Ph+ ALL patients diagnosed at the Department of Pediatrics, The Catholic University of Korea from January 2005 to December 2016, and treated with imatinib and chemotherapy, followed by allogeneic HCT in first CR were included in the study (N=31) (Table 1). Patients who completed treatment without transplant, or who received HCT in second CR or later were excluded. The median age and white blood cell (WBC) count at diagnosis were 12.7 years and 72,200/µL, respectively. None of the patients had central nervous system (CNS) involvement at diagnosis. Twenty-four (77%) of the patients had additional cytogenetic abnormalities besides the Ph chromosome according to Giemsa band karyotyping (Supplementary Table 1).

Remission induction and subsequent chemotherapy until transplant, was based on an institutional regimen [9]. Consolidation chemotherapy duration in our regimen was 8 weeks (Supplementary Table 2). Patients diagnosed until December 2008 were administered dexamethasone during all phases of steroid treatment (CMCP-ALL2005); subsequent patients received prednisolone (CMCP-ALL2009). Notably, the initial 7 patients here were administered 1–5 courses of anthracycline (idarubicin or daunorubicin) and high dose cytarabine combination after remission induction instead of our ALL post-induction treatment. All but 4 patients started imatinib after initial remission induction at a dose of 340 mg/m²/day and were treated with the TKI continuously until conditioning for HCT; the remaining 4 patients were administered imatinib during remission induction. Five of the patients (16%) failed to achieve CR after initial remission induction and achieved CR after second induction chemotherapy or consolidation. Routine TKI therapy post-transplant was not done; only patients who showed an increase in the BCR-ABL1 RQ-PCR transcript post-HCT re-started TKI therapy.

Ph+ ALL was diagnosed by reverse-transcription polymerase chain reaction (RT-PCR) for the fusion transcript and bone marrow (BM) karyotyping. Leukemic burden was assessed by RQ-PCR measurement of the BCR-ABL1 transcript at diagnosis, post-consolidation, just prior to HCT, and at 1, 3, 6, 9, and 12 months post-HCT with a BM specimen, and utilizing a quantification kit for each fusion transcript (BioSewoom Inc., Seoul, Korea). The relative expression level of the gene was calculated using the 2^−ΔΔCt method by normalizing to the geometrical mean of ABL1 as a stable reference gene and presenting it relative to the control.

The first objective of our study was to determine the 5-year EFS, overall survival (OS), incidence of relapse and non-relapse mortality (NRM) post-transplant of our study group. We also calculated incidence of molecular relapse; while considering an increase in BCR-ABL1 RQ-PCR value post-transplant which required the re-initiation of TKI therapy, regardless of BM morphology, an event. Isolated extramedullary relapse was also considered an event in the incidence of molecular relapse calculation. EFS was defined as the time from HCT to last follow-up in CR, or the first event, which included relapse or death from any cause; OS was defined as the time from HCT to last follow-up, or death from any cause.

Additionally, we attempted to define clinical and chemotherapy-related (age and WBC count at diagnosis, presence of additional cytogenetic abnormalities, steroid response, chemotherapy protocol), transplant-related [donor type, human leukocyte antigen (HLA) disparity, infused TNC and CD34+ cell dose, acute and chronic graft-versus-host disease (GVHD)], and RQ-PCR-based MRD factors [values at diagnosis, log decrement post-induction, post-consolidation, just prior to HCT,and achievement of molecular remission (RQ-PCR of ‘0’)] which significantly influenced either the EFS or incidence of molecular relapse.

Continuous variables were dichotomized according to respective median values in order to analyze their impact on outcomes. Disparity in RQ-PCR decrement according to the type of chemotherapy was calculated using the Mann-Whitney test. Survival rates were calculated according to Kaplan-Meier method and comparisons were done with the Log-rank test. The impact of acute and chronic GVHD on EFS were analyzed using GVHD as a time dependent covariate in the Cox proportional hazard regression. Incidence of relapse, molecular relapse, NRM, and acute and chronic GVHD were calculated by the cumulative incidence function with consideration of the competing risks, and comparisons were done with Gray's test. Patient follow-up was done until July 19th, 2018. P-values <0.05 were considered significant.

