Key Points

  • MRD at time of allo-HCT was an important risk factor in Ph+ ALL patients during both CR1 and CR2.

  • No significant difference in relapse rate was observed between CR1 MRD and CR2 MRD.

Abstract

Although measurable residual disease (MRD) at the time of allogeneic hematopoietic cell transplantation (allo-HCT) has been reported to be an important prognostic factor for Philadelphia chromosome (Ph)–positive acute lymphoblastic leukemia (ALL) during first complete remission (CR1), the prognostic impact of MRD is unclear during second CR (CR2). To clarify the impact of MRD for both CR1 and CR2, we analyzed data from a registry database including 1625 adult patients with Ph+ ALL who underwent first allo-HCT during either CR1 or CR2 between 2002 and 2017. Adjusted overall and leukemia-free survival rates at 4 years were 71% and 64%, respectively, for patients undergoing allo-HCT during CR1 with MRD, 55% and 43% during CR1 with MRD+, 51% and 49% during CR2 with MRD, and 38% and 29% during CR2 with MRD+. Although survival rates were significantly better among patients with CR1 MRD than among patients with CR2 MRD, no significant difference was observed in survival rate between patients with CR1 MRD+ and CR2 MRD. Relapse rates after 4 years were 16% in patients with CR1 MRD, 29% in CR1 MRD+, 21% in patients with CR2 MRD, and 46% in patients with CR2 MRD+. No significant difference was identified in relapse rate between patients with CR1 MRD and CR2 MRD. CR2 MRD was not a significant risk factor for relapse in multivariate analysis (hazard ratio, 1.26; 95% confidence interval, 0.69-2.29; P = .45 vs CR1 MRD). MRD at time of allo-HCT was an important risk factor in patients with Ph+ ALL during both CR1 and CR2.

Introduction

Measurable residual disease (MRD) has become more important in Philadelphia chromosome (Ph)–positive acute lymphoblastic leukemia (ALL) because tyrosine kinase inhibitors (TKIs) have facilitated the achievement of deep molecular responses.1,2  MRD can predict treatment responses and serve as a prognostic factor.3,4  In the development of new treatment drugs, such as blinatumomab and inotuzumab ozogamicin (Ino), MRD has become an important end point, and treatment strategies for ALL cannot be considered without thorough MRD evaluation.5-7 

Allogeneic hematopoietic cell transplantation (allo-HCT) remains a popular treatment option for Ph+ ALL in the era of TKIs.8-12  Although MRD status at time of allo-HCT performed during first complete remission (CR1) has attracted attention,9,10,13-15  the prognostic value of MRD during second CR (CR2) remains unclear. Understanding how MRD at time of allo-HCT affects outcomes of patients during CR2 is critical, particularly when considering that the number of cases for which deep molecular remission can be achieved during CR2 is likely to increase with development of new drugs.16  Therefore, in this study, we evaluated the impact of MRD at time of allo-HCT in patients undergoing transplantation during either CR1 or CR2.

Patients and methods

Collection of data and data source

Patients’ clinical data were provided by the Japan Society for Hematopoietic Cell Transplantation (JSHCT) and the Japanese Data Center for Hematopoietic Cell Transplantation using the Transplant Registry Unified Management Program as described previously.17  Data regarding survival, disease status, and long-term complications, including chronic graft-versus-host disease (GVHD) and secondary malignancies, are updated annually. Opt-out is permitted for observational studies using existing data, according to current ethical guidelines. Those who declined registration were not included among patients in this study. This study was approved by the data management committees of JSHCT as a study performed by the Adult ALL Working Group of JSHCT and the Nagoya University Hospital Institutional Review Board.

Patients

Data for 1865 patients who were at least age 16 years and who had undergone first allo-HCT for Ph+ ALL during either CR1 or CR2 between 2002 and 2017 were available in the JSHCT registration database. After excluding 240 patients for whom no pretransplantation TKI administration data were available, we analyzed data for 1625 adult patients with Ph+ ALL.

Definitions

Ph+ ALL was diagnosed by presence of Ph through chromosome and/or fluorescence in situ hybridization analysis and detection of BCR-ABL fusion transcripts using real-time quantitative polymerase chain reaction (qPCR) analysis. Timing and procedure of allo-HCT, including conditioning regimen, GVHD prophylaxis, and assessment of BCR-ABL fusion transcripts, were determined by each institution. In most laboratories, BCR-ABL transcript copy numbers were normalized against copy numbers of glyceraldehyde-3-phosphate dehydrogenase and then converted into molecules per microgram of RNA. Threshold for quantification was 50 copies per microgram of RNA, which corresponded to minimal sensitivity of 10−5.8  In the present study, MRD was defined when the MRD result was reported as not detected by real-time qPCR. Of the 110 patients whose real-time qPCR values were not registered in the database, those whose status at time of allo-HCT was registered as molecular CR by the transplantation center were also defined as MRD. MRD at time of allo-HCT was evaluated by results of real-time qPCR a median of 21 days before transplantation (interquartile range, 14-36 days; MRD was assessed within 60 days before allo-HCT in 1572 [97%] of 1625 patients).9  Determination of whether to prescribe TKIs after allo-HCT was also made by each institution.

Acute and chronic GVHD were diagnosed and graded according to existing consensus criteria.18-20  Relapse was defined as hematological leukemia recurrence. Nonrelapse mortality (NRM) was defined as death during continuous remission. For analyses of overall survival (OS), failure was defined as death resulting from any cause, and surviving patients were censored at date of last contact. We defined a reduced-intensity regimen as having the following dose levels: <9 mg/kg busulfan, <140 mg/m2 melphalan, and total body irradiation at ≤500 cGy (single) or 500 to 800 cGy (fractionated).21  HLA matching of cord blood (CB) was performed using low-resolution typing for HLA-A, -B, and -C and high-resolution molecular typing for HLA-DRB1. HLA matching of unrelated donors was performed using high-resolution typing for HLA-A, -B, -C, and -DRB1.22-24  For unrelated donors, well matched was defined as no known disparity in HLA-A, -B, -C, or -DRB1, partially matched was defined as presenting 1 locus that demonstrated disparity with donor, and mismatched was defined as disparities in ≥2 loci. For CB, well matched was defined as no known disparity in HLA-A, -B, -C, or -DRB1, partially matched was defined as at least 4 loci matches, and mismatched was defined as ≤3 loci matches.9,25-27 

