Prognostic Significance of Bcl-2 and p53 Protein Expressions and Ki67 Proliferative Index in Diffuse Large B-cell Lymphoma

Objective: Diffuse large B-cell lymphoma (DLBCL) is a high-grade neoplasm that has heterogeneous properties in clinical, morphological, and immunophenotypic aspects. In the present study the effects of p53, Bcl-2, and Ki67 on prognosis and their relationships with clinical parameters were examined.   Materials and Methods: Thirty-five patients who had been diagnosed with nodally located DLBCL at İzmir Atatürk Training and Research Hospital between January 1999 and June 2006 were included in the study. The Ann Arbor classification system was used to determine the stage of the patients. The patients were evaluated according to age, sex, stage, B symptoms, extranodal involvement, and lactate dehydrogenase (LDH) level as well as immunohistochemically. P53 protein and Bcl-2 oncoprotein expressions and Ki67 proliferation index were assessed immunohistochemically. Results: High Bcl-2 expression was found in 9 patients (25.7%), high p53 expression was found in 10 patients (28.6%), and high Ki67 was observed in 23 patients (65.7%). There was no significant correlation between p53 expression, Bcl-2 expression, or Ki67 proliferation index and age, sex, stage, B symptoms, extranodal involvement, LDH level, and overall survival (p>0.05). We did not find a relationship among p53 expression, Bcl-2 expression, Ki67 proliferation index, and prognosis (p>0.05). There was no significant relationship between overall survival and age, sex, stage, B symptoms, extranodal involvement, or LDH level (p>0.05). Our results revealed that Bcl-2 and p53 protein expressions and Ki67 proliferation index have no effect on overall survival of patients with DLBCL. Conclusion: The prognostic importance of p53 and Bcl-2 protein expressions and Ki67 proliferation index in DLBCL, which has biological and clinical heterogeneity, can be understood in a large series of studies that have subclasses and immunohistochemical markers with optimal cut-off values.


Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma, comprising 30%-40% of all non-Hodgkin's lymphoma (NHL). It has heterogeneous clinical features and varies markedly in response to treatment and in prognosis [1,2]. The response rate is 60%-80% in NHL after acceptable therapy. The 5-year overall survival rate is higher than 55%. Patients who responded to chemotherapy and were cured after therapy could be estimated using clinic and laboratory results. Therapy plans can be determined using these prognostic factors [3,4]. Although survival can be estimated on the basis of clinical parameters, molecular abnormalities in a panel of suppressor proteins and oncogenic proteins have also been reported related with prognosis [5,6,7]. In the present study we investigated the effects of p53 (tumor suppressor protein), which is a cell cycle regulator; Bcl-2 oncoprotein, which is an inhibitor of apoptosis; and Ki67, which is a cell proliferation marker on prognosis, and their relationships with clinical parameters.

Materials and Methods
Patients: Thirty-five patients who had been diagnosed with nodally located DLBCL at İzmir Atatürk Training and Research Hospital between January 1999 and June 2006 were included in the study. The patients were evaluated according to age, sex, stage, B symptoms, extranodal involvement, and lactate dehydrogenase (LDH) level as well as immunohistochemically. p53 and Bcl-2 oncoprotein expressions and Ki67 proliferation index were assessed immunohistochemically. The clinical parameters and the outcome were reviewed retrospectively. All clinical and laboratory data, along with the follow-up data, were obtained from the hospital records or patients' charts. Overall survival (OS) was calculated from the date of diagnosis until death or last follow-up. Affected lymph nodes from all of the patients were examined by biopsy ( Figure 1) and lymphoma was diagnosed according to WHO lymphoma classification.

Immunohistochemistry:
Paraffin sections were immunostained by the labeled streptavidin-avidinbiotin method with the antibodies for Ki67 (clone SP6, Neomarkers), Bcl-2 (clone 124, Dako), and p53 (clone DO7, Dako). Positive staining of small reactive lymphocytes for Bcl-2 was provided as an internal control. A previously known positive case was used as an external control in order to evaluate p53 reactivity. A known positive control (normal tonsil) was used to evaluate Ki67 reactivity.
Statistical Analysis: Survival curves were drawn according to the Kaplan-Meier method and compared by log-rank test. The relationship between p53, Bcl-2, and Ki67 expressions and clinicopathological parameters was evaluated by chi-square test for data qualification and Fisher's exact test for data categorization. Differences were considered as significant if the p value was less than 0.05. The study was approved by the ethics committee.

