diffuse large b - cell lymphoma ( dlbc ) is the most common type of non - hodgkin lymphoma ( nhl ) accounting for 50% of nhl in thailand . although the outcomes of treatment were markedly improved by adding rituximab to standard chop regimen ( r - chop ) , the 5-year event - free survival rate was only 47% . 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 . however , it remains inconclusive whether the p53 expression is an independent outcome predictor in patients with non - hodgkin lymphoma ( nhl ) . 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 ) . 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 [ 911 ] . in this study , we evaluated p53 and p - gp expression , as well as clinical parameters in patients with dlbc . 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 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 . all patients with stage ii  iv were treated with a standard chemotherapy of cyclophosphamide , doxorubicin , vincristine , and prednisolone ( chop ) for at least six cycles . after completion of treatment , all patients were regularly followed up at intervals every few months for at least 5 years or until death . 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 . 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 4c 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 . 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 . 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 1024% , 2549% , and 50% positive reactions were found , respectively . paraffin - embedded placenta was used as a positive control for p - gp ( figure 1(c ) ) . baseline categorical characteristics were compared among patients with and without overexpression of p53 using chi - square . 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 . multivariate analyses for os and dfs were performed using a cox regression model with backward elimination . critical p - values for entry and removal were 0.2 and 0.5 , respectively . to test the main hypothesis hazard ratios ( hrs ) , 95% confidence intervals ( 95% ci ) and p - values were obtained from the best - fit model . . 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 1884 years ) . the median os and dfs were 27.9 months ( range 168 months ) and 30.9 months ( range 074 months ) , respectively with a median follow - up time of 19.7 months ( range 0.366.2 months ) . 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 ) . all tissue sections from patients with newly diagnosed and relapsed dlbc were negative for p - gp expression ( figure 1(d ) ) . 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 ( p = 0.79 ) , os ( p = 0.73 ) , or dfs ( p = 31 ) between the p53-positive ( 3 + ) and p53-negative groups ( 02 + ) , even we tried to change the cut - off values to 1 + or 2 + ( data not shown ) . 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 24 was significantly associated with lower cr rate ( or 14.7 , 95% ci 4.845.3 , p < 0.001 ) and shorter os ( hr 5 , 95% ci 2.89 , p < 0.001 ) . moreover , the advanced stage was a significant predictor of dfs ( p = 0.03 ) . patients with stage iii had hr 3.1 ( 95% ci 1.37.5 ) while patients with stage iv had hr 2.7 ( 95% ci 1.17.1 ) . 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 . 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 . 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 = 03.4% and 018.5% 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 , 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 . in addition , chang et al .   demonstrated 58% of patients with high clinical stage dlbc stained positive for p53 using the cut - off value of 20% as a positive expression . .   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 . regarding the prognostic significance of p53 , no significant relationship between p53 protein expression and any of the clinical variables and outcomes 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 .
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 ( p = 0.79 ) , overall survival ( os ) ( p = 0.73 ) , or disease - free survival ( dfs ) ( p = 31 ) between the p53-positive and p53-negative groups . 
 the final model from multivariate analysis that revealed poor performance status was significantly associated with cr ( p < 0.001 ) and os ( p < 0.001 ) . 
 moreover , the advanced stage was a significant predictor of dfs ( p = 0.03 ) . 
 this study demonstrated no impact of the expression of p53 on either response or survival rates .