant postoperative treatment. A search for a new prognostic factor such as biomolecular marker is important to identify the risk of recurrence in this group of patients. The bcl-2 oncogene (B cell lymphoma/leukemia-2 gene) was originally detected in B cell lymphomas with a balanced chromosomal translocation t(14;18) (q32;21) [15]. It was later shown in subsequent studies that bcl-2 expression is not only specific for this translocation but is also found in normal tissues, long-lived stem cells demonstrating apoptotic turnover, postmitotic cells and in certain tumor [16,17]. The role of Bcl-2 for inhibition of programmed cell death or apoptosis is well known [18,19]. Overexpression of bcl-2 can prevent or markedly reduce cell killing induced by various stimuli such as chemotherapeutic drugs, gamma and UV radiation, heat shock, some viruses, free radicals, etc. [20]. This mechanism of action may explain why the response to chemotherapy and radiation is reduced in tumors which express bcl-2 [21]. This was also seen in many studies of cervical cancer patients who were treated by radiation, tumors which expressed bcl-2 tended to have poorer prognosis [22–24]. However, the prognostic role of bcl-2 in surgical-treated cervical cancers are somewhat different from those treated by radiation. Some studies found that bcl-2 expression was a good prognostic factor in this group of patients [25–28]. This favorable prognostic role of bcl-2 was explained by the association of bcl-2 and lower cellularity or lower proliferative activity of tumor [29,30]. Moreover, some authors reported the association of bcl-2 expression with favorable prognostic factors, such as tumor stage [28,31], tumor grade [32,33], tumor size [34], depth of invasion [35], and lymph node metastasis [35,36]. However, some other studies could not demonstrate the relationship between bcl-2 expression and good outcome in surgical-treated cervical cancer [37–39]. Hence, the prognostic significance of bcl-2 in cervical cancer patients treated by surgery remained unclear. Furthermore, some previous studies include the patients who received multimodalities of treatment. This even added more questions rather an answer. There was only one study that enrolled the cervical cancer patients treated by surgery alone [25]. This study did not include some important prognostic factors in their multivariable analysis. Hence, the question whether bcl-2 is an independent prognostic factor in surgical-treated cervical cancer remained unanswered. If bcl-2 is a dependent prognostic factor, being associated with other good prognostic factors such as tumor stage, tumor grade, tumor size, depth of invasion, the need to demonstrate its expression at high cost may not yield further benefit than these obvious clinico-pathological factors.

To examine the association between bcl-2 expression and tumor recurrence in node negative surgical-treated cervical cancer, we conducted a matched case-control study in order to control all available and known prognostic factors affecting recurrence, such as patients' age, tumor stage, histology, and tumor size. Other possible associated factors such as tumor grade, tumor depth, and LVSI were also evaluated for the association with tumor recurrence. We used conditional logistic regression to adjust for these uncontrolled associated factors to better evaluate whether bcl-2 was independently associated with tumor recurrence. Materials and methods Patients To study the association between bcl-2 expression and tumor recurrence in cervical cancer patients who were treated by radical hysterectomy and had negative node, we used the matched case-control study with two controls for each case. The study was conducted after an approval from the ethics committees and the directors of Maharaj Nakorn Chiangmai Hospital and Bangkok Metropolitan Administration Medical College and Vajira Hospital. The medical records including follow-up information of all stage Ib and IIa cervical cancer patients, who were primarily treated by radical hysterectomy with pelvic lymph node dissection between January 1992 and June 1998, in both hospitals were reviewed. The exclusion criteria were (1) past or present history of other cancers (two primary cancers), (2) those with rare histologic subtypes such as neuroendocrine carcinoma, lymphoma, etc., (3) receiving any form of preoperative or postoperative adjuvant treatment, (4) lymph node metastasis or parametrial or surgical margin involvement, (5) died from other causes, (6) unavailable paraffin blocks of the tumor. Any patient who was lost to follow-up before a 5-year period was contacted via telephone or letter. All patients who had tumor recurrence within 5 years after surgery were defined as cases. During the study period, 36 recurrent cases met eligible criteria. Four cases were excluded from the study due to unavailable pathological paraffin blocks. Overall, 32 recurrent cases were included in this study. Controls were those who had no recurrence after the follow-up of at least 5 years. Controls must be within 10 years of age different from cases, had the same stage disease (Ib or IIa), and underwent surgery in the same hospital. The tumors of cases and controls had the same histology (squamous or non-squamous) and were about the same size or within one cm difference of their maximal dimensions. Two controls who were operated on the nearest surgical date of cases were chosen for each case according to the eligible criteria. We were able to find two matching controls for 31 cases. Only one case had only one matching control. Altogether, we recruited 63 controls. More than 80% of controls were operated within 2 years from the date of surgery of their matching cases. Clinical data of cases and controls were recorded.

