Comparative Analysis of Flow Cytometry and Real-Time Quantitative Polymerase Chain Reaction for the Detection of Minimal Residual Disease in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia before Hematopoietic Stem Cell Transplantation

ABSTRACT

Background：The aim of this study was to examine the value of MRD detection by multiparameter flow cytometry (MFC) and RQ-PCR before hematopoietic stem cell transplantation (HSCT) for predicting relapse and leukemia-free survival (LFS) in Philadelphia chromosome-positive ALL (Ph⁺ ALL).

Methods：A retrospective study (n=280) was performed. MRD was determined using multiparameter flow cytometry and RQ-PCR.

Results：MRD analysis with MFC and RQ-PCR of the BCR-ABL fusion transcript showed a strong correlation before transplantation. The positive rates of MRD detected by MFC and RQ-PCR before transplantation were 25.7% and 60.7% respectively. Ph⁺ ALL patients with MFC MRD positive (MRDpos) had a higher 3-year cumulative incidences of relapse (CIR) compared to those with MFC MRD negative (MRDneg) (23.6% vs. 8.6%, P < 0.001). However, the RQ-PCR MRDpos group had similar rates of 3-year OS, LFS and NRM compared with those in the RQ-PCR MRDneg group. Moreover, compared to RQ-PCR MRD＜1% group, patients with RQ-PCR MRD≥1% experienced higher 3-year CIR（23.1% vs. 11.4%， P = 0.032）, lower LFS(53.8% vs.74.1%，P = 0.011) and OS (57.7% vs.79.1%，P = 0.009). Multivariate analyses confirmed the association of MFC MRD status and RQ-PCR MRD≥1% with outcomes (P ˂ 0.05). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of MFC detection MRD to predict recurrence were 48.50%, 77.56%, 23.62%, 87.16%. When RQ-PCR MRD≥1% was used to predict recurrence, the sensitivity, specificity, PPV, and NPV were 23.0%, 88.59%, 17.15% and 91.84%, respectively. Moreover，MRD-positive status before transplantation (MFC MRDpos or RQ-PCR MRD ≥ 1%) was used to predict recurrence after transplantation, the sensitivity, specificity, PPV, and NPV were 54.29%, 73.88%, 45.7%, and 91.87%.

Conclusion：Both MFC and RQ-PCR detection of pre-transplant MRD levels can predict the prognosis of Ph⁺ B-ALL patients eceiving Allo-HSCT. MFC MRD positive status and RQ-PCR MRD ≥ 1% before transplantation are the risk factors for recurrence after transplantation. The combined use of the two methods can improve the sensitivity of predicting recurrence and help to better screen high-risk patients for intervention, thereby improving clinical efficacy.

Keywords: acute lymphocytic leukemia, minimal residual disease, pre-transplant MRD levels, flow cytometry, RQ-PCR, BCR/ABL, relapse