  glucocorticoids were among the first drug classes used in the treatment of patients with all and are still essential components of treatment .
the glucocorticoid receptors ( gr ) can form dimers , translocate to the nucleus , and interact with glucocorticoid - response elements to transactivate gene expression , or they can remain as monomers and repress the activity of transcription factors such as the activating protein-1 ( ap-1 ) and nuclear factor-b ( nfb ) .   traditionally , prednisone has been the glucocorticoid 's most commonly used drug in the treatment of patients with acute lymphoblastic leukemia ( all ) .
it is typically given for 4 consecutive weeks in combination with vincristine , anthracycline , asparaginase , and intrathecal chemotherapy .
in the past few years , dexamethasone , another glucocorticoid , has been increasingly used to treat all .
however , there have been investigations with respect to glucocorticoid receptors , distribution of gr isoforms , and genetic polymorphisms .  
polymorphisms at the promoter region of il10 gene are associated with several diseases , including autoimmune , infectious , cancer , alzheimer 's disease ( ad ) , and lymphoblastic leukemia .
subsequently , pleiotropic inhibitory and stimulatory effects on various types of blood cells were described for il-10 , including its role as a survival and differentiation factor for b cells .
a polymorphism at -1082 position , within the il-10 gene promoter region , is known to have an influence on il-10 plasma levels .
have shown that the binding capacity for dexamethasone can be upregulated by high il-10 production .   tumor necrosis factor ( tnf )
is a cytokine with pleiotropic biological activities , including the induction of programmed cell death ( apoptosis ) and the regulation of immune cell proliferation and differentiation , and may influence the transcriptional activity induced by the glucocorticoid of the glucocorticoid receptor gene .  
although genetic polymorphisms in the tnf locus have been implicated in the severity of several b - cell lymphoproliferative diseases [ 12 , 13 ] , to date , only a few studies have found an association among the rare tnf aa of g308a polymorphism , the increased production of tnf protein , and the non - hodgkin 's lymphomas ( nhl ) susceptibility .   
currently , there are few studies related to the overall survival of patients with acute lymphoid leukemia and g1082a and g308a polymorphisms . in this study
, the association of tnf ( g308a ) and il10 ( g1082a ) polymorphisms was analyzed in relation to the overall survival of brazilian children with all , observed for a long time according to the gbtli99 treatment protocol .
  the study group was composed of children with acute lymphoblastic leukemia , aged 0  18 years , who were being evaluated at the oncohematology pediatric center oswaldo cruz hospital ( huoc ) , recife , brazil , since january 2004 to december 2011 .
we analyzed 105 patients for tnf ( g1082a ) and il10 ( g308a ) polymorphisms according to the overall survival , presenting clinical and laboratory diagnosis for acute lymphoblastic leukemia .
the treatment was in accordance with the protocol of brazilian group for the treatment of lymphoblastic leukemia in childhood ( gbtli99protocol of brazilian group for treatment of lymphoblastic leukemia in childhood ) .  
the primers   used for genotyping were ( forward ) 5-agg caa tag gtt ttg agg gc-3 and ( reverse ) 5-tcc tcc ctg ctc cga ttc cg-3. the cycling included 1 cycle of 95c/6  min ; 40 cycles of 95c/60  s , 62.5c/90  s , and 72c/60  s ; 1 cycle of 72c/7  min .
thirty microliters of pcr mixture comprised 3.0  l buffer ( 10x ) , 1.5  l mgcl2 ( 50  mm ) , 1  u taq polymerase ( 5  u ) , 0.6  l dntp ( 10  mm ) , and 0.6  l primer ( 10  pmol/l ) . the 308 g  a base pair substitution creates a ncoi restriction site .
the pcr product ( 107  bp ) of c308 t was digested for 16 hours at 37c using ncoi and analyzed on agarose gel 4% with ethidium bromide ( 0.4  mg / ml ) in electrophoresis .
the digestion of pcr product with ncoi showed fragments of 107  bp for aa genotype and 87  bp and 23  bp for the gg genotype .  
