allogeneic hematopoietic cell transplantation ( allo - hct ) is a potential curative treatment for children and adults with recurrent or high - risk acute lymphoblastic leukemia ( all ) . however , relapse occurs in approximately 30% of patients with all after hct [ 1 , 2 ] , with a relapse rate of more than 60% in high - risk patients . the outcome of patients who relapse after hct is usually poor despite salvage therapies . outcome of all patients after hct might be improved by identification of markers to predict impending relapse . minimal residual disease ( mrd ) assessment relies on the identification of specific molecular or immunophenotypic markers on the leukemia cells . pcr is used to detect leukemia - specific fusion transcripts ( e.g. , bcr - abl ) or clone specific immunoglobulin ( ig ) or t - cell receptor ( tcr ) genes . mrd detected by pcr has been demonstrated as an independent risk factor for all relapse after induction or consolidation therapy [ 47 ] . a number of studies have also shown clinical significance of mrd , as detected by pcr , in the transplant setting [ 816 ] . pcr methods for detection of mrd have high sensitivity , but are relatively labor intensive and not widely available . an alternative method for mrd measurement is by multiparameter flow cytometry ( mfc ) , based on the detection of leukemia associated immunophenotypes that can be used to distinguish them from normal hematopoietic cells . using 4-color flow cytometry , leukemia - associated immunophenotypes can be identified in more than 90% of all patients , with detection limits of 10 - 10 [ 1825 ] . increasing evidence has demonstrated the prognostic importance of mrd detected by mfc in pediatric and adult patients with all in the nontransplant setting [ 18 , 21 , 24 , 2628 ] . results have indicated that patients with detectable mrd by mfc at the end of induction and during maintenance therapy have a high rate of relapse . however , only a few studies have evaluated the clinical impact of mrd monitoring by mfc in all patients who undergo hct [ 29 , 30 ] . in the present study , we evaluated the value of mrd , detected by seven - color mfc before and after allo - hct , in 160 pediatric and adult patients with all , to identify the impact of pre- and post - hct mrd on relapse and survival posttransplant . patients of all ages , identified from the fred hutchinson cancer research center computerized database , were included in this retrospective study . the study cohort included 160 patients , who underwent allo - hct for treatment of all in remission ( < 5% blasts in marrow by morphology and no evidence of extramedullary disease ) from april 2006 through march 2011 . patients received high - intensity conditioning regimens before hct according to a standard treatment plan or prospective clinical trials at the fred hutchinson cancer research center . 142 patients ( 89% ) received tbi based conditioning , and the rest received regimens consisted of treosulfan and fludarabine , busulfan and cyclophosphamide , or busulfan and fludarabine . all patients provided informed consent for treatment according to transplantation protocols approved by the institutional review board . in addition , separate institutional approval was obtained to gather data from patient records and databases . mfc was performed on bone marrow aspirates as previously described [ 17 , 31 ] . for b - lineage all , the panel consisted of one tube as follows : cd20-fluorescein isothiocyanate ( fitc ) , cd10-phycoerythrin ( pe ) , cd34-percp - cy5.5 , cd19-pe - cy7 , cd38-alexa 594 ( a594 ) , cd58-allophycocyanin ( apc ) , and cd45-apc - h7 . for t - lineage all , the panel consisted of one tube as follows : cd8 v450 , cd2 fitc , cd5 pe , cd34 pe - tr , cd56 pe - cy5 , cd3 pe - cy7 , cd4 a594 , cd7 apc , cd30 apc - a700 , and cd45 apc - h7 . for biphenotypic leukemia ( two patients ) , the same combination of reagents used for b or t cell all was used and supplemented by a combination directed at abnormal myeloid progenitors ( ( 1 ) hla - dr pb , cd15 fitc , cd33 pe , cd19 pe - tr , cd117 pe - cy5 , cd13 pe - cy7 , cd38 a594 , cd34 apc , cd71 apc - a700 , cd45 apc - cy7 , ( 2 ) hla - dr pb , cd64 fitc , cd123 pe , cd4 pe - tr , cd14 pe - cy5.5 , cd13 pe - cy7 , cd38 a594 , cd34 apc , cd16 apc - a700 , cd45 apc - cy7 , and ( 3 ) cd56 a488 , cd7 pe , cd5 pe - cy5 , cd33 pe - cy7 , cd38 a594 , cd34 apc , cd45 apc - cy7 ) . all antibodies were obtained from beckman - coulter ( fullerton , ca ) or becton dickinson ( bd biosciences , san jose , ca ) . up to 1 million events per tube were acquired on a custom - built lsr ii flow cytometer ( bd biosciences ) , and data compensation and analysis were performed by using noncommercial software developed in our laboratory . mrd was identified as a cell population showing deviation from the normal patterns of antigen expression seen on specific