Proliferating cell nuclear antigen (PCNA), p53 and MDM2 expression in Hodgkin’s disease

ABSTRACT CONTEXT AND OBJECTIVE: Tumor cells in Hodgkin’s disease (HD) express cell proliferation markers that are evaluated according to the oncogenes involved or the expression of their proteins. Correlations between the protein expression grade and clinical data are now important for disease prognosis. DESIGN AND SETTING: This was a retrospective analysis on proliferating cell nuclear antigen (PCNA), p53 and MDM2 (murine double minute-2) expression using immunohistochemistry, on formalin-fixed, paraffin-embedded tissues from diagnostic biopsies on 51 patients with HD. The study was conducted at the Division of Hematology and Transfusion Medicine, Hospital São Paulo, Universidade Federal de São Paulo. METHODS: Antigen expression was evaluated as the proportions of positive Hodgkin and Reed-Sternberg (HRS) cells and reactive lymphocytes (L), which were compared using Spearman correlation coefficients. The Friedman test was used for comparisons between the markers. The Pearson test was used to investigate associations between marker expression and clinical and laboratory parameters, marrow involvement, complete remission (CR) and overall survival (OS) rates. RESULTS: There was overexpression of antigen proteins in HRS, in relation to L (p < 0.001). In HRS, MDM2 was higher than p53 and PCNA (p < 0.003), while the latter two were equivalent. In L, p53 was lower than MDM2 and PCNA (p < 0.001), while the latter two were equivalent. There was no relationship between protein expression and clinical and laboratory variables or outcome. CONCLUSIONS: PCNA, p53 and MDM2 are tumor markers for HD, but showed no clinical or prognostic significance in our analysis.


INTRODUCTION
Hodgkin's disease (HD) is a neoplasm that has the characteristic of containing a small number of scattered large multinucleated or mononucleated cells, designated Reed-Sternberg cells and Hodgkin cells respectively, residing in a heterogeneous admixture of infl ammatory and accessory cells. 1,2][6][7][8][9] There are few previous studies in the literature evaluating cell proliferation and this may be explained by the complex nature of this neoplasm and its heterogeneous cell composition. 10It has already been described that, in HD, HRS cells express proliferating cell nuclear antigen (PCNA) and p53 in more than 50% of the cases and that these expressions may play a role in the pathogenesis of the disease. 11CNA is a cell cycle-associated protein that interferes with cell proliferation in normal and tumor cells.It is an essential protein in DNA repair.PCNA is detected by positive reaction for the monoclonal antibody PC-10. 10,11PCNA activity in DNA repair increases resistance to chemotherapy in which the cytotoxicity depends on its integrity. 12PCNA expression in relapsed HD is greater than at diagnosis. 12ild-type p53 protein encoded by the p53 gene acts in the cell cycle to interrupt it at the G1 phase. 13This suppressive activity allows DNA repair in injured cells and avoids apoptosis. 144][15] Mutated p53 has a mean half-life of six to eight hours and no suppressive action, and it is easily detectable by immunohistochemistry methods.Mutated p53 replaces the wild-type p53 that is present CONTEXT AND OBJECTIVE: Tumor cells in Hodgkin's disease (HD) express cell proliferation markers that are evaluated according to the oncogenes involved or the expression of their proteins.Correlations between the protein expression grade and clinical data are now important for disease prognosis.

DESIGN AND SETTING:
This was a retrospective analysis on proliferating cell nuclear antigen (PCNA), p53 and MDM2 (murine double minute-2) expression using immunohistochemistry, on formalinfi xed, paraffi n-embedded tissues from diagnostic biopsies on 51 patients with HD.The study was conducted at the Division of Hematology and Transfusion Medicine, Hospital São Paulo, Universidade Federal de São Paulo.