the primers   used for genotyping were allele a forward-5-cct atc cct act tcc cct-3 and allele g forward 5-cct atc cta ctt ccc cc-3 and reverse 5-agc aac act cct cgt cgc aac-3. the cycling was of 1 cycle of 95c/10  min ; 40 cycles of 95c/60  s , 59c/60  s , and 72c/60  s , 1 cycle of 72c/7  min . for each reaction , 10  l mixture comprised 2  l of each primer ( 10  pmol/l ) , 1x gotaq green master mix ( 2x)-promega ( gotaq dna polymerase is supplied in 2x green gotaq reaction buffer ( ph 8.5 ) , 400  m datp , 400  m dgtp , 400  m dctp , 400  m dttp , and 3  mm mgcl2 ) , and 1  l of dimethyl sulfoxide ( dmso ) . the pcr products ( 139  bp )
were analyzed on agarose gel 2% with ethidium bromide ( 0.4  mg / ml ) in electrophoresis .  
survival rates were estimated using the kaplan - meier method , and survival curves were compared using the log - rank test .
the overall survival analysis was performed using the patient 's follow - up data , according to the statistical models in conjunction with the kaplan - meier log rank ( mantel cox ) to assess the death risk in 10.5 years ( 126 months ) , and the survival time was calculated as the time from diagnosis to date of last followup for patients who were still alive . all results with p
  the ratio found in the genotypes for the g308a ( tnf ) polymorphism was 1(aa )  :  5(ag )  :  2.7(gg ) , and the genotype frequencies were not in hardy - weinberg equilibrium ( p = 0.0351 ; a allele proportion = 0.41 and g allele proportion = 0.59 ) .
the ratio of the genotypes for g1082a ( il10 ) polymorphism was 1(aa )  :  1.9(ag )  :  1.08(gg ) , and the genotypic frequencies were in hardy - weinberg equilibrium ( p = 0.7725 ; a allele proportion = 0.49 and g allele proportion = 0.51 ) ( table 1 ) .   
the median follow - up duration in all patients was 37 months ; our attention was also calledin to the death / living relationship in tnf aa ( 0.71 ) and il10 aa ( 1.0 ) genotypes
. a higher percentage of the il10aa genotype was found in patients with all , and they also showed a high ratio of death / live 1.0 ( table 2 ) . 

table 3 shows the survival rate , stratified at different time intervals ( 25 , 50 , 75 , 100 , and 126 months ) , when compared to allelic frequencies .
the tnf gene showed a small difference between their rates , and the tnfa allele had a greater rate in the intervals analyzed . on the other hand
, the il10 gene showed a greater range wider than the intervals between the rate of their alleles , and the il10a allele had a better rate survival in the intervals .    after analyzing the follow - up results
, we found that up to 75 months , the death estimate decreased , showing a higher percentage up to 25 months ( 18.18% ) for il10a allele , while the il10 g allele had a better life expectancy in all ranges of months analyzed . regarding the tnf gene ,
the tnf a allele had the best life expectancy up to 75 months and tnfg allele demonstrated a higher death risk until 50 months ( table 3 ) .  
patients with il10aa genotype showed a worse survival compared with the others ( p = 0.0089 ) , indicating a survival rate of 44% up to 100 months , while the il10ag and il10gg genotypes had survival rate of 69% up to 126 months ( figure 1 ) .  
patients with tnfaa genotype showed a shorter median survival time ( 60 months ) compared with the others genotypes ( tnfag and tnfgg ) ( graph not shown ) .  
the patients who had il10aa + tnfa genotypes demonstrated a lower survival rate when compared with il10gg + tnfag genotypes ( p = 0.0043 ) .
after the 50th month , the survival percentage for the patients with il10aa + tnfaa genotypes was 38% , while it was 75% for the il10gg + tnfag genotypes ( figure 2 ) .
  nowadays , fewer studies propose to study polymorphisms in tnf and il10 genes in relation to prednisone glucocorticoid in acute leukemia during childhood .
this is regrettable once the prednisone is the main drug given during the induction phase in leukemia .
this study was the second in challenging the -1082 ( il10 gene ) and -308 ( tnf alpha gene ) polymorphisms , together , in leukemia .
the death cause in some patients was due to the patient 's reaction in the development of leukemia , acquiring infections due to individual conditions as well as the nutritional state before the treatment and also accentuated by the toxicity to methotrexate ( mtx ) .   regarding the g1082a ( il10 ) polymorphism , edwards - smith et al
.   showed that patients homozygous for the il10 g allele showed high plasma levels of il10 .
this finding is also suggested in the study of lauten et al .   that evaluated 135 patients , mostly adults , with all in accordance with protocols all - bfm 86 and bfm-90 in multicenter trials , according to their response to prednisone ( based in schrappe et al .