cell lineages at specific stages of maturation compared with either normal or regenerating marrow . when identified , the abnormal population was quantified as a percentage of the total white blood cell events . the software used to help analyzing mfc mrd in this study is woodlist version 2.7.7 , the software used in the clinical hematopathology laboratory in our institution . overt relapse was defined as > 5% blasts by morphology or evidence of extramedullary disease . the hazards of relapse and overall mortality were compared between those with mrd and those without mrd using cox regression . patients were categorized as mrd - positive or mrd - negative pre - hct based on the flow value closest to but before date of transplant , while post - hct flow values were modeled as a time - dependent covariate . the association of pre - hct mrd with outcome was assessed separately from the association of post - hct mrd with outcome . patients were also categorized based on pre- and post - hct mrd as positive / positive , positive / negative , negative / negative , or negative / positive . this variable was modeled as a time - dependent covariate based on the status of the post - hct mrd . estimates of the probability of relapse were summarized by cumulative incidence estimates with death without relapse treated as a competing risk . estimates of the probability of survival and relapse - free survival were obtained using the method of kaplan and meier . the study cohort included 160 patients ( 62 pediatric and 98 adult , 99 males and 61 females ) , who underwent myeloablative allogeneic - hct at the fred hutchinson cancer research center from april 2006 to march 2011 for the treatment of all in remission ( < 5% blasts in marrow by morphology and no evidence of extramedullary disease ) . all patients had less than 5% bone marrow blasts and had no evidence of extramedullary disease and thus met the criterion for cr . there were 134 patients with b - all , 24 patients with t - all , and two patients with biphenotypic leukemia ( one patient had markers of b cells and myeloid cells , and one patient had markers of t cells and myeloid cells ) . ninety - nine patients had normal karyotype ; 31 patients had ph+ all as defined by conventional cytogenetics , fish , or pcr ; 15 patients had other unfavorable abnormalities ( 7 , + 8 , 11q23/mll gene rearrangement ) ; and 15 patients had other karyotypic abnormalities . ninety patients were in first complete remission ( cr-1 ) at time of transplant , 58 patients were in cr-2 , and 12 patients were in cr-3 or beyond . one hundred and fifty - three of the 160 ( 96% ) patients achieved an absolute neutrophil count of 500/mm on 3 consecutive days at a median of 20 days posttransplant ( range : 1248 days ) . the seven patients who failed to achieve this level died between days 18 and 86 due to infection complications . one hundred and thirty - three ( 83% ) patients reached a platelet count of 20,000/mm without transfusions for 7 consecutive days at a median of 20 days ( range : 1199 days ) . there were 66 deaths by last contact ; these include relapse related deaths ( n = 28 ) and nonrelapse mortality ( n = 38 ) . days of death ranged from 13 to 1908 days with a median of 156 days . median follow - up among the patients who have not died by last contact was 1219 days ( range : 1822487 days ) . one hundred and twenty - six ( 79% ) patients developed grades 24 acute gvhd ; 34 ( 21% ) developed grades 3 - 4 gvhd . mrd evaluation posttransplant was performed based on our institution standard practice ( usually on day + 28 , day + 80100 , and one year after transplant ) and patient clinical status and per the discretion of the treating physician . the relationship between the status of mrd before transplant and clinical outcomes was evaluated for 153 patients with an mfc result within 50 days of hct . patients with positive mfc results at time of transplant were assigned to the mrd - positive group , while patients with negative mfc results were assigned to the mrd - negative group . all patients had less than 5% bone marrow blasts by morphology and thus met the morphologic criterion for cr . among 59 patients who were mrd - positive pre - hct , there were 23 relapses compared to 14 of 94 patients who were mrd - negative pre - hct ( hr = 3.64 ; 95% ci , 1.877.09 ; p = .0001 ) . sixty - six patients had pre - hct flow cytometry data and bcr - abl molecular data . among patients with pcr data , the risk of relapse for pre - hct mrd - positive is 3.18 times that of patients who are pre - hct mrd - negative . after adjusting for bcr - abl , thirty - five of the 59 mrd - positive patients have died compared to 30 of the 94 mrd - negative patients ( hr = 2.39 ; 95% ci , 1.463.90 , p = .0005 ) . the hazard ratios were qualitatively unchanged after adjusting for the presence of abnormal cytogenetics or positive bcr - abl