Results
Patient characteristics are summarized in Table 1. Follow-up duration ranged from 0.5 to 68 months, with an average of 17.6 months. Fifteen (42.9%) patients were followed until death, whereas 20 (57.1%) patients were still alive at the end of the study. There were 21 (60%) men and 14 (40%) women in the study. The average age of the patients was 53.6 years. All patients were classified according to the Ann Arbor classification. As such, 12 patients (34.3%) had stage 1-2 and 23 patients (65.7%) had stage 3-4 disease. Elevated serum LDH levels were observed in 26 patients (74.3%). Eighteen (51.4%) patients had B symptoms at the time of diagnosis. Thirteen (37.1%) patients presented with involvement of extranodal sites.
There was no significant correlation between p53 and Bcl-2 expressions or Ki67 proliferation index and age, sex, stage, B symptoms, extranodal involvement, LDH level, and overall survival (p>0.05) ( Table 2). We did not find significant relationships between p53, Bcl-2, and Ki67 expressions and prognosis (p>0.05) ( Figures 5, 6, 7). There was no significant relationship between overall survival and age, sex, stage, B symptoms, extranodal involvement, or LDH level (p>0.05).
Treatment records of 27 patients were attained. Twentythree patients had been treated with 6-8 cycles of CHOP chemotherapy regimens including cyclophosphamide, doxorubicin, vincristine, and prednisolone. Only 4 patients had been treated with 6-8 cycles of a rituximab and CHOP chemotherapy regimen. Therefore, statistical analysis could not be done for the chemotherapy protocols.

Discussion
DLBCL exhibits heterogeneous clinical features and varies markedly in response to treatment and prognosis [1,12]. Although survival can be estimated on the basis of clinical parameters, molecular abnormalities in a panel of suppressor proteins and oncogenic proteins have also been reported to be related to prognosis [5,6,7].    Several studies reported that age [7,12], serum LDH level [5,7,10,13], the involvement of extranodal sites [7,10], the stage of the disease [5,10,13,11,12,13,14,15], and B symptoms [10,15] were significant clinical predictors of survival of patients with DLBCL. In the present study, similar to the other studies, there was no statistically significant relationship between OS and age [5,11,13,14], sex [5,10,14,15], stage [16], B symptoms [13], extranodal involvement [11,13,15], or LDH level [11,15]. p53 can be considered as a tumor suppressor protein. It is involved in the regulation of cell survival by interaction with G-S phase transition within the cell cycle and by induction of apoptotic cell death [17,18]. The incidence of p53 expression in DLBCL varies between 5.0% and 71.0% [5,6,7,10,14,[19][20][21][22]. In the present study, p53 expression was identified in 62.9% of all cases and high p53 expression was identified in 10 patients (28.6%). Although Ichikawa   Piris et al. found a correlation between p53 expression and OS in high-grade lymphomas, and patients with combined expression of Bcl-2 and p53 in tumors had poorer prognosis than those with p53 expression only, which was particularly significant in lymph nodes in DLBCL cases [24]. Kramer et al. showed that p53 expression was only related to a high tumor burden and was not an independent risk factor for survival in patients with DLBCL [6]. Similarly, in the present study, no significant correlation was found between p53 expression and OS or disease-free survival [5,10,23]. It has been found that p53 expression had no significant effect on OS.
Ki67 is a proliferation marker for several human neoplasms. Ki67 detects a nuclear antigen associated with cell proliferation [31]. The prognostic value of Ki67 has been the subject of different studies in the past. Although most of the studies showed a high proliferation index to be an adverse prognostic marker, there were inconsistent results, as well [32,33,34]. High Ki67 expression was observed in 23 patients (65.7%) in the present study. There was no correlation between Ki67 expression level and any studied clinicopathological factors. Miller et al. [32] reported that tumor proliferation of >80.0% was associated with poorer survival in previously untreated patients with aggressive NHL, whereas Hall et al. [35] found that patients who responded well to chemotherapy were less likely to relapse if they had >80.0% tumor proliferation. Jovanovic et al. reported that the patients with a high proliferative fraction (Ki67 > 60.0%) had a worse OS rate with log-rank test analysis [30]. Conversely, Hasselblom et al. found that patients with low Ki67 expression (<49.0%) had significantly worse progression-free survival and OS as independent risk factors [36]. In this study, similar to the other studies, there was no statistically significant relationship between Ki67 expression and prognosis [7,10,11,15].
The effects of p53 and Bcl-2 protein expressions and Ki67 proliferation index on prognosis of DLBCL have been investigated. The results from prior studies are variable and controversial. Methodological differences, different cut-off levels of immunohistochemical markers, and differences in the treatment regimens and follow-up time may account for the variation in results.
The present study revealed that Bcl-2 and p53 protein expressions and Ki67 proliferation index have no effect on the OS of patients with DLBCL. The prognostic importance of p53 and Bcl-2 protein expressions and Ki67 proliferation index in DLBCL, which has biological and clinical heterogeneity, can be understood in a large series of studies that have subclasses and immunohistochemical markers with optimal cut-off values.