Immunohistochemistry study A single paraffin-embedded block with a large volume of tumor tissue was chosen from each case and each control. Three-micrometer-thick sections were cut from paraffin blocks. Paraffin sections were dewaxed with xylene and treated with 95% alcohol. Sections were then treated with 3% H2O2 in phosphate-buffered saline (PBS) to block endogenous peroxidase activity. For antigen retrieval, they were immersed in 10 mM/l citrate buffer (pH 6.0) and microwaved at 750 W power for 10 min. After the buffer had cooled, sections were treated with anti-human bcl-2 oncoprotein mouse monoclonal antibody clone 124 (Dako code No. M0887) diluted 1:50 for 60 min at room temperature. Sections were treated for another 10 min with the biotinylated link antibody (Dako LSAB code no. K0675 bottle 1). After being rinsed in phosphate-buffered saline (PBS), the sections were coated with streptavidin–HRP (Dako LSAB code no. K0675 bottle 2) for 10 min. The reaction product was developed with diaminobenzidine solution for 10 min. Sections were then counterstained with Harry hematoxylin, dehydrated through 95% alcohol and absolute alcohol, and

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were mounted. Intratumor lymphocytes were used as positive control. The negative control consisted of sections that were treated with the same technique with the primary antibody omitted.

Staining evaluation Positive bcl-2 staining tumor cells were counted when they have the same staining intensity as internal positive control (lymphocyte). Bcl-2 staining was scored depending on the proportion of positively stained tumor cells (excluding areas of necrosis) relative to the overall number of tumor cells. Staining of ≤5% positive tumor cells was defined as negative. Positive immunoreactivity in this study was classified into 3 categories: 1+, N5–10% positive tumor cells; 2+, N10–30% positive tumor cells; 3+, N30% positive tumor cells.

Interobserver and intraobserver reliability Two skilled pathologists (SS, SK), who were unaware of the clinical information, reviewed all pathological hematoxylin and eosin slides and the bcl2 immunohistochemical staining slides independently. The intraobserver and interobserver reliability was evaluated before conducting the research. The kappa value of nearly all factors evaluated (histology, tumor grade, tumor depth) in intraobserver and interobserver reliability tests was more than 0.7 except for LVSI (0.61–0.73) and bcl-2 (0.40– 0.53). Since the kappa value of bcl-2 was quite low, two pathologists had discussed and clarified the definition of bcl-2 scoring before evaluating all slides in the study. From all 95 patients in this study, the tumor histology was discordantly reported in only one case, bcl-2 positive and negative was reported differently in 14 cases. For other pathologic factors: tumor grade (1 versus 2–3), depth of invasion (≤1/2 versus N1/2), LVSI (presence versus absence), the reports were different in 15, 14, and 12 cases respectively. The two pathologists had reviewed these slides together until arriving the final conclusion with consensus. The kappa value of bcl-2 was acceptable at the level of 0.71, the kappa values of histology and LVSI were also acceptable at the levels of 0.98 and 0.75, respectively, while the kappa values of tumor grade and tumor depth were in moderate levels of 0.64 and 0.68, respectively.

Statistical analysis Stata 7.0 was used for the analysis. The associations of tumor recurrence and bcl-2 expression, together with other pathological characteristics, were analyzed by conditional simple logistic regression for univariable analysis and conditional multiple logistic regression for multivariable analysis. The crude and adjusted odds ratios (ORs) with their 95% confidence intervals were reported. The association between bcl-2 and other pathological factors was evaluated by Chi-squared test for univariable analysis and by multiple logistic regression for multivariable analysis. All associations were calculated using two-sided test and a P value of 0.05 or less was considered as statistical significance.

Fig. 1. Bcl-2 immunohistochemical staining of normal cervical tissue. Expression of bcl-2 in the basal layer of nonneoplastic cervical squamous epithelium.

Results Patients’ basic characteristics The basic characteristics of cases and controls were demonstrated in Table 1. The mean age ± SD of the cases and controls was 41.5 ± 7.4 and 41.3 ± 6.7 years respectively. Approximately 94% of cases and controls were in stage Ib. Squamous cell carcinoma was the most common histology at 63%. The mean tumor size ± SD of cases and controls were 2.7 ± 1.6 cm and 2.6 ± 1.4 cm respectively. Immunoreactivity of bcl-2 expression In normal cervical tissue, the expression of bcl-2 protein was consistently demonstrated in the basal layer of cervical squamous epithelium (Fig. 1). In cervical cancer tissue, the immunoreactivity for bcl-2 was always observed in the cytoplasm of the tumor cells (Fig. 2). In almost all cases, bcl2 expression in the same tumor was heterogeneous with variable degree of staining from strongly positive to negative.