by molecular testing . one- and 3-year estimates of relapse for patients without mrd pre - hct were 12% and 17% , respectively , compared to 32% and 38% among those with mrd pre - hct , respectively ( figure 1(a ) ) . one- and 3-year estimates of survival for the pre - hct mrd - negative group were 75% and 68% , respectively , compared to 44% and 40% in the mrd - positive group , respectively ( figure 1(b ) ) . relapse - free survival estimates at one and three years were 69% and 61% , respectively , in the mrd - negative group and 41% and 34% , respectively , in the mrd - positive group ( figure 1(c ) ) . in order to see if the degree of mrd among patients correlated with outcome among those who were mrd - positive pre - hct , we arbitrarily categorized the flow values as less than or equal to 0.01% ( n = 12 ) , 0.010.1% ( n = 14 ) , 0.11.0% ( n = 16 ) , and 1.09.0% ( n = 11 ) and greater than 9.0% ( n = 6 ) the proportion of posttransplant relapses in these groups were 25% , 50% , 19% , 58% , and 50% , respectively , and the proportion of deaths were 50% , 64% , 50% , 67% , and 67% , respectively . these data provide no convincing evidence to suggest that patients with  more  mrd pretransplant have worse outcome than patients with  less mrd among those who are mrd - positive , although the numbers are too small to definitively draw a conclusion . a statistical test for interaction between pre - hct mrd and t - all versus b - all yields p = .30 , although the number of t - all is small ( n = 24 ) , severely limiting the power to detect a meaningful interaction . the risk of relapse was higher among patients with mrd pre - hct compared to those without mrd among b - all patients ( hr = 2.87 ; 95% ci , 1.376.02 ; p = .005 ) and among t - all patients ( hr = 7.07 ; 95% ci , 1.3138.09 ; p = .02 ) . thus , mrd appears to be correlated with an increased risk of relapse in both b - all and t - all , but the small sample sizes limit our ability to determine if the effect is larger in the t - all patients as compared to the b - all patients . adjusting for age or cr status at time of transplant has no significant effect on the association between mrd pre - hct and post - hct relapse ( age p = .59 ; cr p = .72 ) . one hundred and forty - four patients had at least one mfc flow sample post - hct , all of these with at least one sample within 78 days of transplant . one hundred and thirty patients had at least one value within 35 days of transplant , and one hundred and forty patients had at least one value within 50 days of transplant . all of those patients were in remission by marrow morphology ( < 5% blasts ) and with no evidence of extramedullary disease at that time . modeling post - hct flow values as a time - dependent covariate showed an increased risk of relapse ( hr = 7.47 ; 95% ci , 3.3016.92 , p < .0001 ) and death ( hr = 3.00 ; 95% ci , 1.446.28 , p = .004 ) among patients with mrd - positive compared to patients with mrd - negative . if the flow values are restricted to those that occurred within 100 days ( to loosely assess the impact of  early  mrd on outcome ) , the results are qualitatively the same ; that is , the relapse rate is higher in mrd - positive patients compared to mrd - negative patients ( hr = 4.64 ; 95% ci , 2.0410.55 , p = .0003 ) as is the rate of mortality ( hr = 2.36 ; 95% ci , 1.144.91 , p = .02 ) . among the 17 patients with post - hct mrd - positive , 15 had cytogenetic data . all those 15 patients had normal conventional cytogenetics , but one patient had abnormal fish . seventeen patients were in morphological remission , but with evidence of mrd by mfc within 50 days posttransplant . four of those 17 patients had evidence of mrd in their last marrow evaluation pretransplant . six patients had mrd level of 0.01% or less , seven patients had mrd level between 0.01% and 0.1% , and four patients had mrd level between 0.1% and 1.0% . preemptive therapy for patients with mrd - positive after transplant included rapid immunosuppression taper in 3 patients , tyrosine kinase inhibitor ( tkis ) in two patients , and rapid immunosuppression taper plus dli in one patient ; the other 11 patients received no intervention . five of the 11 patients who received no intervention have relapsed , and none of the three patients who had rapid immunosuppression taper had relapsed . the one patient who received dli had relapsed ; a follow - up marrow evaluation 12 days after dli demonstrated no evidence of disease by morphology , flow cytometry , and cytogenetics ; however , the patient developed mrd again two months after dli , which progressed to overt relapse two months later . nonetheless , both patients subsequently relapsed , on day 81 post - hct and on day 152 post - hct , respectively . among the 17 patients who were mrd - positive early ( i.e. , within 50 days ) post - hct , 8 patients relapsed during the follow - up