ISRN OncologyVolume 2011 (2011), Article ID 670358, 6 pagesdoi:10.5402/2011/670358

Clinical Study

No Prognostic Impact of p53 and P-Glycoprotein Expression in Patients with Diffuse Large B-Cell Lymphoma

Pairaya Rujirojindakul,¹ Kumpol Aiempanakit,² Kanita Kayasut,¹ Arnuparp Lekhakula,² and Hutcha Sriplung³

¹Department of Pathology, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand²Department of Internal Medicine, Faculty of Medicine, Prince of Songkla University, Songkhla 90110, Thailand³Epidemiology Unit, Faculty of Medicine, Prince of Songkla University, Songkhla 90110, Thailand

Received 15 August 2011; Accepted 18 September 2011

Academic Editor: G. Schiavon

Copyright © 2011 Pairaya Rujirojindakul et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The aim of this study was to determine the clinical significances of p53 and p-glycoprotein (P-gp) expression on outcome predictors for patients with DLBC. We assessed the immunohistochemical expression of p53 and P-gp using formalin-fixed, paraffin-embedded specimens in 108 patients diagnosed with de novo DLBC. A high expression of p53 was found in 53.7% of the patients. No expression of P-gp was demonstrated in any of the specimens. There were no significant differences in the complete remission (CR) rate (), overall survival (OS) (), or disease-free survival (DFS) () between the p53-positive and p53-negative groups. The final model from multivariate analysis that revealed poor performance status was significantly associated with CR () and OS (). Moreover, the advanced stage was a significant predictor of DFS (). This study demonstrated no impact of the expression of p53 on either response or survival rates.

1. Introduction

Diffuse large B-cell lymphoma (DLBC) is the most common type of non-Hodgkin lymphoma (NHL) accounting for 50% of NHL in Thailand [1]. Although the outcomes of treatment were markedly improved by adding rituximab to standard CHOP regimen (R-CHOP) [2], the 5-year event-free survival rate was only 47% [3]. Stratifying newly diagnosed patients according to risk will provide invaluable information to achieve the optimal risk-adapted strategy. Since the International Prognostic Index (IPI) was introduced in 1993, the strong prognostic predictability has been proved [4, 5]. However, patients with high-risk IPI may be unsuitable for intensive therapy due to old age or poor performance status (PS). Therefore, additional prognostic factors reflecting tumor biology are needed to identify patients who might benefit from dose intensification or new targeted therapy.

The p53 tumor suppressor gene is located on the short arm of chromosome 17 (17p13.1). It plays a role in the regulation of cell survival acting as a cell-cycle checkpoint protein and apoptotic cell death. Therefore, the p53 protein contributes to preventing the replication of cells suffering from DNA damage. Loss of the p53 function may cause resistance to apoptosis that leads to treatment failure to DNA-damaging agents [6]. Thus, p53 inactivation may be associated with a poorer prognosis. However, it remains inconclusive whether the p53 expression is an independent outcome predictor in patients with non-Hodgkin lymphoma (NHL) [7].

In addition, another major reason for treatment failure is drug resistance. Several biological mechanisms are responsible for this problem. One of the most important reasons that has been extensively investigated is the multidrug resistance (MDR) gene. The classical MDR is related to the expression of the MDR-1 gene, which is located at chromosome 7 and encodes a 170-kDa membrane-associated P-glycoprotein (P-gp) [8]. The P-gp functions as an energy-dependent drug efflux pump and causes a reduction in intracellular accumulation of the drug. In NHL, varying incidences of P-gp expression were reported from 0 to 49% and its impacts on the response are controversial [9–11].

In this study, we evaluated p53 and P-gp expression, as well as clinical parameters in patients with DLBC. The purpose was therefore to verify their impacts on treatment outcomes.

2. Materials and Methods

From January 2003 to December 2006, 122 patients were enrolled at Songklanagarind Hospital, but only 108 patients had available tissue sections. The eligibility criteria were older than 18 years, newly diagnosed with DLBC, and had stage II–IV diseases. The patients with human immunodeficiency virus or primary extranodal lymphomas were excluded. For lymphoma immunophenotyping, monoclonal antibodies targeting CD3, CD5, CD20, and CD79a (Dako, Glostrup, Denmark) were used to determine the T- or B-cell lineage. This study was approved by the Ethics Committee of Prince of Songkla University.

Clinical stage was performed using the Ann Arbor staging system. All patients with stage II–IV were treated with a standard chemotherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP) for at least six cycles. Rituximab was not routinely administered in Thailand. After completion of treatment, all patients were regularly followed up at intervals every few months for at least 5 years or until death.

2.1. Immunohistochemical Staining

Tumor samples were obtained by tissue biopsy at the time of initial diagnosis. Eighteen samples at the time of relapse for additional P-gp study were also included. The expressions of p53 and P-gp were analyzed by immunohistochemistry using the Envision technique. The immunohistochemistry was performed in formalin-fixed paraffin-embedded tissue sections. The 5-μm-thick sections were cut on aminopropyltriethoxysilane-coated slides. They were deparaffinized in xylene and rehydrated through graded alcohol. Antigen retrieval was carried out by high-temperature and- pressure cooking in Tris EDTA pH 9 for 30 seconds. After rinsing in distilled water, the endogenous peroxidase activity was blocked by treating the slides with 3% hydrogen peroxide for 5 minutes. To inhibit nonspecific antibody binding, the tissue samples were reacted with nonimmune serum for half an hour in a moisture chamber. The sections were incubated overnight at 4°C with 1 : 250 and 1 : 40 dilutions of the monoclonal antibody of p53 (DO-7, DAKO, Glostrup, Denmark) and P-gp (Novocastra Laboratories, UK), respectively. After rinsing in phosphate-buffered saline solution, the sections were incubated with Envision for 30 min and then were rinsed in phosphate-buffered saline solution again. The color was developed using diaminobenzidine (DAB) solution for 5 min and counterstained with hematoxylin. Any brown nuclear and membrane staining of the tumor cells were taken as positive for p53 and P-gp, respectively.

2.2. Immunohistological Scoring

The scoring was analyzed in the area of highest protein expression scanning under low magnification (×100). Then, at least 500 cells under high-power field (×1000) were evaluated for a final score. Negative staining was made after carefully examining the entire section with high magnification. Expression of p53 and P-gp was semiquantitatively assessed without knowledge of the clinical outcome. The staining was graded on a scale of 1+, 2+, and 3+ when 10–24%, 25–49%, and 50% positive reactions were found, respectively [12]. Paraffin-embedded placenta was used as a positive control for P-gp (Figure 1(c)).

(a)

(b)

(c)

(d)

Figure 1: Immunohistochemical detection showing (×400) (a) negative for p53 staining in DLBC, (b) strongly positive for p53 staining in DLBC (+++), (c) placenta as a positive control for P-gp staining, (d) negative for P-gp staining in DLBC.

2.3. Statistical Analysis

Baseline categorical characteristics were compared among patients with and without overexpression of p53 using Chi-square. Logistic regression model was used to predict complete remission (CR). Univariate analysis of survival was performed with the Kaplan-Meier method. Overall survival (OS) was calculated as the time interval from the date of diagnosis to death or last follow-up. Disease-free survival (DFS) was defined as the time interval between CR and first progression/relapse or death from any cause. Kaplan-Meier methods were used to estimate time-to-event endpoints, including OS and DFS. Survival data between subgroups were compared using the log-rank test. Multivariate analyses for OS and DFS were performed using a Cox regression model with backward elimination. Critical -values for entry and removal were 0.2 and 0.5, respectively. To test the main hypothesis, we forced the p53 into the model. Hazard ratios (HRs), 95% confidence intervals (95% CI) and -values were obtained from the best-fit model. All the statistic analyses were calculated using the R program. A significance level of 0.05 was used throughout all statistical tests in the study.

3. Results

There were 122 DLBC patients diagnosed during the study period. However, only 108 patients with adequate paraffin-embedded tumor tissue sections were analyzed. To avoid bias, we compared the clinical characteristics and outcomes between the 108 and 14 patients and found no statistically significant differences (data not shown). There were 56 males and 52 females with a median age of 53.8 years (range 18–84 years). The majority of patients were treated with CHOP chemotherapy. Rituximab was administered in eight patients (7.4%). The median OS and DFS were 27.9 months (range 1–68 months) and 30.9 months (range 0–74 months), respectively with a median follow-up time of 19.7 months (range 0.3–66.2 months).

3.1. Expression of p53 and P-gp

Assessment of p53 expression was performed in 108 patients. Of these, 30 (27.8%) were p53− (Figure 1(a)), 5 (4.6%) were p53+, 15 (13.9%) were p53++ (Figure 1(b)), and 58 (53.7%) were p53+++ (Figure 1(a)). A comparison between clinical characteristics and p53 overexpression showed no significances in any of the variables (Table 1).

Table 1: Clinical characteristics of the 108 patients according to p53 expression.

Variables
Immunostaining for p53 (no.)
-value

0–2+
3+

Age group

0.69

    ≤60 years
20
47

    >60 years
10
31

Sex

0.68

    Male
17
39

    Female
13
39

B symptoms

0.67

    Absent
16
47

    Present
14
31

Bulky mass (>6 cm)

0.39

    Absent
25
57

    Present
5
21

Extranodal involvement

0.86

    Absent
11
29

     Present
19
49

Serum LDH level

0.90

    Normal
13
31

    High
17
47

Ann Arbor staging system

0.12

    II
11
37

     III
12
16

     IV
7
25

ECOG performance status

0.46

    0-1
25
58

    2–4
5
20

International prognostic index

0.85

    Low
8
15

    Low intermediate
15
28

    High intermediate
8
21

    High
5
14

All tissue sections from patients with newly diagnosed and relapsed DLBC were negative for P-gp expression (Figure 1(d)).

3.2. Univariate Analysis

Patients with bulky mass, poorer performance status (PS), and higher IPI were less likely to achieve CR. The results from univariate analysis showed that those who presented with at least one of the variables (more advanced stage, B symptoms, bulky mass, high LDH, poorer performance status, and/or higher IPI) were significantly associated with a shorter overall survival (OS). Simultaneously, only stage, B symptom, LDH, and IPI were significantly associated with the DFS (Table 2). There were no significant differences in the CR rate (), OS (), or DFS () between the p53-positive (3+) and p53-negative groups (0–2+), even we tried to change the cut-off values to 1+ or 2+ (data not shown).

Table 2: Univariate analysis of CR, OS and DFS for 107 patients treated at Songklanagarind hospital.

Variables
Total
Univariate analysis

CR no. (%)
OS HR (95% CI)
DFS HR (95% CI)

Age group, y

    ≤60

45 (68.2)
1.0
1.0

    >60

26 (63.4)
1.4 (0.9–2.4)
1.2 (0.6–2.5)

Sex

    Male

39 (70.9)
1.0
1.0

    Female

32 (61.5)
1.1 (0.6–1.8)
0.9 (0.4–1.8)

Stage

    II

35 (72.9)
1.0
1.0

    III

18 (64.3)
2.0 (1.1–3.7)
3.2 (1.3–7.7)

    IV

18 (58.1)
2.1 (1.1–3.9)*
2.5 (1.0–6.3)*

B symptoms

    Absent

45 (72.6)
1.0
1.0

    Present

26 (57.8)
1.9 (1.1–3.1)*
2.6 (1.2–5.4)*

Bulky mass

    Absent

59 (72.0)
1.0
1.0

    Present

12 (48.0)*
2.0 (1.1–3.6)*
0.5 (0.1–1.9)

Extranodal

    Absent

30 (75.0)
1.0
1.0

    Present

41 (61.2)
1.5 (0.9–2.7)
1.4 (0.7–3.0)

LDH

    Normal

34 (77.3)
1.0
1.0

    High

37 (58.7)
1.9 (1.1–3.3)*
2.4 (1.1–5.2)*

PS

    0-1

66 (79.5)
1.0
1.0

    2–4

5 (20.8)**
4.8 (2.7–8.3)**
1.5 (0.4–4.8)

p53

    0–(2+)

36 (72)
1.0
1.0

    3+

35 (61.4)
1.2 (0.7–2.0)
0.9 (0.4–1.9)

IPI

    Low

20 (87.0)
1.0
1.0

    Low Int.

29 (78.4)
1.5 (0.6–3.4)
1.2 (0.4–3.6)

    High Int.

16 (57.1)
3.6 (1.6–8.2)
3.3 (1.1–9.7)

    High

6 (31.6)**
5.3 (2.2–12.6)**
3.4 (0.9–13.0)*

* , **.

3.3. Multivariate Analysis

The preliminary regression model included the following variables: p53, sex, age group, stage, B symptoms, bulky mass, extranodal involvement, LDH and performance status. The final model revealed PS 2–4 was significantly associated with lower CR rate (OR 14.7, 95% CI 4.8–45.3, ) and shorter OS (HR 5, 95% CI 2.8–9, ). Moreover, the advanced stage was a significant predictor of DFS (). Patients with stage III had HR 3.1 (95% CI 1.3–7.5) while patients with stage IV had HR 2.7 (95% CI 1.1–7.1).

4. Discussion

This study was undertaken to investigate the impact of p53 and P-gp expression as well as clinical parameters on treatment outcomes in patients with de novo DLBC. From multivariate analysis, our results indicate that only poor PS was independently associated with both CR and OS while the advanced stage was the independent predictor of DFS. Contrary to our expectations, the expression of p53 shows no impact on either response or survival rates. Furthermore, both initial and relapsed specimens were negative for P-gp expression.

Although there have been several studies on these markers, conflicting results have still existed. Moreover, independent associations between the individual markers and known clinical prognostic factors were not assessed. To address this, the relationship of the p53 expression with clinical endpoints was independently determined with clinical characteristics. We included only patients who were ethnic Thais and diagnosed with DLBC subtype to avoid confounding effects. In addition, they were homogeneously treated with the same anthracycline-containing regimen, and the treatment outcomes were analyzed based on complete follow-up data.

In our study, no expression of P-gp in any of the specimens was demonstrated. We tested three possible explanations to confirm the negative immunohistochemical results. First, we tried different concentrations of the P-gp antibody. Second, a few specimens with faint staining were repeated using an automatic immunostaining device (DAKO). These two hypotheses about the methods are unlikely to be responsible in the negative staining due to that the paraffin-embedded placenta showed positive staining. Another reasonable assumption is that the sample size was too small to detect the low positive expression rate of P-gp (95% confidence interval = 0–3.4% and 0–18.5% in the newly diagnosed and relapsed patients, respectively). Based on our knowledge, the negative result of P-gp from the immunohistochemical method has never been reported. One explanation may be from publication bias. Though the evidence shows that both RT-PCR and immunohistochemistry are reliable methods to detect MDR expression in lymphomas [10], the fresh specimens were not obtained to assess the expression of MDR-1 mRNA and P-gp that could strongly confirm the negative expression in this study.

Based on a cut-off value greater than 50% of positive immunostaining for p53, the positive rate of 53.7% is consistent with a report from Italy [13]. In addition, Chang et al. [14] demonstrated 58% of patients with high clinical stage DLBC stained positive for p53 using the cut-off value of 20% as a positive expression. Paik et al. [15] also showed overexpression of p53 in 56% of nodal DLBC with germinal centre phenotype based on nuclear staining more than 10%. However, our positive rate is higher than a population-based study using the cut-off value of 50% that shows that only 13% of 364 cases with B-cell lymphomas were positive [16]. Regarding the prognostic significance of p53, no significant relationship between p53 protein expression and any of the clinical variables and outcomes was confirmed in this study. These results were in concordance with a population-based study [16]. Similarly, nothing significant in survival was observed in previous studies [17, 18]. On the other hand, our results contradicted to the study that demonstrated the impact of p53 on survival [19, 20].

Altogether, this study provides evidence that both of the well-known biomarkers, p53 and P-gp, are not the independent predictors for treatment outcomes in these patients with DLBC. Nowadays, gene expression signatures provide evidences to predict the prognosis in patients with DLBC [21, 22]. Nevertheless, given the limitations of routine clinical utility, gene expression profiling in daily practice has allowed the immunohistochemistry to be of great practical value.

Acknowledgments

The authors are indebted to Ms. K. Tongmitr for her technical assistance in immunohistochemical techniques. This work was supported by grants from the Thai Society of Hematology and Faculty of Medicine, Prince of Songkla University.

References

S. Sukpanichnant, “Analysis of 1983 cases of malignant lymphoma in Thailand according to the World Health Organization classification,” Human Pathology, vol. 35, no. 2, pp. 224–230, 2004.

B. Coiffier, E. Lepage, J. Briere et al., “Chop chemotherapy plus rituximab compared with chop alone in elderly patients with diffuse large-B-cell lymphoma,” The New England Journal of Medicine, vol. 346, no. 4, pp. 235–242, 2002.

P. Feugier, A. Van Hoof, C. Sebban et al., “Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the groupe d'etude des lymphomes de l'adulte,” Journal of Clinical Oncology, vol. 23, no. 18, pp. 4117–4126, 2005.

“A predictive model for aggressive non-Hodgkin's lymphoma. The international non-Hodgkin's lymphoma prognostic factors project,” The New England Journal of Medicine, vol. 329, no. 14, pp. 987–994, 1993.

M. Ziepert, D. Hasenclever, E. Kuhnt et al., “Standard International prognostic index remains a valid predictor of outcome for patients with aggressive CD20+ B-cell lymphoma in the rituximab era,” Journal of Clinical Oncology, vol. 28, no. 14, pp. 2373–2380, 2010.

S. A. Amundson, T. G. Myers, and A. J. Fornace, “Roles for p53 in growth arrest and apoptosis: putting on the brakes after genotoxic stress,” Oncogene, vol. 17, no. 25, pp. 3287–3299, 1998.

C. Nieder, S. Petersen, C. Petersen, and H. D. Thames, “The challenge of p53 as prognostic and predictive factor in Hodgkin's or non-Hodgkin's lymphoma,” Annals of Hematology, vol. 80, no. 1, pp. 2–8, 2001.

V. Ling, “P-glycoprotein and resistance to anticancer drugs,” Cancer, vol. 69, no. 10, pp. 2603–2609, 1992.

A. R. Yuen and B. I. Sikic, “Multidrug resistance in lymphomas,” Journal of Clinical Oncology, vol. 12, no. 11, pp. 2453–2459, 1994.

Q. Liu, K. Ohshima, and M. Kikuchi, “High expression of MDR-1 gene and P-glycoprotein in initial and re-biopsy specimens of relapsed B-cell lymphoma,” Histopathology, vol. 38, no. 3, pp. 209–216, 2001.

M. Ohsawa, Y. Ikura, H. Fukushima et al., “Immunohistochemical expression of multidrug resistance proteins as a predictor of poor response to chemotherapy and prognosis in patients with nodal diffuse large B-cell lymphoma,” Oncology, vol. 68, no. 4–6, pp. 422–431, 2005.

K. Leroy, C. Haioun, E. Lepage et al., “p53 gene mutations are associated with poor survival in low and low-intermediate risk diffuse large B-cell lymphomas,” Annals of Oncology, vol. 13, no. 7, pp. 1108–1115, 2002.

C. Visco, F. Canal, C. Parolini et al., “The impact of P53 and P21waf1 expression on the survival of patients with the germinal center phenotype of diffuse large B-cell lymphoma,” Haematologica, vol. 91, no. 5, pp. 687–690, 2006.

C. C. Chang, Y. C. Liu, R. P. Cleveland, and S. L. Perkins, “Expression of c-Myc and p53 correlates with clinical outcome in diffuse large B-cell lymphomas,” American Journal of Clinical Pathology, vol. 113, no. 4, pp. 512–518, 2000.

J. H. Paik, Y. K. Jeon, S. S. Park et al., “Expression and prognostic implications of cell cycle regulatory molecules, p16, p21, p27, p14 and p53 in germinal centre and non-germinal centre B-like diffuse large B-cell lymphomas,” Histopathology, vol. 47, no. 3, pp. 281–291, 2005.

M. H. H. Kramer, J. Hermans, J. Parker et al., “Clinical significance of bcl2 and p53 protein expression in diffuse large B-cell lymphoma: a population-based study,” Journal of Clinical Oncology, vol. 14, no. 7, pp. 2131–2138, 1996.

M. Llanos, H. Alvarez-Argüelles, R. Alemán, J. Oramas, L. Díaz-Flores, and N. Batista, “Prognostic significance of ki-67 nuclear proliferative antigen, bcl-2 protein, and p53 expression in follicular and diffuse large b-cell lymphoma,” Medical Oncology, vol. 18, no. 1, pp. 15–22, 2001.

L. Colomo, A. López-Guillermo, M. Perales et al., “Clinical impact of the differentiation profile assessed by immunophenotyping in patients with diffuse large B-cell lymphoma,” Blood, vol. 101, no. 1, pp. 78–84, 2003.

M. A. Piris, F. Pezella, J. C. Martinez-Montero et al., “p53 and bcl-2 expression in high-grade B-cell lymphomas: correlation with survival time,” British Journal of Cancer, vol. 69, no. 2, pp. 337–341, 1994.

L. Stefancikova, M. Moulis, P. Fabian, et al., “Prognostic impact of p53 aberrations for R-CHOP-treated patients with diffuse large B-cell lymphoma,” International Journal of Oncology, vol. 39, no. 6, pp. 1413–1420, 2011.

A. A. Alizadeh, M. B. Eisen, R. E. Davis et al., “Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling,” Nature, vol. 403, no. 6769, pp. 503–511, 2000.

A. Rosenwald, G. Wright, W. C. Chan et al., “The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma,” The New England Journal of Medicine, vol. 346, no. 25, pp. 1937–1947, 2002.