Table 1 Basic characteristics of cases and controls Cases (n = 32) Number Age (years) mean ± SD Tumor size (cm) mean ± SD Stage Ib IIa Histology Squamous Adenocarcinoma Adenosquamous

Controls (n = 63) %

41.5 ± 7.4 2.7 ± 1.6

Number

%

41.3 ± 6.7 2.6 ± 1.4

30 2

93.7 6.3

59 4

93.7 6.3

20 9 3

62.5 28.1 9.4

40 19 4

63.5 30.2 6.3

Fig. 2. Bcl-2 immunohistochemical staining of cervical cancer tissue. Expression of bcl-2 was observed in the cytoplasm of the tumor cells.

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Table 2 Bcl-2 expression and pathological findings of cases and controls Adjusted a

Case (n = 32)

Controls (n = 63)

Crude

No.

%

No.

%

OR

95% CI

P value

OR

95% CI

P value

Bcl-2 Negative Positive 1+ 2+ 3+

18 14 5 6 3

56.3 43.7 15.6 18.7 9.4

31 32 14 8 10

49.2 50.8 22.2 12.7 15.9

1b 0.7

0.3–1.6

0.378

0.6

0.2–1.7

0.304

Tumor grade 1 2–3

7 25

21.9 78.1

30 33

47.6 52.4

1b 8.1

1.8–36.9

0.007

6.9

1.4–35.0

0.019

Lymph-vascular space invasion (LVSI) Absence 9 28.1 Presence 23 71.9

35 28

55.6 44.4

1b 3.8

1.3–10.6

0.013

5.4

1.4–20.1

0.012

Depth of invasion ≤1/2 8 N1/2 24

26 37

41.3 58.7

1b 2.1

0.8–5.4

0.129

1.7

0.6–5.1

0.320

25.0 75.0

OR = odds ratio. a Adjusted for the remaining variables in the table. b Reference group.

Association between bcl-2 expression, other pathological factors and tumor recurrence Bcl-2 expression was found in 43.7% (95% CI, 39.4–48.4%) of cases compared to 50.8% (95% CI, 47.7–53.9%) of controls. The difference was not statistically significant different (P value = 0.378; Table 2). Among other pathological factors which were studied as the prognostic factors for cervical cancer (Table 2), we found that higher tumor grade and presence of LVSI were significantly associated with tumor recurrence (P value = 0.007 and 0.013 respectively). Patients with depth of invasion more than half of cervical thickness also had a trend to have higher risk of recurrence but did not reach statistical significance (P

value = 0.129). In multivariable analysis, tumor grade and presence of LVSI were still significantly associated with tumor recurrence. Patients with grade 2–3 tumors had 6.9 times risk of recurrence compared to those with grade 1 tumors (P value = 0.019). Those tumors with LVSI had 5.4 times risk of recurrence compared to those without LVSI (P value = 0.012: Table 2). Association between bcl-2 expression and other prognostic factors for tumor recurrence To assess the association between bcl-2 expression and other prognostic factors, bcl-2 expression was considered as a dependent variable (Table 3). From univariable and

Table 3 Association between bcl-2 expression and other prognostic factors No. (n = 95)

Bcl-2 number

37 58

Adjusted a

Crude OR

95%CI

P value

OR

95%CI

P value

23 (62.2) 23 (39.7)

1b 0.4

0.2–0.9

0.034

0.4

0.1–0.9

0.023

Lymph-vascular space invasion (LVSI) Absence 44 17 (38.6) Presence 51 29 (56.9)

1b 2.1

0.9–4.8

0.078

2.2

0.9–5.1

0.078

Depth of invasion ≤1/2 N1/2

1b 1.9

0.8–4.5

0.140

1.9

0.8–4.7

0.151

Tumor grade 1 2–3

34 61

13 (38.2) 33 (54.1)

OR = odds ratio. a Adjusted for the remaining variables in the table. b Reference group.