METHODS:
Antigen expression was evaluated as the proportions of positive Hodgkin and Reed-Sternberg (HRS) cells and reactive lymphocytes (L), which were compared using Spearman correlation coeffi cients.The Friedman test was used for comparisons between the markers.The Pearson test was used to investigate associations between marker expression and clinical and laboratory parameters, marrow involvement, complete remission (CR) and overall survival (OS) rates.

RESULTS:
There was overexpression of antigen proteins in HRS, in relation to L (p < 0.001).In HRS, MDM2 was higher than p53 and PCNA (p < 0.003), while the latter two were equivalent.In L, p53 was lower than MDM2 and PCNA (p < 0.001), while the latter two were equivalent.There was no relationship between protein expression and clinical and laboratory variables or outcome.

CONCLUSIONS:
PCNA, p53 and MDM2 are tumor markers for HD, but showed no clinical or prognostic signifi cance in our analysis.

OBJECTIVE
The present study had the aims of evaluating p53, PCNA and MDM2 protein expression using immunohistochemical methods, on formalin-fi xed, paraffi n-embedded preserved tissue samples used for HD diagnosis, and correlating these expressions with clinical and laboratory parameters in order to evaluate their impact on HD outcome.

Patients Patients
Our sample consisted of 126 patients with HD that were followed up over the period from Janaury 1992 to December 1996 at Hematology Division of Unifesp/EPM.The admissions for the fi rst-line treatment of these patients were from December 1976 to December 1996.Suffi cient paraffi n-embedded blocks of diagnostic tissues were available in relation to 51 patients from this sample for performing new histological analyses with hematoxylin-eosin staining and for making immunohistochemistry slides de Oliveira for PCNA, p53 and MDM2 analysis.Fifty-one other patients out of the remaining 75 were randomly selected as a control group.The paraffi n-embedded blocks were obtained from the Pathology Department of Unifesp.
The patients' records were retrospectively reviewed to collect data on gender, age, Ann Arbor clinical stage, B symptoms (fever, weight loss and night sweating), histological subtype, hemoglobin level, white blood count (WBC), erythrocyte sedimentation rate (ESR), alkaline phosphates and bone marrow involvement.First complete remission (CR) and overall survival (OS) were also evaluated (Table 1).Nine of these patients (six in the study group and three in the control group) were not evaluated with regard to the achievement of fi rst CR and OS, but they had diagnostic and staging data available and therefore they were included because of these criteria.
The fi rst patient was treated and followed up starting in 1976 and the remainder from 1980 onwards.Subsequently, the treatment protocols changed over the course of time.Patients treated before 1985 received chemotherapy using the MOPP (mechlorethamine, vincristine, prednisone and procarbazine) protocol, while those treated after that year received either the MOPP/ABV (MOPP plus doxorubicin, bleomycin and vincristine) hybrid protocol or the MOPP/ABVD (ABV plus dacarbazine) alternative regimen. 24Involved-fi eld radiation therapy was performed on all stage I and II pa-tients and on most stage III patients, except those with bulky disease, who received extended-fi eld radiation therapy. 24here were no signifi cant differences between the control and study groups with regard to the following variables: age (p = 0.19), hemoglobin level (p = 0.99), WBC (p = 0.53), ESR (p = 0.37) A and B symptoms (p = 0.14), bone marrow infi ltration (p = 0.46), likelihood of achieving the fi rst complete remission (CR) (p = 0.68), overall survival (OS) (p = 0.83) and stages (I + II, III + IV) (p = 0.99).In relation to histological subtypes (LP + NE), (MC + LD) (LP = lymphocyte predominance; NE = nodular sclerosis; MC = mixed cellularity; LD = lymphocyte depletion) (p = 0.03), greater frequency of NE in the study group than in the control group was observed.
Forty-fi ve out of 51 patients in the study group and 48 out of 51 in the control group could be evaluated regarding achievement of fi rst CR and OS.There were 12 deaths: 7/48 (14%) and 5/45 (11%) in the control and studied groups, respectively.
This study was approved by the Ethics Committee of Universidade Federal de São Paulo, and informed consent was obtained from all subjects who were still alive.