Statistical analysis

A 2-sided χ2 test was used to compare categorical variables, and Mann-Whitney or Kruskal-Wallis test was used to compare continuous variables. OS and leukemia-free survival (LFS) were estimated by Kaplan-Meier method, and P values were calculated using log-rank test.28,29  Holm-Sidak test was used to compare 2 groups, for post hoc comparisons. Cumulative incidence of relapse, NRM, and GVHD was calculated by Gray’s method.30,31  For relapse, death without relapse was the competing event; for NRM, relapse was the competing event; and for GVHD, relapse and death, without GVHD, were the competing events. Cox proportional hazards models were used to perform multivariate analyses of OS and LFS.32  Fine-Gray proportional hazards models were used for multivariate analyses of events with competing risks. Adjusted probabilities of OS and LFS were estimated using Cox proportional hazards regression models, with consideration of other significant clinical variables in final multivariate models. Backward stepwise procedure was used to develop a final model based on a threshold value of P < .05. Covariates considered included disease status at allo-HCT (CR1 with MRD vs CR1 with MRD+ vs CR2 with MRD vs CR2 with MRD+), age at allo-HCT (<55 vs ≥55 years), performance status (PS; 0 vs 1-4), white blood cell (WBC) count at diagnosis (<30 × 103/μL vs ≥30 × 103/μL), additional chromosomal abnormalities, breakpoint cluster region (p190 vs p210 vs other), TKI use (imatinib vs dasatinib vs other), cytomegalovirus status, sex matching (match vs male to female vs female to male), donor (related vs unrelated bone marrow or peripheral blood vs unrelated CB), HLA disparity (well matched vs partially matched vs mismatched), preparative regimen (myeloablative conditioning vs reduced-intensity conditioning; etoposide + cyclophosphamide + total-body irradiation33  vs other), GVHD prophylaxis (cyclosporine A ± other vs tacrolimus ± other vs other), posttransplantation TKI (before hematological relapse, as a time-dependent covariate; no vs yes), and year of allo-HCT (2002-2012 vs 2013-2017). Significance level of P < .05 was used for all analyses.

Results

Patient characteristics

Patient characteristics, according to stage and MRD, are listed in Table 1. MRD was negative in 1111 (73%) of 1523 patients undergoing allo-HCT during CR1 and 61 (60%) of 102 patients undergoing allo-HCT during CR2. Age at allo-HCT was higher and PS worse among patients undergoing allo-HCT during CR2 compared with those undergoing allo-HCT during CR1. WBC counts were lower in patients with CR1 MRD compared with other groups. Posttransplantation TKI was administered to 23% of patients. Although more patients with MRD received posttransplantation TKI compared with those without MRD, frequency was comparable between patients with CR1 MRD and CR2 MRD.

Table 1.

Characteristics of patients with Ph+ ALL according to disease status

CR1CR2P
MRD (n = 1111)MRD+ (n = 412)MRD (n = 61)MRD+ (n = 41)
n%n%n%n%
Median (range) patient age at allo-HCT, y 46 (16-71) 44 (16-71) 50 (16-69) 51 (16-67) .009 
PS         .002 
 0 675 61 243 59 22 36 19 46  
 1-2 419 38 158 38 37 61 22 54  
 3-4 10  
 Missing  
Median WBC count at diagnosis, × 103/μL 19.6 31.7 39.95 33.0 <.001 
 <30 638 57 201 49 28 46 18 44 .005 
 ≥30 473 43 221 54 33 54 23 56  
Additional chromosomal abnormality positive 202 18 76 18 13 12 29 .22 
BCR breakpoint         .53 
 p190 859 77 281 68 47 77 31 76  
 p210 193 17 87 21 11 18 20  
 Other 26  
 Missing 33 36  
Pretransplantation TKI*         <.001 
 Imatinib 671 60 251 61 11 18 14 34  
 Dasatinib 425 38 149 36 44 72 22 54  
 Other 15 12 10 12  
CMV antibody positive 842 76 300 73 50 82 29 71 .46 
Sex matching         .50 
 Match 573 52 207 50 32 52 25 61  
 Male to female 257 23 105 25 14 23 10  
 Female to male 241 22 85 21 14 23 10 24  
 Missing 40 15  
Donor         .002 
 Related 335 30 139 34 16 26 12 29  
 UBM 531 48 169 41 19 31 16 39  
 UCB 243 22 104 25 26 43 13 32  
 Missing  
HLA matching         .07 
 Well matched 556 50 192 47 18 30 17 41  
 Partially matched 404 36 163 40 29 48 16 39  
 Mismatched 147 13 53 13 13 21 17  
 Missing  
Median time from diagnosis to CR1, d 40 41 42 41 .48 
Median time from CR1 to allo-HCT, d 132 118 332 206 <.001 
Preparative regimen         <.001 
 MAC 792 71 305 74 28 46 27 66  
 CY + TBI 443 40 156 38 15 11 27  
 VP + CY + TBI 120 11 51 12 10  
 CA + CY + TBI 108 10 46 11 15  
 Other TBI regimen 65 24 15 10  
 BU + CY 24 13  
 Other non-TBI regimen 32 15  
 RIC 317 29 107 26 33 54 14 34  
 Missing  
GVHD prophylaxis         .03 
 Cyclosporine A ± other 411 37 161 39 12 20 18 44  
 Tacrolimus ± other 676 61 242 59 49 80 21 51  
 Other 22  
 Missing  
Posttransplantation TKI 222 20 121 29 13 21 11 27 <.001 
 Imatinib 80 54 13 10  
 Dasatinib 130 12 58 14 10 16  
 Other 12 10  
Year of allo-HCT         <.001 
 2002-2012 608 55 267 65 27 44 27 66  
 2013-2017 503 45 145 35 34 56 14 34  
CR1CR2P
MRD (n = 1111)MRD+ (n = 412)MRD (n = 61)MRD+ (n = 41)
n%n%n%n%
Median (range) patient age at allo-HCT, y 46 (16-71) 44 (16-71) 50 (16-69) 51 (16-67) .009 
PS         .002 
 0 675 61 243 59 22 36 19 46  
 1-2 419 38 158 38 37 61 22 54  
 3-4 10  
 Missing  
Median WBC count at diagnosis, × 103/μL 19.6 31.7 39.95 33.0 <.001 
 <30 638 57 201 49 28 46 18 44 .005 
 ≥30 473 43 221 54 33 54 23 56  
Additional chromosomal abnormality positive 202 18 76 18 13 12 29 .22 
BCR breakpoint         .53 
 p190 859 77 281 68 47 77 31 76  
 p210 193 17 87 21 11 18 20  
 Other 26  
 Missing 33 36  
Pretransplantation TKI*         <.001 
 Imatinib 671 60 251 61 11 18 14 34  
 Dasatinib 425 38 149 36 44 72 22 54  
 Other 15 12 10 12  
CMV antibody positive 842 76 300 73 50 82 29 71 .46 
Sex matching         .50 
 Match 573 52 207 50 32 52 25 61  
 Male to female 257 23 105 25 14 23 10  
 Female to male 241 22 85 21 14 23 10 24  
 Missing 40 15  
Donor         .002 
 Related 335 30 139 34 16 26 12 29  
 UBM 531 48 169 41 19 31 16 39  
 UCB 243 22 104 25 26 43 13 32  
 Missing  
HLA matching         .07 
 Well matched 556 50 192 47 18 30 17 41  
 Partially matched 404 36 163 40 29 48 16 39  
 Mismatched 147 13 53 13 13 21 17  
 Missing  
Median time from diagnosis to CR1, d 40 41 42 41 .48 
Median time from CR1 to allo-HCT, d 132 118 332 206 <.001 
Preparative regimen         <.001 
 MAC 792 71 305 74 28 46 27 66  
 CY + TBI 443 40 156 38 15 11 27  
 VP + CY + TBI 120 11 51 12 10  
 CA + CY + TBI 108 10 46 11 15  
 Other TBI regimen 65 24 15 10  
 BU + CY 24 13  
 Other non-TBI regimen 32 15  
 RIC 317 29 107 26 33 54 14 34  
 Missing  
GVHD prophylaxis         .03 
 Cyclosporine A ± other 411 37 161 39 12 20 18 44  
 Tacrolimus ± other 676 61 242 59 49 80 21 51  
 Other 22  
 Missing  
Posttransplantation TKI 222 20 121 29 13 21 11 27 <.001 
 Imatinib 80 54 13 10  
 Dasatinib 130 12 58 14 10 16  
 Other 12 10  
Year of allo-HCT         <.001 
 2002-2012 608 55 267 65 27 44 27 66  
 2013-2017 503 45 145 35 34 56 14 34  