2000 ) and with stratification into risk groups , which was made , predominantly , according to the leukaemic cell mass estimate ( the so - called risk factor , the composite variable calculated from the initial blast count in the peripheral blood , and the sizes of liver and spleen below the costal margin ) and the initial treatment response , event - free survival ( efs ) , and relapse risk . in our study ( based on gbtli99 protocol ) there is no definition for response to prednisone , because there has been no study for this purpose .
however , no study ( based on gbtli99 protocol  initial dose of prednisone 40  mg / m / day ) established a background of prednisone response .  
( 2002 )   found that patients showing poor response were stratified into the high - risk group and therefore underwent a more intensive treatment , suggesting that patients with il10gg may have a lower risk when there is a poor response to prednisone .    in terms of overall survival
, we found in this study that older patients ( > 15 years ) showed worse survival rates , although it has not been statistically significant and that the il10aa genotype had worse survival under protocol gbtli99 .    in this study ,
the tnfga genotype was present in a higher ratio among patients with the cd10 + ( calla ) marker ( p = 0.0296 ) .
once the cd10 + marker is a variable of good prognosis and the tnf ga genotype presented best survival , we suggest that these patients with these markers ( cd10 + and tnf ga ) have a better performance in the treatment of all .  
the monitoring of survival , through the divisions of the months , showed that both alleles il10a and tnf g had the highest rate for survival during the period of 126 months , and that the il10a allele , up to 25 months of followup , showed the highest percentage of risk estimate ( 18.18% ) in the occurrence of deaths .    on the other hand ,
when the g1082a and g308a polymorphisms were correlated with respect to survival , the patients with both genotypes tnfga + il10gg showed high overall survival , as opposed to lauten et al . , which found no statistical significance for this comparison .  
in other related studies [ 20 , 21 ] , we found no associations with the g308a ( tnf ) polymorphism , perhaps due to the tnf expression that can be modulated by several interleukins ( not evaluated in this study ) .
however , we suggest that the tnf a allele be involved in worse survival in all during childhood .   
we also suggest that patients presenting il10aa and tnfaa genotypes have high risk that could lead to poor survival .
the results showed no association for tnf gene , but the tnfaa genotype indicated a high rate of death ( 0.71 ; table 1 ) and worse overall survival .   
however , until today , few studies have found an association between the rare tnfaa ( -308 ) genotype involved in an increased production of tnf protein and the worse survival .  

bel hadj jrad et al .   evaluated 194 patients with non - hodgkin 's lymphomas ( nhl ) and 160 controls , associating a risk increase in the development of nhl for the tnfaa genotype .
the tnfaa genotype can be related to low survival of patients with all due to the adenine position ( -308 ) at the tnf gene , and a correlation with a higher protein expression of tnf [ 22 , 23 ] has been shown .
an increased tnf level could impair the efficiency of the antitumor cellular immune response , and tnf can induce the apoptosis by natural killer cells .  
the knowledge of these polymorphisms in the development of leukemic diseases could optimize , in the future , the innovative therapies using mirs to silence genes according to the worst response to prednisone and enhance studies such as that of zhang et al .   who analyzed the expression of different genes , using eight mirs for the stratification of pediatric patients with all in relation to relapse risk .  
the knowledge of the pathogenesis and survival monitoring of patients with all is still unclear .
it is necessary to analyze other studies , using other chemotherapeutic protocols to approach this issue .
 interleukin 10 ( il10 ) is a pleiotropic cytokine that stimulates various hematopoietic cells . 
 the tumor necrosis factor alpha ( tnf ) is a cytokine that may influence the transcriptional activity induced by glucocorticoids . 
 this study examined the impact of tnf ( g308a ) and il10 ( g1082a ) polymorphisms at promoter regions in relation to the overall survival of 105 children ( 0  18 years ) with acute lymphoblastic leukemia ( all ) for a period of 126 months , treated according to the protocol gbtli99 . 
 the g1082a and g308a polymorphisms were identified by allele - specific pcr and pcr - rflp , respectively . 
 patients with il10aa genotype had a higher death ratio ( 44% , p = 0.0089 ) . 
 patients with both il10aa and tnfaa genotypes showed the worst survival when compared with the il10gg and tnfga genotypes ( p = 0.0043 ) . 
 the results of this study revealed a lower survival among patients with il10aa genotype and the concomitant occurrence of il10aa and tnfaa genotypes . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion