Background: Droplet digital PCR (ddPCR) is widely applied to monitor measurable residual disease (MRD). However, there are limited studies on the feasibility of ddPCR-MRD monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially targeting multiple molecular markers simultaneously. Methods: Our study collected samples from patients with acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS) in complete remission after allo-HSCT between January 2018 and August 2021 to evaluate whether posttransplant ddPCR-MRD monitoring can identify patients at high risk of relapse. Results: Of 152 patients, 58 (38.2%) were MRD positive by ddPCR within 4 months posttransplant, with a median variant allele frequency of 0.198%. The detectable DTA mutations (DNMT3A, TET2, and ASXL1 mutations) after allo-HSCT were not associated with an increased risk of relapse. After excluding DTA mutations, patients with ddPCR-MRD positivity had a significantly higher cumulative incidence of relapse (CIR, 38.7% vs. 9.7%, P < 0.001) and lower rates of relapse-free survival (RFS, 55.5% vs. 83.7%, P < 0.001) and overall survival (OS, 60.5% vs. 90.5%, P < 0.001). In multivariate analysis, ddPCR-MRD positivity of non-DTA genes was an independent adverse predictor for CIR (hazard ratio [HR], 4.02; P < 0.001), RFS (HR, 2.92; P = 0.002) and OS (HR, 3.12; P = 0.007). Moreover, the combination of ddPCR with multiparameter flow cytometry (MFC) can further accurately identify patients at high risk of relapse (F+/M+, HR, 22.44; P < 0.001, F+/M-, HR, 12.46; P < 0.001 and F-/M+, HR, 4.51; P = 0.003). Conclusion: ddPCR-MRD is a feasible approach to predict relapse after allo-HSCT in AML/MDS patients with non-DTA genes and is more accurate when combined with MFC. Trial registration: ClinicalTrials.gov identifier: NCT06000306. Registered 17 August 2023 –Retrospectively registered (https://clinicaltrials.gov/study/NCT06000306?term=NCT06000306&rank=1).
Keywords: MRD; Allo-HSCT; ddPCR; MFC; Relapse
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1186/s12967-024-05114-w.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered the only cure for acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS), but the high mortality rate from relapse after transplantation remains a concern. Data from the CIBMTR show that over 30% of deaths following allo-HSCT in patients are caused by relapse [[
An optimal tool for posttransplant MRD monitoring requires high sensitivity, great repeatability, and the ability to accurately predict prognosis. MFC technology is widely used for MRD detection to predict patient outcomes [[
Published reports have shown that ddPCR-MRD monitoring is effective in predicting relapse when using specific single genes as MRD markers, such as BCR-ABL, NPM1, PML-RARA, IDH1/2, or WT1 [[
The study reviewed 646 patients with AML/MDS who underwent allo-HSCT in three medical centers (the First Affiliated Hospital of Zhejiang University School of Medicine, Children's Hospital of Zhejiang University School of Medicine and Shanghai Rui Jin Hospital) from January 2018 to August 2021. The inclusion criteria for this study were as follows: (a) the presence of at least one myeloid neoplasm-associated mutation or fusion gene detected at diagnosis by NGS or real-time qPCR provided for posttransplant MRD monitoring; (b) received myeloablative conditioning regimen; (c) successful stem cell engraftment; and (d) received at least one bone marrow MRD detection by ddPCR in + 30 days to + 120 days after HSCT. Patients who relapsed or died before the first ddPCR monitoring or patients with only germline mutations were excluded. A total of 152 patients who met the criteria were included in this study, comprising those with persistent MRD positivity of core binding factor (CBF) fusion genes and high-risk MDS before transplantation (ClinicalTrials.gov identifier: NCT06000306, a multicenter retrospective cohort study). The grouping process of the whole study is shown in Fig. 1. All patients signed written informed consent for this study. The entire study procedure was conducted according to the Declaration of Helsinki and was approved by the Ethics Review Committee of each center.
Graph: Fig. 1 Consort diagram
The ddPCR primers and probes used for MRD detection mentioned in this article were designed and tested by Shanghai Dishuo Beken Biotechnology Co., Ltd. The primers, probe design, and detection were consistent with previously published studies of the company [[
MFC was performed on bone marrow samples to detect MRD at every follow-up after allo-HSCT. MRD detection by MFC used an 8- to 10-color assay according to previous publications [[
The cumulative incidence was estimated for relapse (CIR), being competing risks with non-relapse mortality (NRM). The competing risk model using Gray test was applied as there are a piece of competing risks. OS and relapse-free survival (RFS) curves were plotted using the Kaplan‒Meier method. The log-rank test was applied for univariate comparisons of groups in OS and RFS.
Continuous variables were compared using the Mann‒Whitney U test or t test, while the comparisons of categorical variables were determined by the chi-square or Fisher's exact test. All variables with P < 0.1 in univariate analysis were further included in multivariate analysis. In multivariate analysis, Cox proportional hazard regression model was adopted for OS and RFS, while the Fine-Gray proportional hazard regression model was constructed for CIR and NRM because of the competing risks.
The positive predictive value (PPV) was calculated by the ratio of true-positive patients / (true-positive + false-positive patients), and the negative predictive value (NPV) was calculated by the ratio of true-negative patients / (true-negative + false-negative patients) [[
P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 26 (Chicago, IL, USA) and R software (version 4.2.1, R Foundation for Statistical Computing, Vienna, Austria).
A total of 152 patients, all of whom had at least one molecular marker and underwent ddPCR-MRD detection within 120 days after allo-HSCT, were included in our study cohort. In these patients, 525 ddPCR-MRD analyses were performed after allo-HSCT, with a median of 4 ddPCR-MRD analyses per patient (range 1 to 6 times). The median age of these patients was 44 years (range 12 to 67 years). The primary diseases included acute myeloid leukemia (AML; 83.6%, 127/152) and myelodysplastic syndrome (MDS; 16.4%, 25/152). There was no significant difference in baseline characteristics between the DTA MRD-positive group and DTA MRD-negative group. However, among patients with non-DTA genes, a higher risk level of the refined disease risk index (DRI-R) [[
Table 1 Comparison of clinical characteristics between MRD + and MRD- patients according to DTA genes and non-DTA genes
Non- MRD positive (n = 13) MRD negative (n = 27) MRD positive (n = 52) MRD negative (n = 98) 44(12–67) 50(21–64) 48(23–61) 0.194 43(13–64) 43(12–67) 0.442 0.577 0.742 Male 78(51.3) 7(53.8) 12(44.4) 28(53.8) 50(51.0) Female 74(48.7) 6(46.2) 15(55.6) 24(46.2) 48(49.0) 0.736 0.881 AML 127(83.6) 10(76.9) 22(81.5) 44(84.6) 82(83.7) MDS 25(16.4) 3(23.1) 5(18.5) 8(15.4) 16(16.3) 0.201 0.269 CR1 103(67.8) 6(46.1) 18(66.7) 31(59.6) 71(72.5) ≥CR2 27(17.8) 4(30.8) 2(7.4) 11(21.2) 15(15.3) No CR 22(14.4) 3(23.1) 7(25.9) 10(19.2) 12(12.2) 0.564 0.038 Low/Intermediate 94(61.8) 8(61.5) 14(51.9) 26(50.0) 66(67.3) High/Very high 58(38.2) 5(38.5) 13(48.1) 26(50.0) 32(32.7) 0.807 0.210 MMRD/MMUD 127(83.6) 11(84.6) 22(81.5) 41(78.8) 85(86.7) MRD/MUD 25(16.4) 2(15.4) 5(18.5) 11(21.2) 13(13.3) 0.090 0.145 Male 103(67.8) 11(84.6) 15(55.6) 39(75.0) 62(63.3) Female 49(32.2) 2(15.4) 12(44.4) 13(25.0) 36(36.7)
Abbreviations DTA, DNMT3A, TET2, and ASXL1 mutations; MRD, measurable residual disease; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; CR, complete remission; CR1, first complete remission; CR2, second complete remission; DRI-R, refined disease risk index; MMRD, mismatched related donor, MMUD, mismatched unrelated donor; MRD, matched related donor; MUD, matched unrelated donor
There were 54 myeloid neoplasm-associated mutations or fusion genes as trackable molecular targets after allo-HSCT (Supplementary Table 1), with a median number of 2 molecular targets per patient (range 1 to 7). Among the 152 patients, the most frequently detected genes at diagnosis were WT1 (n = 36), NRAS (n = 25), FLT3-ITD (n = 24), NPM1 (n = 23), DNMT3A (n = 20), CEBPA (n = 18), TET2, RUNX1-RUNX1T or IDH2 (n = 17), TP53 (n = 13), RUNX1 (n = 10), IDH1, MLL-PTD or c-KIT (n = 9), BCOR or U2AF1 (n = 8), CBFβ-MYH11 or GATA2 (n = 7), KRAS or ASXL1 (n = 6), PTPN11, FLT3-TKD, EZH2, CSF3R, MLL-ELL or STAG2 (n = 5).
Fifty-eight patients (38.2%) were MRD positive within 4 months after allo-HSCT, with a median VAF of 0.198%. The most commonly detected posttransplant MRD + targets were RUNX1-RUNX1T1 (n = 11), TET2 (n = 10), TP53 (n = 6), WT1 (n = 5), RUNX1 or CBFβ-MYH11 (n = 4), and NRAS, NPM1 or U2AF1 (n = 3). Additionally, among targets with a frequency ≥ 5, genes with a relatively high posttransplant MRD + detection ratio (posttransplant /diagnosis) included RUNX1-RUNX1T1 (detection ratio: 65%), TET2 (58%), CBFβ-MYH11 (57%), TP53 (46%), RUNX1 (40%), U2AF1 (38%), and ASXL1 (33%).
The median follow-up time of our cohort was 23.1 months (range 5.9–49.6 months) after allo-HSCT. Through analysis of a competing risk model, the overall CIR for patients who were MRD positive after allo-HSCT was higher than for MRD negative patients (33.5% vs. 10.3%, P < 0.001), while the NRM of these two groups was similar (6.7% vs. 4.4%, P = 0.541). With respect to RFS and OS, we found that MRD-positive patients had significantly inferior OS and RFS compared to MRD-negative patients (RFS: 59.8% vs. 83.8%, P < 0.001; OS: 64.3% vs.91.0%, P < 0.001). The comparison of CIR, RFS, and OS in the two groups of patients is shown in Supplementary Fig. 1.
We further analysed the prognostic impact of DTA and non-DTA genes to determine whether DTA mutations are suitable as MRD markers for predicting outcome after allo-HSCT. A total of 40 patients had at least one DTA mutation, while 150 patients had at least one non-DTA gene. The DTA and non-DTA genes were then grouped separately. Among patients with DTA genes, 13 patients (32.5%) were detected as MRD + after HSCT, including 10 TET2 mutations, 2 DNMT3A mutations, and 2 ASXL1 mutations, while in the non-DTA gene group, 52 patients (34.7%) were detected as MRD + after allo-HSCT. The CIR (23.1% vs. 14.8%, P = 0.571), RFS (69.2% vs. 81.5%, P = 0.453), and OS (76.9% vs. 85.2%, P = 0.537) (Supplementary Fig. 2) after allo-HSCT were not significantly different between the DTA MRD + group and the DTA MRD- group, suggesting that DTA genes may not have a predictive effect and are not suitable as monitoring markers after transplantation. However, in the non-DTA group, patients with MRD + had a higher CIR (38.7% vs. 9.7%, P < 0.001) and inferior RFS (55.5% vs. 83.7%, P < 0.001) and OS (60.5% vs. 90.5%, P < 0.001) (Fig. 2) compared to MRD- patients.
Graph: Fig. 2 CIR, NRM, RFS, and OS for patients who were MRD positive compared with MRD negative in non-DTA genes by ddPCR after allo-HSCT (150 patients). (A) CIR and NRM by competing risk analysis for MRD-positive (n = 52) and MRD-negative (n = 98) patients. (B, C) RFS and OS by Kaplan-Meier method for MRD-positive (n = 52) and MRD-negative (n = 98) patients
The univariate analysis revealed that ddPCR-MRD + of non-DTA genes and high/very high DRI-R were linked to increased CIR, inferior RFS and inferior OS. The remission status of ≥ CR2 at allo-HSCT was associated with increased CIR and inferior RFS, and no CR was associated with an increased risk of CIR and NRM, along with inferior RFS and OS (Table 2). In patients with DTA genes, the univariate analysis revealed no significant risk factors associated with these clinical outcomes (Supplementary Table 2). The multivariate analysis model (Fig. 3; Table 3) showed that ddPCR-MRD + in non-DTA genes was an independent prognostic factor for CIR (HR, 4.02; 95% CI, 1.80 to 8.96; P < 0.001), RFS (HR, 2.92; 95% CI, 1.51 to 5.68; P = 0.002) and OS (HR, 3.12; 95% CI, 1.36 to 7.15; P = 0.007). Additionally, a higher risk level of DRI-R significantly correlated with higher CIR (HR, 3.39; 95% CI, 1.41 to 8.14; P = 0.006), worse RFS (HR, 4.16; 95% CI, 1.86 to 9.34; P = 0.001) and worse OS (HR, 4.00; 95% CI, 1.44 to 11.13; P = 0.008). The PPV and NPV of the ddPCR-MRD status for predicting relapse in patients with non-DTA genes were 38.5% and 90.8%, respectively, with an overall accuracy of 64.6%. The lower PPV compared to NPV is generally considered to be the fact that patients who tested positive for MRD after transplantation received preemptive treatment to eliminate remaining disease, which can prevent relapse in some patients.
Table 2 Univariate analysis for CIR, NRM, RFS, and OS in patients with non-DTA genes
Variables n CIR NRM RFS OS HR (95% CI) HR (95% CI) HR (95% CI) HR (95% CI) Negative 98 1.00[Reference] 1.00[Reference] 1.00[Reference] 1.00[Reference] Positive 52 3.97(1.81–8.71) < 0.001 1.14(0.27–4.75) 0.860 3.58(1.87–6.87) < 0.001 3.80(1.67–8.61) 0.001 < 0.001 0.011 F-/M- 93 1.00[Reference] 1.00[Reference] 1.00[Reference] 1.00[Reference] F-/M+ 43 5.73(2.23–14.70) < 0.001 1.49(0.36–6.25) 0.583 3.60(1.72–7.54) 0.001 3.88(1.61–9.38) 0.003 F+/M- 6 9.83(2.54-38.00) < 0.001 N 4.83(1.36–17.13) 0.015 1.74(0.22–13.98) 0.600 F+/M+ 8 25.30(7.24–88.50) < 0.001 N 12.80(4.77–34.36) < 0.001 5.54(1.47–20.92) 0.012 < 0.001 0.002 CR1 102 1.00[Reference] 1.00[Reference] 1.00[Reference] 1.00[Reference] ≥CR2 26 2.66(1.12–6.28) 0.026 1.93(0.18–20.70) 0.590 2.42(1.07–5.48) 0.034 1.29(0.42-4.00) 0.661 No CR 22 3.36(1.38–8.16) 0.008 12.37(2.36–64.90) 0.003 5.27(2.53–10.99) < 0.001 4.50(1.89–10.73) 0.001 MMRD/MMUD 126 1.00[Reference] 1.00[Reference] 1.00[Reference] 1.00[Reference] MRD/MUD 24 0.85(0.30–2.43) 0.760 N 0.60(0.21–1.69) 0.332 0.66(0.20–2.22) 0.506 Low/Intermediate 92 1.00[Reference] 1.00[Reference] 1.00[Reference] 1.00[Reference] High/Very high 58 3.97(1.81–8.71) < 0.001 11.60(1.47–92.10) 0.020 5.51(2.67–11.35) < 0.001 5.76(2.30-14.43) < 0.001
Abbreviations ddPCR, droplet digital PCR; MRD, measurable residual disease; CIR, cumulative incidence of relapse; NRM, non-relapse mortality; RFS, relapse-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; F+/M+, MFC MRD-positive and non-DTA MRD-positive; F-/M+, MFC MRD-negative and non-DTA MRD-positive; F+/M-, MFC MRD-positive and non-DTA MRD-negative; F-/M-, MFC MRD-negative and non-DTA MRD-negative; CR, complete remission; CR1, first complete remission; CR2, second complete remission; MMRD, mismatched related donor, MMUD, mismatched unrelated donor; MRD, matched related donor; MUD, matched unrelated donor; DRI-R, refined disease risk index
Graph: Fig. 3 Forest plot showing the first (A) and second (B) multivariate analysis for CIR and OS
Table 3 Multivariate analysis for CIR, NRM, RFS, and OS in patients with non-DTA genes
Variables n CIR NRM RFS OS HR (95% CI) HR (95% CI) HR (95% CI) HR (95% CI) Negative 98 1.00[Reference] 1.00[Reference] 1.00[Reference] Positive 52 4.02(1.80–8.96) < 0.001 2.92(1.51–5.68) 0.002 3.12(1.36–7.15) 0.007 0.052 0.366 CR1 102 1.00[Reference] 1.00[Reference] 1.00[Reference] 1.00[Reference] ≥CR2 26 2.44(0.93–6.40) 0.071 2.41(0.27–21.30) 0.430 2.28(1.00-5.23) 0.051 1.30(0.42–4.08) 0.652 No CR 22 1.52(0.58–3.98) 0.390 5.16(0.67-40.00) 0.120 2.27(1.03–5.01) 0.043 1.97(0.77–5.04) 0.157 Low/Intermediate 92 1.00[Reference] 1.00[Reference] 1.00[Reference] 1.00[Reference] High/Very high 58 3.39(1.41–8.14) 0.006 5.78(0.68–49.30) 0.110 4.16(1.86–9.34) 0.001 4.00(1.44–11.13) 0.008
Abbreviations ddPCR, droplet digital PCR; MRD, measurable residual disease; CIR, cumulative incidence of relapse; NRM, non-relapse mortality; RFS, relapse-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; CR, complete remission; CR1, first complete remission; CR2, second complete remission; DRI-R, refined disease risk index
We further established internal validation at the First Affiliated Hospital of Zhejiang University School of Medicine and external validation at Other Hospitals to better prove feasibility of using ddPCR to detect non-DTA genes for predicting prognosis after transplantation. The results of internal validation showed that MRD positivity were significantly associated with higher CIR, lower RFS and OS (P < 0.001) (Supplementary Fig. 3). In the external validation cohort, it was observed that MRD-positive patients showed a higher CIR compared to MRD-negative patients (P = 0.049), and their RFS demonstrated a borderline significant difference compared to MRD-negative patients (P = 0.067). The absence of obviously differences (P = 0.325) in OS between the two groups may be attributed to the relatively small sample size in this subset (Supplementary Fig. 4). Overall, the findings from both validation groups strongly indicate a significant association between MRD-positive and higher CIR, in line with previous observations.
To more accurately identify patients who are at high risk of relapse, we performed a tandem assessment of MRD detection using both MFC and ddPCR in patients with non-DTA genes. We divided these patients into four groups: ①MFC MRD-positive and non-DTA MRD-positive (F+/M+, n = 8), ②MFC MRD-negative and non-DTA MRD-positive (F-/M+, n = 43), ③MFC MRD-positive and non-DTA MRD-negative (F+/M-, n = 6), and ④MFC MRD-negative and non-DTA MRD-negative (F-/M-, n = 93). Our analysis showed that patients with F+/M + had the highest CIR (F+/M + 75.0% vs. F+/M- 50.0% vs. F-/M + 32.9% vs. F-/M- 6.9%, P < 0.001), the worst RFS (F+/M + 25.0% vs. F+/M- 50.0% vs. F-/M + 60.1% vs. F-/M- 86.1%, P < 0.001) and the worst OS (F+/M + 57.1% vs. F+/M- 83.3% vs. F-/M + 60.4% vs. F-/M- 91.1%, P = 0.004) compared to the other groups, while patients with F-/M- had the most favorable outcomes in terms of relapse, RFS and OS (Fig. 4). NRM did not differ between the four groups. We conducted a multivariate analysis again on the tandem assessment, DRI-R and remission status at the time of HSCT, and the results showed that the F+/M + group (HR, 22.44; 95% CI, 7.31 to 68.91; P < 0.001), the F+/M- group (HR, 12.46; 95% CI, 3.22 to 48.21; P < 0.001), and the F-/M + group (HR, 4.51; 95% CI, 1.69 to 12.07; P = 0.003) had a significantly increased risk of relapse after allo-HSCT compared to the F-/M- groups (Fig. 3; Table 4). The DRI-R level remained statistically significant after this multivariate analysis: a higher risk level of DRI-R was associated with increased CIR and inferior RFS and OS. The PPV and NPV of MFC alone for predicting relapse were 64.3% and 85.3%, with an overall accuracy of 74.5%. While the PPV and NPV for combined detection exhibited higher PPV and NPV at 75.0% and 93.5%, resulting in an improved overall accuracy of 84.3%. Notably, the false negative rate in the F-/M + group was 10.3%, which was lower than the 14.7% observed in the F- group. This combined approach demonstrated superior diagnostic performance compared to either of the techniques used in isolation.
Graph: Fig. 4 CIR, RFS, and OS for patients who were F+/M+, F-/M+, F+/M-, and F-/M- in non-DTA genes after allo-HSCT by tandem assessment (150 patients). (A, B) CIR and NRM by competing risk analysis for F+/M+ (n = 8), F-/M+ (n = 43), F+/M- (n = 6), and F-/M- (n = 93) patients. (C, D) RFS and OS by Kaplan-Meier method for F+/M+ (n = 8), F-/M+ (n = 43), F+/M- (n = 6), and F-/M- (n = 93) patients
Table 4 Multivariate analysis for CIR, RFS, and OS in patients with non-DTA genes after introduction of MFC for tandem assessment
Variables n CIR RFS OS HR (95% CI) HR (95% CI) HR (95% CI) < 0.001 0.048 F-/M- 93 1.00[Reference] 1.00[Reference] 1.00[Reference] F-/M+ 43 4.51(1.69–12.07) 0.003 2.85(1.39–6.30) 0.005 3.51(1.43–8.60) 0.006 F+/M- 6 12.46(3.22–48.21) < 0.001 7.19(1.84–28.02) 0.004 3.02(0.34–26.80) 0.321 F+/M+ 8 22.44(7.31–68.91) < 0.001 9.97(3.52–28.22) < 0.001 3.06(0.78-12.00) 0.108 0.169 0.358 CR1 102 1.00[Reference] 1.00[Reference] 1.00[Reference] ≥CR2 26 1.95(0.72–5.30) 0.190 1.94(0.82–4.59) 0.132 1.17(0.36–3.79) 0.795 No CR 22 1.14(0.47–2.75) 0.780 1.86(0.82–4.21) 0.135 2.01(0.77–5.25) 0.152 Low/Intermediate 92 1.00[Reference] 1.00[Reference] 1.00[Reference] High/Very high 58 4.76(1.74–12.99) 0.002 5.32(2.26–12.55) < 0.001 4.11(1.45–11.66) 0.008
Abbreviations CIR, cumulative incidence of relapse; RFS, relapse-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; F+/M+, MFC MRD-positive and non-DTA MRD-positive; F-/M+, MFC MRD-negative and non-DTA MRD-positive; F+/M-, MFC MRD-positive and non-DTA MRD-negative; F-/M-, MFC MRD-negative and non-DTA MRD-negative; CR, complete remission; CR1, first complete remission; CR2, second complete remission; DRI-R, refined disease risk index
This retrospective cohort study was designed to assess the prognostic value of MRD detection after allo-HSCT in AML/MDS based on ddPCR. Our study provides evidence that ddPCR applied to MRD detection for non-DTA genes after allo-HSCT can effectively identify patients who are at high risk of relapse. In addition, to our knowledge, this study is the first comprehensive investigation to evaluate MRD by combining MFC with ddPCR, and this combination is proven to further improve the accuracy of predicting relapse after HSCT. Patients with negativity of both MFC and ddPCR were identified to have the most favorable outcomes after allo-HSCT.
The selection of ddPCR for MRD monitoring is often driven by its ability to perform absolute quantification of nucleic acids, resulting in increased sensitivity and accuracy of detection with a lower limit of detection of 0.001% compared to MFC and NGS and without the requirement of a standard curve compared to qPCR [[
The prognostic impact of DTA mutations as posttransplant MRD markers is still uncertain. Our study found that 32.5% (n = 13) of patients with DTA mutations had persistent DTA MRD + in posttransplant, especially TET2 mutation, and that persistent DTA mutations had little effect on prognosis. In contrast, after excluding DTA mutations, we observed a stronger association between non-DTA MRD + and negative prognosis after transplantation. At the end of follow-up, 3 of the 13 patients with DTA MRD + relapsed, while 4 of the remaining 27 patients with DTA MRD- relapsed. Among the DTA MRD - patients who relapsed, three were at a higher risk of DRI-R prior to HSCT and were found to be non-DTA MRD + before relapse. Of note, some patients with DTA MRD + had relatively high VAF values (> 5%) in CR status, in line with prior reports [[
We also found that 62.5% (15/24) of patients with CBF AML could still be detected with CBF MRD + after transplantation, with a median VAF of 0.011% (RUNX1-RUNX1T1: 0.010%, CBFβ-MYH11: 0.026%). Nevertheless, the incidence of relapse in these patients with CBF MRD + after allo-HSCT was low (RUNX1-RUNX1T1: 18.2%, 2/11; CBFβ-MYH11: 25%, 1/4). Meanwhile, we dynamically monitored the CBF levels of these patients and found that a majority of patients persistently had low VAF values of CBF at multiple time points (30–120 days) after transplantation, with a downward trend in VAF values. This seems to imply that extremely low VAF levels of CBF after early posttransplant period are not associated with relapse [[
Although MFC as the posttransplant MRD monitoring assay is less sensitive than ddPCR, some studies have revealed that MFC can indeed identify patients at increased risk of relapse after transplantation [[
In conclusion, our study provides a viable option for MRD monitoring in most patients with AML/MDS following allo-HSCT. MRD positivity of non-DTA genes detected by ddPCR after transplantation was associated with an increased risk of relapse. Besides, our study is pioneering in the field of posttransplant MRD monitoring, as it represents the first concerted effort to explore the combined use of ddPCR and MFC for prognostic prediction. When ddPCR is combined simultaneously with MFC to monitor posttransplant MRD, it is more accurate in identifying patients at high risk of relapse.
Sincere thanks to Shanghai Dishuo Beken Biotechnology Co., Ltd that provided ddPCR technology for this study.
YMZ, XXH and HH designed the study. WHC, JTH, YQZ, LH and MEG collected the data. WHC, JTH and ZYY analysed the data and wrote the manuscript. CHB, XH, and ZZZ performed experiments. XJX, JMS, YL, JY, XYL, LZL, and HRF contributed to the patient recruitment and treatment.
This work was supported by grants from the National Key Research and Development Program of China (grant nos. 2022YFA1103500) and the National Natural Science Foundation of China (grant nos. 82170210).
The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.
All patients signed written informed consent for this study. The entire study procedure was conducted according to the Declaration of Helsinki and was approved by the Ethics Review Committee of each center (IIT20230588C-R1).
The authors declare no competing interests.
Below is the link to the electronic supplementary material.
Graph: Supplementary Material 1: Supplementary Fig. 1. CIR, NRM, RFS, and OS for patients who were MRD positive compared with MRD negative by ddPCR after allo-HSCT (152 patients). (A) CIR and NRM by competing risk analysis for MRD-positive (n = 60) and MRD-negative (n = 92) patients. (B, C) RFS and OS by Kaplan-Meier method for MRD-positive (n = 60) and MRD-negative (n = 92) patients
Graph: Supplementary Material 2: Supplementary Fig. 2. CIR, NRM, RFS, and OS for patients who were MRD positive compared with MRD negative in DTA genes by ddPCR after allo-HSCT (40 patients). (A, B) CIR and NRM by competing risk analysis for MRD-positive (n = 13) and MRD-negative (n = 27) patients. (C, D) RFS and OS by Kaplan-Meier method for MRD-positive (n = 13) and MRD-negative (n = 27) patients
Graph: Supplementary Material 3: Supplementary Fig. 3. Internal verification cohort for ddPCR-MRD at the First Affiliated Hospital of Zhejiang University School of Medicine.
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By Weihao Chen; Jingtao Huang; Yeqian Zhao; Luo Huang; Zhiyang Yuan; Miner Gu; Xiaojun Xu; Jimin Shi; Yi Luo; Jian Yu; Xiaoyu Lai; Lizhen Liu; Huarui Fu; Chenhui Bao; Xin Huang; Zhongzheng Zheng; He Huang; Xiaoxia Hu and Yanmin Zhao
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