BU, busulfan; CA, cytarabine; CMV, cytomegalovirus; CY, cyclophosphamide; MAC, myeloablative conditioning; RIC, reduced-intensity conditioning; TBI, total-body irradiation; UBM, unrelated bone marrow or peripheral blood; UCB, unrelated cord blood; VP, etoposide.

*

TKI used just before allo-HCT.

First TKI used after allo-HCT.

Survival

OS.

Median follow-up period for survivors was 49 months (range, 1.9-198 months). OS rates at 4 years were 70% (95% confidence interval [CI], 67-73) for patients with CR1 MRD, 54% (95% CI, 49-59) for patients with CR1 MRD+, 47% (95% CI, 33-60) for patients with CR2 MRD, and 31% (95% CI, 17-46) for patients with CR2 MRD+. Although OS was significantly better for CR1 MRD patients than for CR2 MRD patients (P < .001), no significant difference in OS was observed between patients with CR1 MRD+ and CR2 MRD (P = .52) when post hoc analyses were performed. In multivariate analysis, disease status was a significant prognostic factor, as were age, PS, WBC count, and year of allo-HCT (Table 2). Adjusted OS rates at 4 years were 71% for patients with CR1 MRD, 55% for patients with CR1 MRD+, 51% for patients with CR2 MRD, and 38% for patients with CR2 MRD+ (Figure 1A).

Table 2.

Impact of disease status among adult Ph+ ALL patients undergoing unrelated allo-HCT during CR1 or CR2: multivariate analyses

CovariateHR95% CIP
OS    
 Age ≥55 y at allo-HCT (vs ≥16 and <55 y) 1.51 1.27-1.81 <.001 
 PS 1-4 (vs 0) 1.31 1.11-1.54 .002 
 WBC count ≥30 × 103/μL at diagnosis (vs <30 × 103/μL) 1.25 1.06-1.47 .01 
 Year of allo-HCT 2013-2017 (vs 2002-12) 0.79 0.65-0.95 .01 
 Status at allo-HCT (vs CR1 MRD   
  CR1 MRD+ 1.80 1.50-2.16 <.001 
  CR2 MRD 2.01 1.38-2.94 <.001 
  CR2 MRD+ 2.37 1.59-3.55 <.001 
LFS    
 Age ≥55 y at allo-HCT (vs ≤16 and < 55 y) 1.39 1.13-1.72 .002 
 WBC count ≥30 × 103/μL at diagnosis (vs <30 × 103/μL) 1.35 1.16-1.58 <.001 
 Preparative regimen of VP + CY + TBI (vs other) 0.64 0.48-0.85 .002 
 Year of allo-HCT 2013-2017 (vs 2002-2012) 0.84 0.71-0.996 .045 
 Status at allo-HCT (vs CR1 MRD   
  CR1 MRD+ 1.82 1.54-2.16 <.001 
  CR2 MRD 1.64 1.14-2.36 .008 
  CR2 MRD+ 2.61 1.77-3.85 <.001 
Relapse    
 WBC count ≥30 × 103/μL at diagnosis (vs <30 × 103/μL) 1.43 1.14-1.80 .002 
 Donor (vs related)    
  UBM 0.68 0.52-0.88 .003 
  UCB 0.62 0.46-0.85 .003 
 Preparative regimen of VP + CY + TBI (vs other) 0.47 0.30-0.75 .001 
 Posttransplantation TKI (vs none) 2.34 1.70-3.21 <.001 
 Status at allo-HCT (vs CR1 MRD   
  CR1 MRD+ 1.82 1.42-2.33 <.001 
  CR2 MRD 1.27 0.69-2.32 .44 
  CR2 MRD+ 3.87 2.31-6.49 <.001 
NRM    
 Age ≥55 y at allo-HCT (vs ≤16 and <55 y) 1.67 1.34-2.08 <.001 
 PS 1-4 (vs 0) 1.35 1.09-1.66 .005 
 Donor (vs related)    
  UBM 1.54 1.19-2.00 .001 
  UCB 1.38 1.02-1.87 .04 
 Posttransplantation TKI (vs none) 0.50 0.29-0.86 .01 
 Year of allo-HCT 2013-2017 (vs 2002-2012) 0.76 0.60-0.95 .02 
 Status at allo-HCT (vs CR1 MRD   
  CR1 MRD+ 1.54 1.22-1.95 <.001 
  CR2 MRD 1.66 1.04-2.66 .04 
  CR2 MRD+ 1.23 0.65-2.34 .53 
CovariateHR95% CIP
OS    
 Age ≥55 y at allo-HCT (vs ≥16 and <55 y) 1.51 1.27-1.81 <.001 
 PS 1-4 (vs 0) 1.31 1.11-1.54 .002 
 WBC count ≥30 × 103/μL at diagnosis (vs <30 × 103/μL) 1.25 1.06-1.47 .01 
 Year of allo-HCT 2013-2017 (vs 2002-12) 0.79 0.65-0.95 .01 
 Status at allo-HCT (vs CR1 MRD   
  CR1 MRD+ 1.80 1.50-2.16 <.001 
  CR2 MRD 2.01 1.38-2.94 <.001 
  CR2 MRD+ 2.37 1.59-3.55 <.001 
LFS    
 Age ≥55 y at allo-HCT (vs ≤16 and < 55 y) 1.39 1.13-1.72 .002 
 WBC count ≥30 × 103/μL at diagnosis (vs <30 × 103/μL) 1.35 1.16-1.58 <.001 
 Preparative regimen of VP + CY + TBI (vs other) 0.64 0.48-0.85 .002 
 Year of allo-HCT 2013-2017 (vs 2002-2012) 0.84 0.71-0.996 .045 
 Status at allo-HCT (vs CR1 MRD   
  CR1 MRD+ 1.82 1.54-2.16 <.001 
  CR2 MRD 1.64 1.14-2.36 .008 
  CR2 MRD+ 2.61 1.77-3.85 <.001 
Relapse    
 WBC count ≥30 × 103/μL at diagnosis (vs <30 × 103/μL) 1.43 1.14-1.80 .002 
 Donor (vs related)    
  UBM 0.68 0.52-0.88 .003 
  UCB 0.62 0.46-0.85 .003 
 Preparative regimen of VP + CY + TBI (vs other) 0.47 0.30-0.75 .001 
 Posttransplantation TKI (vs none) 2.34 1.70-3.21 <.001 
 Status at allo-HCT (vs CR1 MRD   
  CR1 MRD+ 1.82 1.42-2.33 <.001 
  CR2 MRD 1.27 0.69-2.32 .44 
  CR2 MRD+ 3.87 2.31-6.49 <.001 
NRM    
 Age ≥55 y at allo-HCT (vs ≤16 and <55 y) 1.67 1.34-2.08 <.001 
 PS 1-4 (vs 0) 1.35 1.09-1.66 .005 
 Donor (vs related)    
  UBM 1.54 1.19-2.00 .001 
  UCB 1.38 1.02-1.87 .04 
 Posttransplantation TKI (vs none) 0.50 0.29-0.86 .01 
 Year of allo-HCT 2013-2017 (vs 2002-2012) 0.76 0.60-0.95 .02 
 Status at allo-HCT (vs CR1 MRD   
  CR1 MRD+ 1.54 1.22-1.95 <.001 
  CR2 MRD 1.66 1.04-2.66 .04 
  CR2 MRD+ 1.23 0.65-2.34 .53 

CY, cyclophosphamide; HR, hazard ratio; TBI, total body irradiation; UBM, unrelated bone marrow or peripheral blood; UCB, unrelated cord blood; VP, etoposide.

Figure 1.

Adjusted survival according disease status. (A) OS. Results adjusted for age at allo-HCT, PS, WBC count at diagnosis, and year of transplantation. (B) LFS. Results adjusted for age at allo-HCT, WBC count at diagnosis, preparative regimen (etoposide + cyclophosphamide + total-body irradiation vs other), and year of transplantation.

Figure 1.

Adjusted survival according disease status. (A) OS. Results adjusted for age at allo-HCT, PS, WBC count at diagnosis, and year of transplantation. (B) LFS. Results adjusted for age at allo-HCT, WBC count at diagnosis, preparative regimen (etoposide + cyclophosphamide + total-body irradiation vs other), and year of transplantation.

LFS.

LFS rates at 4 years were 63% (95% CI, 60-66) for patients with CR1 MRD, 42% (95% CI, 37-48) for patients with CR1 MRD+, 46% (95% CI, 32-58) for patients with CR2 MRD, and 25% (95% CI, 12-39) for patients with CR2 MRD+. Although LFS was significantly better for patients with CR1 MRD patients than for patients with CR2 MRD (P = .007), no significant difference in LFS was observed between patients with CR1 MRD+ and CR2 MRD (P = .77) when post hoc analyses were performed. In multivariate analysis, disease status was a significant prognostic factor, as were age, WBC count, preparative regimen, and year of allo-HCT (Table 2). Adjusted LFS rates at 4 years were 64% for patients with CR1 MRD, 43% for patients with CR1 MRD+, 49% for patients with CR2 MRD, and 29% for patients with CR2 MRD+ (Figure 1B).

Relapse.

Relapse rates after 4 years were 16% (95% CI, 14-19) for patients with CR1 MRD, 29% (95% CI, 25-34) for patients with CR1 MRD+, 21% (95% CI, 12-32) for patients with CR2 MRD, and 46% (95% CI, 29-61) for patients with CR2 MRD+ (Figure 2A). No significant difference in relapse rate was observed between patients with CR1 MRD and CR2 MRD (P = .36) when post hoc analyses were performed. In multivariate analysis, disease status was a significant prognostic factor, as were WBC count, donor, preparative regimen, and posttransplantation TKI (Table 2).

Figure 2.

Relapse and NRM according to disease status. (A) Relapse. (B) NRM.