period and 7 of them died seven patients who were mrd - positive early after transplant converted to mrd - negative and were alive with no evidence of relapse at last contact at median of 726 days after transplant . mfc levels for these patients were 0.0014% to 0.8% , and mrd cleared between day 35 and day 357 post - hct . three of the patients who converted to mrd - negative had rapid taper of immunosuppression and four had no intervention . all seven patients who cleared their mrd and have not relapsed developed grade ii - iii acute gvhd , and three developed chronic gvhd . among patients with early posttransplant mrd who eventually relapsed , two patients cleared their mrd prior to progressing to overt relapse . disease progression and outcome after hct for all 17 patients with mrd - positive early post - hct is shown in table 2 and figure 2 . combining the early post - hct data with the pre - hct flow status revealed that mrd positivity at any time is associated with worse outcome relative to patients who were mrd - negative before and after hct . with post - hct mrd status modeled as a time - dependent covariate for the post - hct closest to day 28 , patients who were mrd - negative pre - hct and became mrd - positive post - hct had an increased risk of relapse compared to negative / negative patients ( hr = 8.08 ; 95% ci , 1.7936.36 , p = .007 ) . patients who were mrd - positive pre - hct but mrd - negative post - hct were also more likely to relapse relative to negative / negative ( hr = 3.61 ; 95% ci , 1.697.73 , p = .0009 ) as were patients who were mrd - positive pre - hct and mrd - positive post - hct ( hr = 5.86 ; 95% ci , 2.3914.39 , p = .0001 ) . there were only 3 patients who were mrd - negative pre - hct but became mrd - positive at some point post - hct . negative / positive versus negative / negative yielded hr = 3.75 ( 95% ci , 0.8915.81 , p = .07 ) ; positive / negative yielded hr = 2.41 ( 95% ci , 1.414.12 , p = .001 ) ; and positive / positive yielded hr = 2.87 ( 95% ci , 1.356.12 , p = .006 ) . in this study , we investigated the prognostic value of mrd , as detected by mfc before and after myeloablative allo - hct in a large cohort of patients with all . a number of reports have shown that mrd prior to hct is a predictive factor for relapse after hct [ 816 , 29 , 30 , 3234 ] . in these studies , the efs for the mrd - negative groups prior to hct was often more than 70% , while for the mrd - positive group the efs fell to approximately 1750% . the majority of those studies evaluated mrd by ig / tcr gene rearrangement , included relatively small numbers of all patients , and evaluated the effect of mrd prior to transplant . only a few studies have demonstrated the impact of mfc - based mrd screening in the allogeneic transplant setting . in one study , bone marrow samples were prospectively taken from 24 all patients at certain time points before and after transplantation . the data showed that pretransplant mrd detected by mfc was a significant predictor of outcome , and indicated that mrd monitoring after hct could be used to identify patients with a high risk of relapse . zhao et al . also demonstrated that a mrd - positive after hct is correlated with poor efs and high cumulative incidence of relapse ( cir ) in both high - risk and standard - risk groups of all patients . the results of our study support those prior findings in a large cohort and demonstrate the importance of mfc as a tool to evaluate all mrd before and after transplant . mfc is less labor intensive and more widely available than pcr , and the results of our study demonstrate that mfc may be used to risk stratify all patients before and after hct . first , patients with all who were in cr without flow cytometric evidence of mrd before myeloablative allogeneic hct had a better outcome than patients with evidence of mrd . second , patients with evidence of mrd after transplant ( and in particular , early after transplant ) had significantly worse outcomes than patients without evidence of mrd after transplant . third , not all patients who became mrd - positive after transplant relapsed , with approximately one - third reverting to mrd - negative status . as shown in table 2 , patient number 3 had no evidence of relapse more than 4 years after transplant , patient number 5 had no evidence of relapse more than 3 years after transplant , patient number 6 had no evidence of relapse 2 years and 8 months after transplant , patient number 7 had no evidence of relapse 2 years after transplant , and patient number 13 had no evidence of relapse 1 years and 7 months after transplant . thus , four out of the seven patients who converted from mrd - positive to mrd - negative after transplant had been in remission for at least two years posttransplant , and one patient had been in remission for more than 18 months posttransplant . patients number 9 , number 