MRD Monitoring of Non-DTA Genes By Digital Droplet PCR in the Early Period after Allogeneic Hematopoietic Stem Cell Transplantation Can Effectively Predict Patients at High Risk of Relapse

Background: Hematological malignancies relapse is the major cause of death after allogeneic hematopoietic stem cell transplantation (HSCT). The detection of measurable residual disease (MRD) after HSCT is the effective and important predictor of relapse. Digital droplet polymerase chain reaction (ddPCR), as the third generation PCR, is a novel technology for absolute quantification of nucleic acids developed in recent years. Compared with real-time quantitative PCR (qPCR, first generation) and next-generation sequencing (NGS), ddPCR has better sensitivity, specificity and repeatability, which provide a new platform for the detection of MRD. The purpose of this study was to evaluate whether early MRD monitoring by ddPCR after HSCT can effectively predict the recurrence of primary disease.

Methods: We retrospectively analyzed patients with hematological malignancies who underwent allogeneic HSCT and monitored post-transplant MRD by ddPCR at the BMT Center of the First Affiliated Hospital of Zhejiang University School of Medicine from August 2018 to February 2021. Patients who met the following criteria were included in our study: (1) at least one hematological tumor-associated mutations or fusion genes for MRD monitoring at the time of diagnosis; (2) underwent allogeneic HSCT and the stem cell engraftment was successful; (3) received bone marrow MRD detection by ddPCR at least once in the early period (+30 days to +120 days) after HSCT; (4) bone marrow morphology was in remission status within +30 days to +120 days.

Results: 100 patients were included in our study. The median follow-up time of patients was 20 months (range 5-46), and the median age was 41 years (range 14-67). Among them, 59 (59.0%) were male, and the most frequent primary diseases were AML (N=59, 59.0%), ALL (N=21, 21.0%), MDS (N=16, 16.0%), respectively.

A total of 43 (43.0%) patients were found to be MRD⁺ after HSCT by early ddPCR detection. The difference in the 2-year cumulative incidence of relapse (2y CIR, 32.5% vs. 19.6%, P= 0.103), 2-year relapse-free survival (2y RFS, 67.0% vs. 79.8%, P=0.110), and overall survival (OS, 63.0% vs. 84.1%, P=0.566) between the MRD⁺ and MRD^- groups were not statistically significant.

30 patients had at least one mutation related to clonal hematopoiesis gene: DNMT3A, TET2, and ASXL1 (DTA), whereas 97 patients had at least one non-DTA gene. When DTA group and non-DTA group were considered separately, MRD⁺ were 33.3% in DTA gene group and MRD⁺ were 36.1% in non-DTA gene group.

The 2y CIR (0% vs. 21.5%, P=0.160), 2y RFS (100% vs. 78.5%, P=0.170) and OS (90.0% vs. 85.0%, P=0.737) after HSCT was not statistically different between DTA MRD⁺ group and DTA MRD^- group, suggesting that DTA was not suitable as monitoring genes for relapse after transplantation. However, in the non-DTA group, patients with MRD⁺ had higher 2y CIR (39.8% vs. 18.4%, P=0.014) and inferior 2y RFS (60.2% vs. 80.8%, P=0.017) compared with MRD^- patients.

To explore whether ddPCR and multiparameter flow cytometry (MFC) can complement each other and further improve the prediction accuracy of relapse, we conducted a joint analysis on the bone marrow MRD detected simultaneously by non-DTA ddPCR and MFC, and divided into four parts: ①MFC MRD⁺ and non-DTA MRD⁺ (F+/M+, n=7); ②MFC MRD^- and non-DTA MRD⁺ (F-/M+, n=26); ③MFC MRD⁺ and non-DTA MRD^- (F+/M-, n=8); ④MFC MRD^- and non-DTA MRD^- (F-/M-, n=56). The sensitivity of ddPCR was higher than that of MFC. The 2y CIR of the four groups was (F+/M+ 71.4% vs. F-/M+ 34.5% vs. F+/M- 53.1% vs. F-/M- 12.2%, P<0.001), and the 2y RFS was (F+/M+ 28.6% vs. F-/M+ 65.5% vs. F+/M- 46.9% vs. F-/M- 87.3%, P<0.001) and OS was (F+/M+ 31.3% vs. F-/ M+ 61.8% vs. F+/M- 87.5% vs. F-/M- 83.8%, P=0.219). Through multivariate analysis, F+/M+ is an independent risk factor for relapse after HSCT (hazard ratio, 9.531, 95%CI, 2.884 to 31.496). It suggested that F+/M+ detected in the early stage after transplantation had higher CIR and inferior RFS.

Conclusion: MRD monitoring for non-DTA gene in the early period after HSCT based on ddPCR has a prognostic effect on the recurrence of primary disease and overall survival after transplantation. When ddPCR is combined with MFC to monitor MRD, it is more accurate in predicting relapse and may help instruct preemptive treatment and then reduce the risk of relapse.

No relevant conflicts of interest to declare.