Figure 2.

Relapse and NRM according to disease status. (A) Relapse. (B) NRM.

NRM, GVHD, and cause of death

NRM.

NRM rates after 4 years were 20% (95% CI, 18-23) for patients with CR1 MRD, 29% (95% CI, 24-33) for patients with CR1 MRD+, 33% (95% CI, 21-46) for patients with CR2 MRD, and 30% (95% CI, 16-45) for patients with CR2 MRD+ (Figure 2B). Although tendency toward lower NRM rate was observed for patients with CR1 MRD compared with patients with CR2 MRD (P = .07), no significant difference in NRM rate was observed between patients with CR1 MRD+ and CR2 MRD (P = .83) when post hoc analyses were performed. In multivariate analysis, disease status was a significant prognostic factor, as were age, PS, donor, posttransplantation TKI, and year of allo-HCT (Table 2).

GVHD.

Cumulative incidence of grade 2 to 4 acute GVHD was not significantly different according to disease status (CR1 MRD, 40%; CR1 MRD+, 40%; CR2 MRD, 39%; and CR2 MRD+, 37% at day 100; P = .98). Similarly, cumulative incidence of grade 3 to 4 acute GVHD was not significantly different according to disease status (CR1 MRD, 10%; CR1 MRD+, 12%; CR2 MRD, 10%; and CR2 MRD+, 12% at day 100; P = .54).

Among evaluable patients who survived for at least 100 days after allo-HCT, no significant difference was observed in incidence of chronic GVHD according to disease status (CR1 MRD, 37%; CR1 MRD+, 34%; CR2 MRD, 35%; and CR2 MRD+, 41% at 2 years; P = .61).

Cause of death.

Relapse was the most common cause of death for patients undergoing allo-HCT during CR1 and those undergoing allo-HCT during CR2, and infection was the most frequent cause of nonrelapse fatality. No significant difference in any nonrelapse cause of death was observed according to disease status (Table 3).

Table 3.

Cause of death according to disease status

CR1CR2
MRDMRD+MRDMRD+
n%n%n%n%
Relapse 68 21 58 30 23 12 46 
Infection 81 25 42 22 13 19 
Organ failure 51 16 23 12 
GVHD 32 10 12 20 
Interstitial pneumonia 12 12 12 
Hemorrhage 11 10 
TMA 15 10 
ARDS 
Graft failure 
SOS 
Secondary malignancy 10 
Other 19 16 10 
CR1CR2
MRDMRD+MRDMRD+
n%n%n%n%
Relapse 68 21 58 30 23 12 46 
Infection 81 25 42 22 13 19 
Organ failure 51 16 23 12 
GVHD 32 10 12 20 
Interstitial pneumonia 12 12 12 
Hemorrhage 11 10 
TMA 15 10 
ARDS 
Graft failure 
SOS 
Secondary malignancy 10 
Other 19 16 10 

ARDS, acute respiratory distress syndrome; SOS, sinusoidal obstruction syndrome; TMA, thrombotic microangiopathy.

Impact of MRD in patients undergoing allo-HCT during CR2

Among 102 patients undergoing allo-HCT during CR2, CR1 duration was longer for patients with MRD compared with patients with MRD+ (193 vs 116 days; P = .01). Sixteen of 33 patients whose CR1 duration was <100 days (48%) were MRD, whereas the remaining 17 (52%) were MRD+. Forty-one of 78 patients whose CR1 duration was <1 year (53%) were MRD, whereas the remaining 37 (47%) were MRD+. In multivariate analysis, MRD was a significant risk factor for LFS, as were PS and conditioning intensity (CR2 MRD+: hazard ratio, 1.73; 95% CI, 1.03-2.91; P = .04), and MRD was also a significant risk factor for relapse, along with HLA disparity (CR2 MRD+: hazard ratio, 2.63; 95% CI, 1.26-5.49; P = .01).

Discussion

This study showed the impact of MRD at time of allo-HCT not only in patients with Ph+ ALL undergoing transplantation during CR1, but also in those undergoing transplantation during CR2. Survival rate was similar between patients with CR1 MRD+ and CR2 MRD. In addition, no significant difference in relapse rate was observed between patients with CR1 MRD and CR2 MRD. CR2 MRD was not a significant risk factor for relapse in multivariate analysis. To our knowledge, this is the first study to demonstrate that MRD is also an important risk factor for patients with Ph+ ALL who undergo allo-HCT during CR2.

Allo-HCT during CR1 remains standard treatment for Ph+ ALL in Japan. Although relatively high survival rates can be achieved, especially in patients with CR1 MRD, long-term impairment of quality life associated with chronic GVHD is a transplantation-specific complication that we aim to avoid, in addition to NRM.34  Four-year OS was 66% without allo-HCT in CR1 in patients who achieved complete molecular remission at 3 months in a previous study,35  similar to our results for patients with MRD undergoing allo-HCT in CR1. This indicates that some patients who achieve early molecular remission may be able to survive for a long time without allo-HCT in CR1. Our results suggest that relatively favorable outcomes may be obtained when allo-HCT is performed for patients with MRD in CR2, even if recurrence will have occurred in such patients who do not undergo allo-HCT in CR1. Further improvement of chemotherapy will be expected with recent advances in ALL, which may change the status of allo-HCT as standard treatment.

This study included data for patients undergoing allo-HCT between 2002 and 2017. Before allo-HCT, a few patients were administered ponatinib, a third-generation TKI showing promising data in Ph+ ALL treatment, including in those with T315I mutation36,37 ; however, ponatinib was only approved for relapsed/refractory Ph+ ALL in September 2016. In addition, because Ino and blinatumomab were approved in 2018 and tisagenlecleucel was approved in 2019 for treatment of relapsed/refractory ALL in Japan, no patients received these drugs before allo-HCT in clinical practice. These new drugs have been reported to achieve high CR rates when used to treat relapsed/refractory ALL (Ino, 57% to 92%; blinatumomab, 36% to 44%; and tisagenlecleucel, 81%), with high probability of obtaining MRD with achievement of CR (Ino, 63% to 93%; blinatumomab, 71% to 88%; and tisagenlecleucel, 100%).38-45  Treatment strategy may be more important when allo-HCT is performed during CR2 instead of during CR1, particularly when CR2 can be achieved with high probability. Low relapse rates associated with patients with CR2 MRD support this strategy, which may help inform the decision of when to perform allo-HCT. More patients undergoing allo-HCT in CR2 received pretransplantation dasatinib than those undergoing allo-HCT in CR1 in this study. Although type of TKI before allo-HCT was not identified as a significant prognostic factor in multivariate analyses, different activity of pretransplantation TKIs could affect transplantation outcome, considering dasatinib is a more potent multitargeted inhibitor compared with imatinib.46,47  This study demonstrates the importance of MRD at time of allo-HCT, even during CR2.

Several limitations exist because of the retrospective nature of this study, which was performed using database entries. Unfortunately, induction/consolidation treatment data were not available, except for pretransplantation TKI information, in this study. This is a major limitation of transplantation registry–based studies.48-52  In addition to the small number of patients undergoing allo-HCT during CR2, backgrounds of these patients were considered heterogeneous because eligibility criteria for allo-HCT depended on individual transplantation centers. Although data on pre- and posttransplantation TKIs were available in our database, treatment was not controlled as in prospective trials. Therefore, examination of effects of posttransplantation treatment was difficult to perform, even though posttransplantation TKI administration could represent an important option for posttransplantation therapeutic intervention.53  NRM might have been underestimated in patients who received posttransplantation TKI because of technical issues in competing risk analyses; of 367 patients receiving posttransplantation TKI, NRM was observed in only 14 patients (3.8%), whereas relapse was observed in 257 (70%). Given that the decision to prescribe a TKI after allo-HCT was made by each institution in this study, the TKI might have been administered to patients who were potentially at high risk of relapse.9  Although posttransplantation TKI administration cannot prevent relapse, achieving comparable survival in such patients may be beneficial with the possibility that innovative new technologies will be developed in the near future.

In summary, the importance of MRD at time of allo-HCT was demonstrated in patients undergoing transplantation during both CR1 and CR2. The present study provides helpful data when considering appropriate timing of allo-HCT in the era of emerging useful new drugs, which may expand therapeutic options for ALL beyond allo-HCT. In patients who must undergo allo-HCT for Ph+ ALL, a therapeutic strategy that can achieve MRD before allo-HCT is desirable, regardless of disease stage.

For data sharing requests, e-mail the corresponding author, Satoshi Nishiwaki (n-3104@tf7.so-net.ne.jp).

Acknowledgments

This study was supported, in part, by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (KAKENHI) grant JP 20K08730, and a research grant from the Hori Sciences and Arts Foundation.

Authorship

Contribution: S.N., Y. Akahoshi, S.M., A.S., S.H., and S.K. designed the research, performed the statistical analyses, interpreted the data, and wrote the manuscript; Y.N., T.F., N.U., M.T., M.O., Y. Ozawa, S.O., S.S., Y. Onishi, Y.K., and M.S. provided patient data; J.T., T.F. and Y. Atsuta collected patient data; and all authors reviewed and approved the final draft.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

All group members of the Adult Acute Lymphoblastic Leukemia Working Group of the Japan Society for Hematopoietic Cell Transplantation who contributed to this study were included as authors of this article.

Correspondence: Satoshi Nishiwaki, Department of Advanced Medicine, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 4668560, Japan; e-mail: n-3104@tf7.so-net.ne.jp.

References

References
1.
Hoelzer
D
.
Monitoring and managing minimal residual disease in acute lymphoblastic leukemia
.
Am Soc Clin Oncol Educ Book
.
2013
;
33
:
290
-
293
.
2.
Berry
DA
,
Zhou
S
,
Higley
H
, et al
.
Association of minimal residual disease with clinical outcome in pediatric and adult acute lymphoblastic leukemia: a meta-analysis
.
JAMA Oncol
.
2017
;
3
(
7
):
e170580
.
3.
Lee
S
,
Kim
DW
,
Cho
BS
, et al
.
Impact of minimal residual disease kinetics during imatinib-based treatment on transplantation outcome in Philadelphia chromosome-positive acute lymphoblastic leukemia
.
Leukemia
.
2012
;
26
(
11
):
2367
-
2374
.
4.
Yoon
JH
,
Yhim
HY
,
Kwak
JY
, et al
.
Minimal residual disease-based effect and long-term outcome of first-line dasatinib combined with chemotherapy for adult Philadelphia chromosome-positive acute lymphoblastic leukemia
.
Ann Oncol
.
2016
;
27
(
6
):
1081
-
1088
.
5.
Kantarjian
H
,
Jabbour
E
.
Incorporating immunotherapy into the treatment strategies of B-cell adult acute lymphoblastic leukemia: the role of blinatumomab and inotuzumab ozogamicin
.
Am Soc Clin Oncol Educ Book
.
2018
;
38
(
38
):
574
-
578
.
6.
DeAngelo
DJ
,
Stock
W
,
Stein
AS
, et al
.
Inotuzumab ozogamicin in adults with relapsed or refractory CD22-positive acute lymphoblastic leukemia: a phase 1/2 study
.
Blood Adv
.
2017
;
1
(
15
):
1167
-
1180
.
7.
Keating
AK
,
Gossai
N
,
Phillips
CL
, et al
.
Reducing minimal residual disease with blinatumomab prior to HCT for pediatric patients with acute lymphoblastic leukemia
.
Blood Adv
.
2019
;
3
(
13
):
1926
-
1929
.
8.
Mizuta
S
,
Matsuo
K
,
Nishiwaki
S
, et al
.
Pretransplant administration of imatinib for allo-HSCT in patients with BCR-ABL-positive acute lymphoblastic leukemia
.
Blood
.
2014
;
123
(
15
):
2325
-
2332
.
9.
Nishiwaki
S
,
Imai
K
,
Mizuta
S
, et al
.
Impact of MRD and TKI on allogeneic hematopoietic cell transplantation for Ph+ALL: a study from the adult ALL WG of the JSHCT
.
Bone Marrow Transplant
.
2016
;
51
(
1
):
43
-
50
.
10.
Wang
J
,
Jiang
Q
,
Xu
LP
, et al
.
Allogeneic stem cell transplantation versus tyrosine kinase inhibitors combined with chemotherapy in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia
.
Biol Blood Marrow Transplant
.
2018
;
24
(
4
):
741
-
750
.
11.
Ravandi
F
,
Othus
M
,
O’Brien
SM
, et al
.
US intergroup study of chemotherapy plus dasatinib and allogeneic stem cell transplant in Philadelphia chromosome positive ALL
.
Blood Adv
.
2016
;
1
(
3
):
250
-
259
.
12.
Tanguy-Schmidt
A
,
Rousselot
P
,
Chalandon
Y
, et al
.
Long-term follow-up of the imatinib GRAAPH-2003 study in newly diagnosed patients with de novo Philadelphia chromosome-positive acute lymphoblastic leukemia: a GRAALL study
.
Biol Blood Marrow Transplant
.
2013
;
19
(
1
):
150
-
155
.
13.
Cai
WZ
,
Cen
JN
,
Chen
J
, et al
.
Major molecular response prior to allogeneic hematopoietic stem cell transplantation predicts better outcome in adult Philadelphia-positive acute lymphoblastic leukemia in first remission
.
Bone Marrow Transplant
.
2017
;
52
(
3
):
470
-
472
.
14.
Yoon
JH
,
Min
GJ
,
Park
SS
, et al
.
Minimal residual disease-based long-term efficacy of reduced-intensity conditioning versus myeloablative conditioning for adult Philadelphia-positive acute lymphoblastic leukemia
.
Cancer
.
2019
;
125
(
6
):
873
-
883
.
15.
Litzow
MR
.
Should anyone with Philadelphia chromosome-positive ALL who is negative for minimal residual disease receive a hematopoietic stem cell transplant in first remission?
Best Pract Res Clin Haematol
.
2016
;
29
(
4
):
345
-
350
.
16.
Forman
SJ
,
Rowe
JM
.
The myth of the second remission of acute leukemia in the adult
.
Blood
.
2013
;
121
(
7
):
1077
-
1082
.
17.
Atsuta
Y
,
Suzuki
R
,
Yoshimi
A
, et al
.
Unification of hematopoietic stem cell transplantation registries in Japan and establishment of the TRUMP System
.
Int J Hematol
.
2007
;
86
(
3
):
269
-
274
.
18.
Przepiorka
D
,
Weisdorf
D
,
Martin
P
, et al
.
1994 Consensus Conference on Acute GVHD Grading
.
Bone Marrow Transplant
.
1995
;
15
(
6
):
825
-
828
.
19.
Sullivan
KM
,
Shulman
HM
,
Storb
R
, et al
.
Chronic graft-versus-host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression
.
Blood
.
1981
;
57
(
2
):
267
-
276
.
20.
Filipovich
AH
,
Weisdorf
D
,
Pavletic
S
, et al
.
National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report
.
Biol Blood Marrow Transplant
.
2005
;
11
(
12
):
945
-
956
.
21.
Giralt
S
,
Ballen
K
,
Rizzo
D
, et al
.
Reduced-intensity conditioning regimen workshop: defining the dose spectrum. Report of a workshop convened by the center for international blood and marrow transplant research
.
Biol Blood Marrow Transplant
.
2009
;
15
(
3
):
367
-
369
.
22.
Sasazuki
T
,
Juji
T
,
Morishima
Y
, et al
.
Effect of matching of class I HLA alleles on clinical outcome after transplantation of hematopoietic stem cells from an unrelated donor. Japan Marrow Donor Program
.
N Engl J Med
.
1998
;
339
(
17
):
1177
-
1185
.
23.
Morishima
Y
,
Sasazuki
T
,
Inoko
H
, et al
.
The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors
.
Blood
.
2002
;
99
(
11
):
4200
-
4206
.
24.
Atsuta
Y
,
Morishima
Y
,
Suzuki
R
, et al;
Japan Marrow Donor Program and Japan Cord Blood Bank Network
.
Comparison of unrelated cord blood transplantation and HLA-mismatched unrelated bone marrow transplantation for adults with leukemia
.
Biol Blood Marrow Transplant
.
2012
;
18
(
5
):
780
-
787
.
25.
Ooi
J
,
Takahashi
S
,
Tomonari
A
, et al
.
Unrelated cord blood transplantation after myeloablative conditioning in adults with acute myelogenous leukemia
.
Biol Blood Marrow Transplant
.
2008
;
14
(
12
):
1341
-
1347
.
26.
Matsuno
N
,
Wake
A
,
Uchida
N
, et al
.
Impact of HLA disparity in the graft-versus-host direction on engraftment in adult patients receiving reduced-intensity cord blood transplantation
.
Blood
.
2009
;
114
(
8
):
1689
-
1695
.
27.
Nishiwaki
S
,
Miyamura
K
,
Ohashi
K
, et al;
Adult Acute Lymphoblastic Leukemia Working Group of the Japan Society for Hematopoietic Cell Transplantation
.
Impact of a donor source on adult Philadelphia chromosome-negative acute lymphoblastic leukemia: a retrospective analysis from the Adult Acute Lymphoblastic Leukemia Working Group of the Japan Society for Hematopoietic Cell Transplantation
.
Ann Oncol
.
2013
;
24
(
6
):
1594
-
1602
.
28.
Kaplan
EL
,
Meier
P
.
Nonparametric estimation from incomplete observations
.
J Am Stat Assoc
.
1958
;
53
(
282
):
457
-
481
.
29.
Peto
R
,
Peto
J
.
Asymptotically efficient rank invariant test procedures
.
J R Stat Soc A
.
1972
;
135
(
2
):
185
-
207
.
30.
Gooley
TA
,
Leisenring
W
,
Crowley
J
,
Storer
BE
.
Estimation of failure probabilities in the presence of competing risks: new representations of old estimators
.
Stat Med
.
1999
;
18
(
6
):
695
-
706
.
31.
Scrucca
L
,
Santucci
A
,
Aversa
F
.
Competing risk analysis using R: an easy guide for clinicians
.
Bone Marrow Transplant
.
2007
;
40
(
4
):
381
-
387
.
32.
Cox
D
.
Regression models and life tables
.
J R Stat Soc B
.
1972
;
34
:
187
-
220
.
33.
Shigematsu
A
,
Kondo
T
,
Yamamoto
S
, et al
.
Excellent outcome of allogeneic hematopoietic stem cell transplantation using a conditioning regimen with medium-dose VP-16, cyclophosphamide and total-body irradiation for adult patients with acute lymphoblastic leukemia
.
Biol Blood Marrow Transplant
.
2008
;
14
(
5
):
568
-
575
.
34.
Kurosawa
S
,
Oshima
K
,
Yamaguchi
T
, et al
.
Quality of life after allogeneic hematopoietic cell transplantation according to affected organ and severity of chronic graft-versus-host disease
.
Biol Blood Marrow Transplant
.
2017
;
23
(
10
):
1749
-
1758
.
35.
Short
NJ
,
Jabbour
E
,
Sasaki
K
, et al
.
Impact of complete molecular response on survival in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia
.
Blood
.
2016
;
128
(
4
):
504
-
507
.
36.
Cortes
JE
,
Kim
DW
,
Pinilla-Ibarz
J
, et al;
PACE Investigators
.
A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias
.
N Engl J Med
.
2013
;
369
(
19
):
1783
-
1796
.
37.
Jabbour
E
,
Kantarjian
H
,
Ravandi
F
, et al
.
Combination of hyper-CVAD with ponatinib as first-line therapy for patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia: a single-centre, phase 2 study
.
Lancet Oncol
.
2015
;
16
(
15
):
1547
-
1555
.
38.
Kantarjian
H
,
Thomas
D
,
Jorgensen
J
, et al
.
Inotuzumab ozogamicin, an anti-CD22-calecheamicin conjugate, for refractory and relapsed acute lymphocytic leukaemia: a phase 2 study
.
Lancet Oncol
.
2012
;
13
(
4
):
403
-
411
.
39.
Kantarjian
HM
,
DeAngelo
DJ
,
Stelljes
M
, et al
.
Inotuzumab ozogamicin versus standard therapy for acute lymphoblastic leukemia
.
N Engl J Med
.
2016
;
375
(
8
):
740
-
753
.
40.
Jabbour
E
,
Sasaki
K
,
Ravandi
F
, et al
.
Chemoimmunotherapy with inotuzumab ozogamicin combined with mini-hyper-CVD, with or without blinatumomab, is highly effective in patients with Philadelphia chromosome-negative acute lymphoblastic leukemia in first salvage
.
Cancer
.
2018
;
124
(
20
):
4044
-
4055
.
41.
Topp
MS
,
Gökbuget
N
,
Stein
AS
, et al
.
Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study
.
Lancet Oncol
.
2015
;
16
(
1
):
57
-
66
.
42.
Kantarjian
H
,
Stein
A
,
Gökbuget
N
, et al
.
Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia
.
N Engl J Med
.
2017
;
376
(
9
):
836
-
847
.
43.
Martinelli
G
,
Boissel
N
,
Chevallier
P
, et al
.
Complete hematologic and molecular response in adult patients with relapsed/refractory Philadelphia chromosome-positive B-precursor acute lymphoblastic leukemia following treatment with blinatumomab: results from a phase II, single-arm, multicenter study [published corrections appear in J Clin Oncol. 2017;35(23):2722 and J Clin Oncol. 2017;35(24):2856]
.
J Clin Oncol
.
2017
;
35
(
16
):
1795
-
1802
.
44.
Gökbuget
N
,
Kantarjian
HM
,
Brüggemann
M
, et al
.
Molecular response with blinatumomab in relapsed/refractory B-cell precursor acute lymphoblastic leukemia
.
Blood Adv
.
2019
;
3
(
20
):
3033
-
3037
.
45.
Maude
SL
,
Laetsch
TW
,
Buechner
J
, et al
.
Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia
.
N Engl J Med
.
2018
;
378
(
5
):
439
-
448
.
46.
Tokarski
JS
,
Newitt
JA
,
Chang
CY
, et al
.
The structure of dasatinib (BMS-354825) bound to activated ABL kinase domain elucidates its inhibitory activity against imatinib-resistant ABL mutants
.
Cancer Res
.
2006
;
66
(
11
):
5790
-
5797
.
47.
Olivieri
A
,
Manzione
L
.
Dasatinib: a new step in molecular target therapy
.
Ann Oncol
.
2007
;
18
(
suppl 6
):
vi42
-
vi46
.
48.
Al Malki
MM
,
Yang
D
,
Labopin
M
, et al
.
Comparing transplant outcomes in ALL patients after haploidentical with PTCy or matched unrelated donor transplantation
.
Blood Adv
.
2020
;
4
(
9
):
2073
-
2083
.
49.
Pavlů
J
,
Labopin
M
,
Niittyvuopio
R
, et al
.
Measurable residual disease at myeloablative allogeneic transplantation in adults with acute lymphoblastic leukemia: a retrospective registry study on 2780 patients from the acute leukemia working party of the EBMT
.
J Hematol Oncol
.
2019
;
12
(
1
):
108
.
50.
Kebriaei
P
,
Anasetti
C
,
Zhang
MJ
, et al;
Acute Leukemia Committee of the CIBMTR
.
Intravenous busulfan compared with total body irradiation pretransplant conditioning for adults with acute lymphoblastic leukemia
.
Biol Blood Marrow Transplant
.
2018
;
24
(
4
):
726
-
733
.
51.
Marks
DI
,
Wang
T
,
Pérez
WS
, et al
.
The outcome of full-intensity and reduced-intensity conditioning matched sibling or unrelated donor transplantation in adults with Philadelphia chromosome-negative acute lymphoblastic leukemia in first and second complete remission
.
Blood
.
2010
;
116
(
3
):
366
-
374
.
52.
Mohty
M
,
Labopin
M
,
Volin
L
, et al;
Acute Leukemia Working Party of EBMT
.
Reduced-intensity versus conventional myeloablative conditioning allogeneic stem cell transplantation for patients with acute lymphoblastic leukemia: a retrospective study from the European Group for Blood and Marrow Transplantation
.
Blood
.
2010
;
116
(
22
):
4439
-
4443
.
53.
Warraich
Z
,
Tenneti
P
,
Thai
T
, et al
.
Relapse prevention with tyrosine kinase inhibitors after allogeneic transplantation for Philadelphia chromosome-positive acute lymphoblast leukemia: a systematic review
.
Biol Blood Marrow Transplant
.
2020
;
26
(
3
):
e55
-
e64
.