16 , and number 17 had short follow - up of less than a year , and therefore we can not comment on their long term disease status after transplant . based on this data , at least four of seventeen patients ( 24% ) with mrd - positive before day 50 post - hct have converted to mrd - negative and had been in remission for 2 years or more after transplant . among the seven patients in our cohort who converted their mrd status , three had rapid immunosuppression taper and four had no intervention . clearing of mrd after allo - hct may be a result of graft - versus - leukemia effect . this potential mechanism is supported by the observation that all seven patients in our cohort who did not relapse and had no evidence of mrd developed graft - versus - host disease . it is difficult to draw firm conclusions regarding the association between level of mrd and outcome ( i.e. , whether patients with higher mrd levels have a worse outcome than those with lower levels ) . for pretransplant mrd , our data support the notion that not the mrd level but the presence of mrd increases the probability of relapse with equal proportion of posttransplant relapses ( 50% ) for pretransplant mrd of 0.01%0.1% or mrd of more than 9% . however , the situation may be different for posttransplant mrd . among 17 patients with posttransplant mrd - positive within 50 days after transplant , six patients had mrd level of 0.01 or less , four of whom ( all with mrd level below 0.01% ) did not develop relapse during the follow - up period ( 2 years or more for two patient , and less than one year for two patients ) . on the other hand , three of four patients with mrd higher than 0.1% have relapsed ( the fourth patient with mrd level of > 0.1% had early death due to infection and treatment related complications ) . despite the small numbers , these results may support correlation between posttransplant mrd level and outcome . one may speculate that lower risk of relapse with lower level of mrd after transplant may be due to graft versus leukemia effect , which is more effective when the disease burden is low . a more definitive examination of the correlation between pre- and posttransplant mrd level and outcome will require a larger number of mrd - positive patients . the data were collected retrospectively , the patient population is heterogeneous , patients were treated according to a variety of protocols with different treatment strategies , and methods and timing of follow - up were not standardized . despite these limitations , we believe that this study provides strong evidence that both pre- and post - hct mrd , detected by mfc , are associated with increased relapse and decreased survival after transplant . determination of mrd , early after induction and/or after consolidation chemotherapy , has proven useful to predict relapse and poor outcome in all patients and may help in identifying patients who require allogeneic hct for treatment intensification . our findings suggest that detection of mrd by mfc at the time of hct or early after hct defines a population of all patients who are at higher risk for adverse outcome , even after adjusting for other factors that influence post - hct outcome . these findings support the use of pre- and post - hct mrd assessment by mfc for risk stratification of post - hct outcome . furthermore , these findings provide the rationale for future studies to test whether hct outcome of mrd - positive all patients can be improved through mrd - stratified interventions , for example , preemptive treatment after hct .
in this retrospective study , we evaluated the impact of pre- and posttransplant minimal residual disease ( mrd ) detected by multiparametric flow cytometry ( mfc ) on outcome in 160 patients with all who underwent myeloablative allogeneic hematopoietic cell transplantation ( hct ) . 
 mrd was defined as detection of abnormal b or t cells by mfc with no evidence of leukemia by morphology ( < 5% blasts in marrow ) and no evidence of extramedullary disease . among 153 patients who had pre - hct flow data within 50 days before transplant 
 , mrd pre - hct increased the risk of relapse ( hazard ratio ( hr ) = 3.64 ; 95% confidence interval ( ci ) , 1.877.09 ; p = .0001 ) and mortality ( hr = 2.39 ; 95% ci , 1.463.90 , p = .0005 ) . 
 three - year estimates of relapse were 17% and 38% and estimated 3-year os was 68% and 40% for patients without and with mrd pre - hct , respectively . 
 144 patients had at least one flow value post - hct , and the risk of relapse among those with mrd was higher than that among those without mrd ( hr = 7.47 ; 95% ci , 3.3016.92 , p < .0001 ) . 
 the risk of mortality was also increased ( hr = 3.00 ; 95% ci , 1.446.28 , p = .004 ) . 
 these data suggest that pre- or post - hct mrd , as detected by mfc , is associated with an increased risk of relapse and death after myeloablative hct for all .