Patients received HCT in first CR at a median of 6.4 months from diagnosis (range, 4.2–47.1 mo). The majority of patients underwent unrelated donor HCT (Table 2). All patients underwent total body irradiation (TBI)-based myeloablative conditioning. Median infused cell doses were as follows: total nucleated cell (TNC) 13.6×10⁸/kg (0.5–23.2), mononuclear cell 10.0×10⁸/kg (0.3–19.8), CD34+ cell 5.0×10⁶/kg (0.1–29.3), CD3+ cell 40.3×10⁷/kg (0.6–133.0).

Cumulative incidence of acute GVHD grade II-IV and chronic GVHD were 74.2±8.2% (23/31) and 48.4±9.7% (15/31) respectively, with 13.0±6.2% (4/31) incidence for at least grade III acute GVHD, and 32.4±8.6% (10/31) incidence for at least moderate chronic GVHD. Severe chronic GVHD was diagnosed in 5 patients (16.1%).

The median RQ-PCR value for the BCR-ABL1 transcript at diagnosis was 1.09 (range, 0.21–9.13) (Table 3), and the median decrement of the RQ-PCR value post-consolidation was −3.72 Log. The difference in post-consolidation RQ-PCR decrement according to the type of consolidation chemotherapy (that is, between patients who received institutional CMCP-ALL regimen and the seven patients who received anthracycline and high dose cytarabine) was not significant (P=0.210). Twelve (39%) and 15 (48%) patients achieved molecular remission (RQ-PCR value of ‘0’) post-consolidation and prior to HCT respectively.

Overall, 8 patients (26%) showed an RQ-PCR value increase after transplant at a median time post-HCT of 9.2 months (range, 2.9–51.8 mo), requiring the initiation of imatinib with or without chemotherapy. Mean duration of subsequent TKI therapy was 7.4 months (range, 0.2–20.3). Three of the patients showed morphological relapses concurrent with the increase in the RQ-PCR value and received both imatinib and chemotherapy; two received a second HCT after achieving second CR and currently survive event-free, while the remaining patient showed a second relapse and died. Of the five patients who started imatinib for an RQ-PCR value increase without experiencing overt relapse, three achieved molecular remission and survived event-free; the remaining two patients, however, died from subsequent relapse and refractory disease, and veno-occlusive disease after second HCT, respectively.

With a median duration of 61.8 months follow-up for surviving patients, the 5-year EFS and OS of the study group were 64.5±9.4% [20/31, relapse (N=5) and NRM (N=6)] and 75.0±8.3% (23/31) (Fig. 1A, B). The 5-year incidence of relapse was 18.3±7.9% (5/31); sites of relapse in the 5 patients were BM (N=3), BM+CNS (N=1), and isolated testis (N=1). Three of these patients currently survive disease-free, including 2 who received second transplants. The 5-year incidence of molecular relapse was 30.9±9.1% (9/31); four of 9 patients showed increase in BM RQ-PCR only, without overt evidence of relapse. The 5-year incidence of NRM was 17.3±7.3%; causes of NRM included chronic GVHD (N=2), cerebral aspergillosis (N=1), thrombotic microangiopathy (N=1), veno-occlusive disease (N=1), and pulmonary hemorrhage (N=1).

None of the factors studied proved to significantly influence EFS. Importantly, the steroid type utilized during chemotherapy and donor type for HCT did not affect EFS (5-year EFS 75.0±15.3% for matched related donor vs. 60.2±11.6% for unrelated donor; P=0.443) (Table 4). Patients who received a transplant from an HLA mismatched donor had lower 5-year EFS, although the difference was not significant (HLA matched donor transplant 80.0±8.9% vs. HLA mismatched donor transplant 39.8±16.3%; P=0.100). Also, patients who had additional cytogenetic abnormalities besides Ph had inferior 5-year EFS (56.8±11.7% vs. 85.7±13.2% for those with Ph only; P=0.157). Variables related to RQ-PCR value of the fusion transcript, such as extent of RQ-PCR decrements at any of the time points studied and the achievement of molecular remission prior to HCT, did not have a significant impact on EFS (Table 4).

Regarding the incidence of molecular relapse, MRD status after consolidation chemotherapy was the only significant factor, with those achieving ≥−4 Log reduction of the BCR-ABL1 transcript having significantly lower incidences of molecular relapse compared to those with less reduction (7.7±7.7% vs. 50.0±13.8%; P=0.027) (Table 4). Patients with additional cytogenetic abnormalities had higher incidences of molecular relapse compared to patients with Ph only (36.9±11.6% vs. 14.3±14.3%; P=0.329), although without statistical significance.