unrelated hematopoietic stem cell transplantation is a rapidly evolving field offering a curative therapy for various hematologic diseases . in particular , the proportions of older patients and patients transplanted with unrelated donors have increased over the last decade . one prerequisite was the introduction of reduced intensity conditioning regimens ( ric ) as an alternative to myeloablative conditioning ( mac ) in elderly patients as well as in patients with co - morbidities . there is already a wealth of data showing that ric is a safe and effective treatment form for patients previously not eligible for hematopoietic stem cell transplantation ( hsct ) . as a consequence , therapeutic schemes for elderly patients nevertheless , classical risk factors still apply , and while increasing age did not influence the incidence of acute or chronic graft - versus - host disease ( gvhd ) , transplant - associated morbidity and mortality as well as disease relapse still pose challenges in elderly patients . one study investigating a significant number of transplanted all patients aged over 45 years showed a substantially higher rate for transplant - related mortality ( trm ) in mac - treated patients with hla - mismatches when compared to the ric - treated cohort , prompting the authors to discourage mac conditioning in this patient group altogether . this observation suggests an interaction between transplantation - associated mortality caused by age - associated risk and hla - mismatching . age and hla - matching status are important clinical predictors for the outcome of hsct and are used among others for risk assessment in hsct . we analyzed the relationship between age - risk and hla - risk in a large cohort of patients transplanted with unrelated donors and tested the hypothesis that age - risk varies according to hla - matching status . a total of 3019 adult patients transplanted for malignant hematologic disorders were included in this analysis . all patients received a first allogeneic unrelated transplant from bone marrow ( bm ) or peripheral blood stem cells ( pbsc ) with no more than 2 hla - mismatches on 5-loci ( hla - a , -b , -c , -drb1 and -dqb1 ) . disease stage definitions were adopted from a previous study defining the european group for blood and marrow transplantation ( ebmt ) risk score . mac was defined according to the recommendations of the ebmt central registry office ( medab manual forms ) . treatments with busulfan 16 mg / kg + cyclophosphamide 120200 mg / kg , cyclophosphamide 120 mg / kg fractionated total body irradiation ( tbi ) 12gy , etoposide vp-16 3060 mg / kg + tbi 12gy fractionated/10gy single dose , beam polychemotherapy , cbv polychemotherapy or tbi 1014gy ; busulfan 16 mg / kg are considered as myeloablative . patient and donor consent for hla typing and for the analysis of clinical data were obtained . the study was approved by the ethical review board of the university of ulm ( project number 263/09 ) . all patients and donors were high resolution typed for hla - a , -b , -c , -drb1 and -dqb1 . ambiguities within exons 2 + 3 for hla - class i and exon 2 for hla - class ii alleles were resolved . ambiguities involving non - expressed ( null ) alleles were resolved according to nmdp confirmatory typing requirements . differences in exon 2 and 3 for hla - class i alleles and exon 2 for hla - class ii alleles were considered as hla - mismatch irrespective of the vector of mismatches . patient hla - c kir ligand status was inferred from high resolution hla - c typing ( c1=asn80 ; c2=lys80 ) . resulting phenotypes were c1c1 , c1c2 and c2c2 . for univariate analysis of overall survival ( os ) , the kaplan - meier method and logrank testing was applied . multivariate analysis for os and disease - free survival ( dfs ) was performed using extended cox - proportional hazards models . for trm and ri , univariate competing risks analysis and multivariate competing risks regression for stratified data was used . evaluated covariates were : patient age , hla - matching status , disease stage , conditioning regimen intensity , treatment with antithymocyte globulin ( atg ) , year of transplantation , time to transplantation , graft source , donor - recipient sex combination , kir ligand status , and donor origin ( national vs. international ) . for antithymocyte globulin ( atg ) treatment , some data were missing ( table 1 ) . models were validated by inclusion of missing values as a separate group and by omission of cases with missing values , and no bias was found . violations of the proportional hazards assumption ( pha ) by disease stage , conditioning regimen intensity and transplantation before 2004 were adjusted using time - dependent modeling of these covariates . a significant center effect was adjusted using a frailty term with gamma distribution . to assess the relationship between age and hla - compatibility , subgroups were formed and analyzed as factors : age group 1835 years ( hla - match : 10/10 , 9/10 and 8/10 ) , 3655 years ( hla - match : 10/10 , 9/10 and 8/10 ) , and over 55 years ( hla - match : 10/10 , 9/10 and 8/10 ) . the cut - off value of 55 years for elderly patients has been used in previous studies and the cut - off value of 35 years is close to the arithmetic mean between the age boundaries in the remaining patients . in addition , an interaction model between age and number of hla - mismatches was investigated . the relative risk conferred by age was visualized as age - dependent risk in different hla - match categories relative to an 18-year old patient transplanted with a 10/10 matched donor as baseline . in this model , the covariate age was included as a continuous variable and no violation of the pha was found . a total of 3019 adult patients transplanted for malignant hematologic disorders were included in this analysis . all patients received a first allogeneic unrelated transplant from bone marrow ( bm ) or peripheral blood stem cells ( pbsc ) with no more than 2 hla - mismatches on 5-loci ( hla - a , -b , -c , -drb1 and -dqb1 ) . disease stage definitions were adopted from a previous study defining the european group for blood and marrow transplantation ( ebmt ) risk score . mac was defined according to the recommendations of the ebmt central registry office ( medab manual forms ) . treatments with busulfan 16 mg / kg + cyclophosphamide 120200 mg / kg , cyclophosphamide 120 mg / kg fractionated total body irradiation ( tbi ) 12gy , etoposide vp-16 3060 mg / kg + tbi 12gy fractionated/10gy single dose , beam polychemotherapy , cbv polychemotherapy or tbi 1014gy ; busulfan 16 mg / kg are considered as myeloablative . patient and donor consent for hla typing and for the analysis of clinical data were obtained . the study was approved by the ethical review board of the university of ulm ( project number 263/09 ) . all patients and donors were high resolution typed for hla - a , -b , -c , -drb1 and -dqb1 . ambiguities within exons 2 + 3 for hla - class i and exon 2 for hla - class ii alleles were resolved . ambiguities involving non - expressed ( null ) alleles were resolved according to nmdp confirmatory typing requirements . differences in exon 2 and 3 for hla - class i alleles and exon 2 for hla - class ii alleles were considered as hla - mismatch irrespective of the vector of mismatches . patient hla - c kir ligand status was inferred from high resolution hla - c typing ( c1=asn80 ; c2=lys80 ) . for univariate analysis of overall survival ( os ) , the kaplan - meier method and logrank testing was applied . multivariate analysis for os and disease - free survival ( dfs ) was performed using extended cox - proportional hazards models . for trm and ri , univariate competing risks analysis and multivariate competing risks regression for stratified data evaluated covariates were : patient age , hla - matching status , disease stage , conditioning regimen intensity , treatment with antithymocyte globulin ( atg ) , year of transplantation , time to transplantation , graft source , donor - recipient sex combination , kir ligand status , and donor origin ( national vs. international ) . for antithymocyte globulin ( atg ) treatment , some data were missing ( table 1 ) . models were validated by inclusion of missing values as a separate group and by omission of cases with missing values , and no bias was found . violations of the proportional hazards assumption ( pha ) by disease stage , conditioning regimen intensity and transplantation before 2004 were adjusted using time - dependent modeling of these covariates . a significant center effect was adjusted using a frailty term with gamma distribution . to assess the relationship between age and hla - compatibility , subgroups were formed and analyzed as factors : age group 1835 years ( hla - match : 10/10 , 9/10 and 8/10 ) , 3655 years ( hla - match : 10/10 , 9/10 and 8/10 ) , and over 55 years ( hla - match : 10/10 , 9/10 and 8/10 ) . the cut - off value of 55 years for elderly patients has been used in previous studies and the cut - off value of 35 years is close to the arithmetic mean between the age boundaries in the remaining patients . in addition , an interaction model between age and number of hla - mismatches was investigated . the relative risk conferred by age was visualized as age - dependent risk in different hla - match categories relative to an 18-year old patient transplanted with a 10/10 matched donor as baseline . in this model , the covariate age was included as a continuous variable and no violation of the pha was found . patients over 55 years of age formed the second largest age group ( n=1195 , 39.6% ) . the distribution of diagnoses reflects the current spectrum of indications , with acute myeloid leukemia ( aml ) being the most frequent diagnosis ( n=924 , 30.6% ) . single hla - mismatches were present in 30.2% ( n=911 ) and double mismatches occurred in 8.7% ( n=261 ) of all patients . although the proportion of hla - dq mismatches among double mismatched transplantations was slightly higher in older patients , there was no statistically significant difference in the distribution of 8/10 mismatches . mac was used in 62.1% ( n=1875 ) of the patients , with peripheral blood stem cells ( pbsc ) being the leading graft source ( n=2694 , 89.2% ) . more than half of the transplantations were performed in the years between 2008 and 2011 ( n=1671 , 55.4% ) . table 2 and figure 1 show the results of the univariate os analysis in patients according to their hla - matching status and age group . logrank - testing showed no significant difference between 10/10 , 9/10 and 8/10 matched transplantations in the youngest age group ( aged 1835 years ) . in the intermediate age group ( 3655 years ) a highly significant difference ( p<0.001 ) was found with higher mortality for patients transplanted with single or double mismatches . in patients over 55 years of age , the differences were even more pronounced , showing high mortality , especially in the 8/10 matching group ( p<0.001 ) . kaplan - meier estimates for overall survival according to hla - matching status ( 10/10 black lines , 9/10 blue lines , 8/10 red lines ) in different age categories . in multivariate modeling , these results could be confirmed for os showing no significant differences between single and double mismatched transplantations in the younger age group ( table 3 ) . risk sharply increased with age in the respective mismatch groups , reaching the highest relative risk in the age group over 55 years ( hr : 3.48 , ci 2.494.86 , p<0.001 ) . similar patterns were seen for dfs and trm with hazard ratios spreading with increasing numbers of hla - mismatches and increasing age , thus conferring highest risk for patients aged over 55 years with double hla - mismatches [ dfs : hazard ratio ( hr ) 2.74 , ci 2.003.76 , p<0.001 and trm : hr 3.79 , ci 2.296.30 , p<0.001 ] . modeling an interaction term between age and number of hla - mismatches allowed estimation of age risk within matched , single - mismatched and double mismatched patient groups . age risk showed increasing risk estimates with increasing number of hla - mismatches . in 10/10 matched transplantations , this additional risk per life year at time of transplantation was lowest ( hr : 1.015 , ci 1.0101.020 ; p<0.001 ) . it increased , however , with the decreasing degree of hla - compatibility between donor and patient ( 9/10 , hr : 1.019 , ci 1.0141.024 , p<0.001 and 8/10 hr : 1.026 , ci 1.0201.031 , p<0.001 ) . the interaction term for age and 2 hla - mismatches was significant ( p=0.009 ) . the cox regression model is a multiplicative hazard model . in order to visualize the component of age - risk within the respective hla - match groups , the change of risk contributed to the prognosis by age at the time of transplantation this visualization is based on the different age - associated risk estimates within each hla - match category as observed in the multivariate model for os , and it illustrates the change in risk with increasing age ( figure 2 ) . relative risk contributed by the continuous covariate age at the time of transplantation according to different levels of hla - mismatches ( completely matched 10/10 : black , single mismatched 9/10 : blue , double mismatched 8/10 : red ) . we found a statistically significant interaction between hla - matching status and age - associated risk . this interaction can be interpreted as different levels of age - associated risk according to the number of hla - mismatches . our findings substantiate that transplantation for patients aged over 55 years with two hla - mismatches are particularly risky with a highly significant hazard ratio of 3.48 ( ci 2.494.86 ; p<0.001 ) when compared to 10/10 matched patients younger than 35 years . if compared to 10/10 transplantations within each age category , double mismatches increased mortality risk for os by a factor of 1.14 in the lowest age group , by a factor of 1.40 in the middle age group , and 2.27 in patients aged over 55 years . this disproportional increase and the poor one - year survival rate of only 19% in double mismatched transplantations for elderly patients highlights the importance of hla - matching especially in this group of patients . luckily , donors with 2 hla - mismatches had to be accepted only in a small fraction of patients aged over 55 years ( 6.3% ) . the age cohorts showed expected structural differences in composition with regard to diagnosis and conditioning regimen , as well as graft source . multivariate analysis adjusted for differences in conditioning treatment , while graft source showed no differential impact on survival end points . it is known that older patients tolerate conditioning related toxicity less well than younger patients , which is the reason for the development and the use of conditioning regimes with reduced intensity . treatment - associated toxicity correlates strongly with transplant - related mortality and therefore it greatly influences os . this relationship explains our findings from the perspective of transplant biology , suggesting that older patients tolerate hla - mismatches less well than younger patients as it is also the case for treatment - related toxicity . on the other hand , it can not be deduced from this data whether younger patients benefit less from better - matched donors , as life expectancy is higher and hla - associated risk cumulates over time . this finding was only made possible because of the relatively high proportion of older patients in our dataset . as most of the transplantations were performed in the years between 2008 and 2011 , our dataset reflects the substantial increase in elderly patients transplanted in germany in recent years . other large studies investigating the impact of risk factors in hsct contained significantly fewer older patients , which is why this interaction may have remained unnoticed in these studies . interestingly , in the youngest age group , no significant difference was found between completely 10/10 matched transplantations and single or double mismatched transplantations . however , this age category was the smallest , consisting of only 17.5% of the cases , which limits interpretation of this particular result . testing for proportional hazards assumption in our models showed no significant violation for the covariate age , which was treated as a continuous variable in the interaction model and in the prediction plot ( figure 2 ) . thus , the way we chose to visualize the disproportional increase in hazard ratios for age - risk at the time of transplantation is justified . our results were obtained from a cohort transplanted with allogeneic unrelated pbsc or bone marrow as a graft source . in our analysis , graft source did not differentially impact outcome , which is why no separate analysis for each graft source was made . data on the impact of haploidentical transplantation or cord blood transplantations on the outcome of hsct in elderly patients are very limited , so that a sensible risk - benefit comparison of our data with alternative graft or transplant sources is difficult . however , cord blood transplantation has been reported to result in similar outcomes in a small cohort of single mismatched transplantations in elderly patients treated with ric . in multivariate analysis ( table 4 ) , some predictors showed violation of the proportional hazards assumption ( pha ) . these violations can be explained by a higher early mortality for patients transplanted in advanced disease stage , transplanted before 2004 and treated with mac . to reflect this relationship , an extended cox regression model was fitted to obtain regression estimates for the respective predictors according to time periods where pha is satisfied , as we have shown before . in analysis of os , advanced disease stage showed a substantially higher mortality risk until day 314 but not thereafter . patients treated with ric showed a significantly lower early mortality until day 96 and a non - significantly different risk afterwards . in addition , patients transplanted before 2004 showed a higher mortality risk until day 198 after transplantation but not thereafter . also a patient c2c2 kir - ligand status as well as an international donor status was associated with adverse outcome , which we have reported before . atg treatment was not included in the final models because it did not reach statistical significance . our analysis encompassed some simplifications , namely that any hla - mismatch was considered equally . hla - dpb1 mismatches were not included and the vector of mismatches was also not regarded . we included hla - dqb1 mismatches in this study , because a previous analysis on the same dataset has shown that these mismatches are associated with higher mortality risk . hla - dpb1 mismatches have been shown to influence outcome of hsct , but due to lower linkage disequilibrium , hla - dpb1-mismatches in hla - a , -b , -c , -drb1 and -dqb1 matched and mismatched transplantations are almost equally distributed . therefore , we may assume that our results are not biased by not including hla - dpb1 . the vector of mismatches was not considered , because no significant differences in survival outcome have been seen for unidirectional mismatches when compared to bidirectional mismatches for the end points analyzed in our study . we refrained from including karnofsky performance status and donor - recipient cytomegalovirus status due to the high proportion of missing data for these variables , which is a limitation of our analysis . when selecting donors for elderly patients , the additional risk associated with hla - mismatches in this age group should be considered . especially when only donors with double hla - mismatches are available for such a patient , the substantial risk conferred in this situation must be carefully weighed against the benefit of transplantation . cord blood transplantation might be an alternative in such cases , although data regarding the impact of alternative graft sources for transplantation of elderly patients are still limited .