Methods Methods
All the lymph node biopsies were preserved in formalin and embedded in paraffi n.Samples were then restained using the hematoxylin-eosin method and reviewed by two hematopathologists.The minimum immunohistochemistry panel consisted of anti-CD30 and CD15 antibodies.Once HD had been confi rmed, PCNA, p53 and MDM2 antibodies were also included.The tissue sections underwent routine treatment.Antigenic recovery was performed by immersing the slides in 0.001 M citrate buffer (pH 6.0) and heating in a microwave oven at maximum power for 45 minutes.After cooling for 20 minutes at room temperature, the slides were washed under running water for fi ve minutes and distilled water for a further fi ve minutes.Endogenous peroxidase was then blocked by immersing the slides in a 0.006% hydrogen peroxide solution for two nine-minute periods, each followed by rinsing using distilled water and phosphate buffer solution (PBS).
Monoclonal antibodies were added at the concentrations recommended by the manufacturer, as follows.For PCNA: PC-10 (Dako, catalog no.MO-879) at 1/200 concentration for 18 hours at 4° C, followed by PBS washing; for MDM2: MDM2 (Novocastra, catalog no.NCR-MDM2) at 1/200 concentration; and for p53: DO7 (Novocastra, catalog no.NCL-p53DO7) and BP (Novocastra, catalog no.NCL-p53BP), at 1/50 concentration for both, incubated for 18 hours at 4º C. The secondary antibody was biotinylated rabbit anti-mouse (Dako, catalog no.E-0354), at 1/100 concentration.Development occurred after incubation with the streptavidin-biotinperoxidase-ABC complex (Dako, catalog no.K-377 A-B), at 1/200 concentration for 30 minutes at 37° C, followed by development on a chromogenic substrate of 3,3' diaminobenzidine (PAB-Sigma, catalog no.D-5637) at 0.006% concentration in PBS, adding 100 µl of hydrogen peroxide at 30 volumes for each 10 ml of solution. 25he PCNA samples were counterstained with Fast-Green (Inlab, catalog no.3870) and p53 and MDM2 with Harris hematoxylin.Negative controls were obtained from distinctive parts of the same slide, by omission of the tested monoclonal antibody.Positive controls were obtained from breast cancer slides that were known to be positive for p53, MDM2 and PCNA, which were provided by the pathology department of our institution.The number of positive cells was determined from the HRS and L counts.Positive cells were defi ned as all cells with any pattern of nuclear staining; negative cells were those without this.Two of the present authors performed the counting, using an optical microscope at a magnifi cation of 1000 times, with a 100-dot integrator (Zeiss), in fi ve randomly chosen different fi elds.In every case, a positive-negative ratio was calculated for HRS and L after specifi c and general proportion ratios for positive and negative L and HRS cells had been obtained.

Statistical analysis Statistical analysis
The tests used were the Wilcoxon test and Spearman coeffi cient to compare marker expression between HRS and L, Friedman's test to determine differences between the markers (PCNA, p53 and MDM2) in HRS and L, and Pearson's chisquared test to evaluate clinical and laboratory variables.CR achievement and OS were calculated by the Kaplan-Meier method and the curves were compared using log-rank tests.Multivariate analysis was based on the Cox regression model.Variables were dichotomized for univariate and multivariate analysis regarding CR achievement and OS.Two-tailed p < 0.05 was considered statistically signifi cant.
The fi rst-line therapy was thus the MOPP/ ABV hybrid for 39 patients, MOPP for six patients and the MOPP/ABVD alternative scheme in four cases. 24Involved-fi eld radiation therapy was performed in 28 cases and extended-fi eld radiation therapy in 12 patients.
The median hemoglobin level was 11.7 g/dl (ranging from 4.4 g/dl to 16.3 g/dl), median WBC was 8 x 10 9 /µl, median ESR was 58 mm/h and median alkaline phosphatase was 233 U/l.Bone marrow analysis was negative in 40 patients, positive in eight and not available in three patients.
The mean MDM2 expression was 60% in HRS and 20.7% in L (p < 0.001).For p53, the mean expression was 52.9% in HRS and 5.9% in L (p < 0.001).For PCNA, the mean expression was 53.4% in HRS and 20% in L (p < 0.001) (Graphs 1, 2 and 3 and Tables 1, 2, 3 and 4).The expression of these tumor markers in HRS was predominantly in the nucleus, and was more positive in tumors than in reactive lymphocytes, for all three markers (Figures 1, 2 and 3 and Graphs 1, 2, 3 and 4).
PCNA, p53 and MDM2 were not associated with gender, age, clinical stage, B symptoms, histological subtype, hemoglobin level, WBC, alkaline phosphatase, ESR or bone marrow involvement (Tables 2, 3 and 4).Univariate analysis between the proportions of marker expression in the tumor cells and all other variables investigated showed tendencies to correlate between MDM2 and PCNA (p = 0.07) (Graph 5, Table 2); PCNA and p53 (p = 0.12) (Graph 5 and Tables 3 and 4); alkaline phosphatase index higher than 150 U and MDM2 (p = 0.09); and unfavorable histology and p53 (p = 0.15) (Tables 1, 2, 3 and 4), but there was no association of marker expression between MDM2 and p53 (p = 0.88 in the Kaplan-Meier test) (Graph 5 and Tables 3 and 4).Multivariate statistical analysis also showed no signifi cant differences.
The expression of different markers had no signifi cant infl uence on CR achievement: p53 (p = 0.All the patients without B symptoms (n = 22) and those with ESR less than 20 (n = 7) were still alive at the end of this study.However, these fi ndings made it impossible to input these variables for multivariate analyses for OS.

DISCUSSION
][3] The cell proliferation rate is an important parameter for better understanding of the clinical picture and for guiding the appropriate therapy.1][12] In non-neoplastic small lymphocytes in HD tissues, PCNA expression is always low. [10][11][12]26,27 Schmid et al. 28 evaluated 23 cases of HD by double labeling with PC-10 monoclonal antibody and CD20 (B cells), and PC-10 and CD45RO (T cells), and found positivity in 50.4% of HRS and 4.3% of lymphocytes. HRS clls express PCNA in 5 to 100% of the cases. Ithas been demonstrated that the intensity of PCNA expression has an infl uence over clinical stages and response to treatment.12,27,28 We found PCNA-positive rates for HRS cells that ranged from 36.9% to 73.68% (median of 52.7%), which were similar to previously reported studies.[10][11][12] We found higher values for PCNA in reactive lymphocytes than did most previous researchers (median of 18.8%).12,27,28 These differences are probably due to variations in the methods and discrepancies in manual cell counts between the individuals doing the counting.
There are many studies evaluating p53 and MDM2 expression in neoplasms.21,22 We characterized p53 using two different clones: BP and DO7.p53 expression was similar for these two clones (data not shown).This indicates that p53 expression in the wild type or in the mutated form may be evaluated using immunohistochemical methods either in fresh tissue or in formalin-fi xed, paraffi n-embedded preserved material.The application of this analysis depends on the characteristics and specifi city of the monoclonal or polyclonal antibodies for p53. 15,16,20,26,29atent EBV infection was initially suggested as a possible cause for p53 mutation.This hypothesis has never been confi rmed in clinical studies.The possibility of an association between latent EBV infection and p53 was fi rst evaluated by Neidobitek et al. 30 in 37 patients in 1993.Only seven cases that were positive for p53 were also positive for  Our data show that, in the "benign" infl ammatory component of HD, there are proliferating and differentiating lymphocytes that are morphologically normal but carry positivity for proliferation markers near to HRS cells.These lymphocytes may correspond to precursors of HRS, as proposed by Hell et al. 27 in 1993.[33]   The occurrence of spontaneous apoptosis, as evaluated by the TUNEL (in situ nick-end labeling) technique, ranged from 10 to 60% in HRS cells.The apoptosis index did not correlate with the histological and clinical findings, although a negative association with outcome was reported in 110 cases by Smolewski et al. 32 Its presence is not influenced by the EBV genome in HRS cells.3][34][35] In a smaller group of patients, it was confi rmed that strong expression of PCNA, p53 and BCL-2 is associated with shorter OS and worse response to treatment. 31,33Brink et al. 35 showed that the BCL-2+/p53-immunophenotype presented the worst prognosis, while the BCL-2-/p53-immunophenotype had fi ve-year OS of more than 90% and the p53+/BCL-2+ immunophenotype had the best prognosis. 35High expressions of LMP1-EBV in HRS cells presented better response to therapy and better OS, while Rb-1 had worse prognosis.7][38] We emphasize that MDM2 and PCNA, MDM2 and p53 expression, and also p53 expression versus unfavorable histology were close to reaching signifi cance in the univariate analyses.[34][35]39 B symptoms and WBC counts higher than 10x10 9 /µl were unfavorable independent factors for achieving the first CR.Bone marrow tumor infiltration, B symptoms and hemoglobin levels less than 12 g/dl had a negative influence on OS.These data have also been reported as negative prognostic factors for survival in HD. 24,32,34,39 The present study was a retrospective analysis, but these findings have led us to attempt to be more specific regarding prognostic factors in this disease. 38The treatment for HD changed over the admission period for these patients, but the ratios of first CR and OS remained almost the same, because of the high sensitivity of the tumor to chemotherapy. 34

CONCLUSIONS
The methods we have used showed that p53, PCNA and MDM2 are expressed in HRS cells at different intensities.Further studies with larger series are needed to elucidate the infl uence of Epstein-Barr virus infection, frequency of Rb-1 gene loss and high expression of P21 on the clinical setting and the relationship of these three factors to treatment response in HD patients.The fi nding of no clear association between the antigen expression evaluated and the clinical data may be explained by the limited number of patients, the retrospective nature of the study and the high expression of these proteins, which made stratifi cation and the result interpretation methods used more diffi cult.
49); MDM2 (p = 0.59) and PCNA (p = 0.62).No signifi cant infl uence on OS was found in relation to PCNA (p = 0.13), MDM2 (p = 0.21) or p53 (p = 0.54).The median OS for the study and control groups has not yet been reached.Therefore, we used the whole available population of 93 patients to analyze any possible associations for fi rst CR and OS in relation to the other variables.The univariate analysis for the likelihood of achieving the fi rst CR showed that B symptoms (p < 0.001), WBC ≥ 10 x 10 9 /µl (p = 0.01), stage (III + IV) (p = 0.01) and hemoglobin < 12 g/dl (p = 0.04) negatively infl uenced the possibility of achieving this.The results for bone marrow infi ltration (p = 0.07) and age (p = 0.17) were close to reaching signifi cance.The Cox regression model indicated that B symptoms (p = 0.001) and WBC ≥ 10 x 10 9 /µl (p = 0.01) were independent from the other analyzed variables in relation to the fi rst CR.

Figure 2 .
Figure 2. Photomicrograph of lymph node showing nuclear MDM2 expression in Hodgkin cells (upper right and left arrows) and lymphocytes (lower right arrow), developed by peroxidase; 400x magnifi cation.

Graph 1 .Graph 2 .
Dispersion of proportional values for MDM2 expression between Hodgkin and Reed-Sternberg (HRS) cells and lymphocytes (L).Dispersion of proportional values for p53 expression between Hodgkin andReed-Sternberg (HRS) cells and lymphocytes (L).

Graph 3 .
Dispersion of proportional values for proliferating cell nuclear antigen (PCNA) expression between Hodgkin and Reed-Sternberg (HRS) cells and lymphocytes (L).