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multivariable analysis, tumor grade was significantly associated with bcl-2 expression; positive bcl-2 expression was found in 62.2% of grade 1 tumor compared to 39.7% in grade 2–3 (P value = 0.034 and 0.023 from univariable and multivariable analysis respectively). LVSI had borderline significant association with bcl-2 expression. Positive bcl-2 expression was found in 56.9% and 38.6% of patients with and without LVSI, respectively (P value = 0.078 from both univariable and multivariable analysis). Positive bcl-2 also tended to be found more often in patients with deep invasion (N1/2) but did not reach statistical significance (P value = 0.140 and 0.151 from univariable and multivariable analysis respectively). Discussion Many previous studies reported the association between bcl2 expression and the prognosis of cervical cancer with various results [22–28,37–45]. These could be partly explained by the population heterogeneity and various treatment modalities. Overexpression of bcl-2 specifically prevents cells from initiating apoptosis (programmed cell death) in response to a number of stimuli including radiotherapy and chemotherapy [20]. The antiapoptic function of bcl-2 permits prolonged cell survival without concomitant cell proliferation [19]. Based on these mechanisms of action, it was proposed that the tumor with overexpression of bcl-2 would resist to radiotherapy resulting in worse prognosis. This finding was confirmed by many clinical studies [22–24]. However, some other studies failed to demonstrate the association between bcl-2 expression and prognosis in cervical cancer patients treated by radiotherapy [43,45]. Many studies in various cancers treated by surgery demonstrated that patients with bcl-2 expression had better prognosis [32,46–48]. This good outcome may be explained by the inhibitory effect of bcl-2 on the cell cycle [21]. These opposing actions of bcl-2 in patients treated by radiation/chemotherapy versus surgery may render the difficulty to explain the association of bcl-2 expression and the patients' outcome in those who received multimodalities of treatment (surgery combined with radiotherapy and/or chemotherapy). To date, there were altogether 7 studies reported on the association of bcl-2 and prognosis in surgical-treated cervical cancer patients [25–28,37–39]. Except one study of Dimitrakakis et al. [25], the other 6 studies included patients who also received adjuvant treatment with radiotherapy and/or chemotherapy [26–28,37–39]. Some of these studies failed to detect the association between bcl-2 and prognosis [37–39], while other studies [26–28] reported the association between bcl-2 and longer survival. Although, Dimitrakakis et al., whose patients received only surgical treatment, reported the significant better 5-year survival rate of patients with bcl-2 positive compared to those with bcl-2 negative, 74% versus 49% in both uni- and multivariable analysis. Their study did not include some important prognostic factors such as tumor grade, tumor depth, and LVSI in the multivariable analysis. Hence, the question whether bcl-2 is the independent prognostic factor in

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cervical cancer could not be demonstrated in their study. We expect that our study is the only study that could correctly answer the question about the prognostic significance of bcl-2 expression in surgical-treated cervical cancer because our study included cervical cancer patients who were solely treated by surgery. Furthermore, all possible confounding factors, which might interfere with the prognosis of the patients such as patients' age, tumor size, histology, and stage, were controlled. Yet, we could not support the assumption that surgical-treated cervical cancer with expression of bcl-2 would be associated with good outcome because we could not demonstrate the association of bcl-2 and tumor recurrence. The difference of bcl2 expression in recurrent cases and nonrecurrent controls was only 7% which was not statistically significantly different. It also deemed that this difference was not clinically meaningful. Although bcl-2 expression had been demonstrated to be associated with lower cellularity or lower proliferative activity in soft tissue sarcoma [30] and adenocarcinoma of lung [29], there was still no such report in cervical cancer. Concerning the association of bcl-2 and other favorable prognostic factors in many types of cancer as previously mentioned, we found from this study that bcl-2 expression was significantly associated with lower grade. This association was also reported with borderline significance in cervical cancer in the study of Aletra et al. [49]. However, for the association of bcl-2 expression with LVSI, Tjalma et al. [21] reported a significantly higher fraction of bcl-2 positive cells in tumors showing no lymphatic or vascular space invasion. This is in contrast to our study that bcl2 expression, though not significant, tended to have some association with the presence of LVSI, which is an unfavorable prognostic factor. Other factors we found to be significantly associated with tumor recurrence were tumor grade and LVSI, which were similar to other studies [11,13]. The factor of depth of stromal invasion, which was reported to be a prognostic factor in other studies [11,12], also tended to be associated with recurrence but did not reach statistical significance in our study. This may be due to too small sample size in this study to demonstrate the statistical significance in this issue. Based on two main findings from our study, there was no association between bcl-2 expression and tumor recurrence in surgical-treated cervical cancer patients who had negative node and free surgical margin, while tumor grade and LVSI clearly showed this association. We rather conclude that there is no need to detect the bcl-2 expression by the costly immunohistochemical study in cervical cancer patients who underwent radical hysterectomy with negative pelvic nodes. Other oncogenes should be studied in the same fashion, to see whether any oncogene would serve as a better prognostic factor in this group of patients. Acknowledgments The authors would like to thank Associate Professor Sompop Limpongsanurak, Assistant Professor Somrat Lertmaharit, and Doctor Chulaluk Komoltri for their invaluable comments and suggestions; Ms. Lakana Eienleng for doing all

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