Monitoring of T-cell acute lymphoblastic leukemia by flow cytometry

Abstract

<jats:title>Abstract</jats:title><jats:p>Minimal residual disease (MRD) predicts the outcome of acute lymphoblastic leukemia (ALL). Flow cytometry (FC) is one of the most sensitive and most applicable methods for MRD diagnostics, but there is still no agreement on the “gold standard” of the method. We tried to optimize flow cytometric MRD detection in T-ALL. Fourteen adults and 11 children with T-ALL and 12 normal bone marrow (BM) donors were enrolled in the study. We found that the most common phenotypic aberrations in T-ALL were TdT and CD99 coexpression on T-cells in BM. Therefore for MRD detection we developed a limited four-color marker panel (TdT/CD7/cCD3/CD19 and CD99/CD7/cCD3/CD2) and a standard analysis strategy. This assay was evaluated on BM of healthy controls. Less than 0.01% TdT+ or CD99 bright T-cells were found in normal BM. MRD was detected in 9 adult patients and 1 child at different time-points of treatment. The average TdT and CD99 mean fluorescence intensity (MFI) value of residual blasts fluctuated during therapy, but it still remained higher than MFI of normal T-cells. Our established MRD detection method differentiated leukemic lymphoblasts with sensitivity in the range of 0.01% and did not give any false positive results in normal BM.</jats:p>

FCM marker importance for MRD assessment in T‐cell acute lymphoblastic leukemia: An AIEOP‐BFM‐ALL‐FLOW study group report

“…In line with [18, 20] a decline of CD99 on leukemic cells during therapy occurs. However, similar to the findings in [39] CD99 still retains enough separability on treatment Days 15 and 33 such that it, together with CD48, influences the direction of greatest separation the most, therefore remaining the strongest separators throughout all analyzed time points. While the results in Figure 4 illustrated, that CD3 and CD5 do have the strongest hyperplane coefficients if CD45, CD48, and CD99 are omitted from the default marker set, we see in Figure 6 that the separation in this case leads on Day 33 to poor (≤0.2 median F1 default marker set) median F1 fitting scores.…”

“…In line with [18,20] a decline of CD99 on leukemic cells during therapy occurs. However, similar to the findings in [39] CD99 still retains enough separability on extending established panels with CD48. However, when a choice must be made, CD48 should be chosen in favor of CD45.…”

“…While the occurrence of phenotypic shifts during leukemia treatment is well described for B‐cell acute lymphoblastic leukemia (B‐ALL) [37, 38], less literature exists on early phenotypic shifts in T‐ALL [18, 39]: Roshal et al [20] reports that markers of immaturity such as TdT and CD99 decline on leukemic cells during therapy. In contrast, Janeliūnienė et al [39] observed that the TdT and CD99 mean fluorescence intensity (MFI) of blast cells fluctuate but are always higher than in normal T‐cells. Several markers and their combination, including TdT, CD99, CD7, CD4 & CD8, CD3, CD5, and CD45 have been established as suitable for MRD assessment in T‐ALL [9, 20].…”

“…Phenotypic shifts can occur, which urges the use of broader panels to avoid false negative MRD results [20,36]. While the occurrence of phenotypic shifts during leukemia treatment is well described for B-cell acute lymphoblastic leukemia (B-ALL) [37,38], less literature exists on early phenotypic shifts in T-ALL [18,39]: Roshal et al [20] reports that markers of immaturity such as TdT and CD99 decline on leukemic cells during therapy. In contrast, Janeli unienė et al [39] observed that the TdT and CD99 The deployment of TdT is technically complicated by requiring intracellular staining and is, therefore, less often used compared to surface-only approaches [9,40,41].…”

“…While the occurrence of phenotypic shifts during leukemia treatment is well described for B-cell acute lymphoblastic leukemia (B-ALL) [37,38], less literature exists on early phenotypic shifts in T-ALL [18,39]: Roshal et al [20] reports that markers of immaturity such as TdT and CD99 decline on leukemic cells during therapy. In contrast, Janeli unienė et al [39] observed that the TdT and CD99 The deployment of TdT is technically complicated by requiring intracellular staining and is, therefore, less often used compared to surface-only approaches [9,40,41]. In addition, TdT is not applicable for all T-ALL patients for MRD assessment [18,42].…”