T-cell acute lymphoblastic leukemia (T-ALL) patients have increased risk for treatment resistance and early relapse. The precise bone marrow evaluation for the presence of minimal residual disease (MRD) is essential for guiding treatment options. This requires techniques more sensitive than the level of sensitivity of light microscopic technique such as multicolour flow cytometry (FCM). Immunophenotypic alterations called leukemia associated immunophenotypic patterns (LAIP) (i.e.aberrant myeloid markers) and ectopic phenotypic expression (i.e. appearance of immature phenotypes such as TdT, CD1a and CD3 outside their normal site in the thymus) are of benefit to track the residual leukemic cells in T-ALL.
A retrospective data analysis of MRD was done comprising T-ALL patients diagnosed and followed-up at the Institute of Cancer Research/Royal Marsden Hospital by means of 3-colour flow cytometry (3C FCM).The aim was to answer a question whether the 3C FCM can reliably split patients into two groups (positive, MRD+ and negative, MRD-) and predict a subsequent relapse and to define a right time point for performing MRD tests. Eight patients were enrolled in the study following the inclusion criteria: (i) complete remission after 1st induction phase of chemotherapy, (ii) presence of LAIP or an ectopic phenotypic expression, and (iii) monitored at defined time points after initial treatment. MRD was measured during the first year of treatment as follows: at the end of phase 1 induction (day 29–35, MRD1), before the start of consolidation (3 months, MRD2), after consolidation (MRD3), during the maintenance therapy (12 months, MRD4). Immunophenotyping was performed on lysed-washed bone marrow samples using CD45 gating strategy and originally defined blast gates at diagnosis. The phenotypes to be followed-up included: TdT+/CytCD3+, CD34+/CYTCD3+, TdT+/CD2+, CD8+/CD10+, CD2+/CD10+, CD7+/CD10+, CD7+/CD33+, CD7+CD34+. Patients were divided into 2 groups in relation to subsequent relapse. Group 1 included 6 patients without relapse. Patient characteristics of the group were: male:female 5:1, mean age 17.7 years, overall survival (OS) 59 months, relapse free survival (RFS) 85 months. Group 2, relapsed patients, included 2 men, mean age 56 years, OS 13 months, RFS 8.5 months. According to the EGIL classification system the 2 men in Group 2 were with an early T-precursor phenotype, whilst Group 1 was heterogenous but cortical-T-ALL predominated. Cytogenetics/FISH and RQ-PCR studies were performed at diagnosis and showed normal karyotype in only one of the Group 2 patients.
MRD results showed a difference between the two groups as regards MRD1 and MRD2 time points. Group 1 patients had negative or low MRD levels (below 0.18%) in their MRD1 bone marrow - MRD-, n=4 and MRD+,n=2 (0.18% and 0.12% respectively, sensitivity 0.04%). Those of them who were tested at MRD2 and MRD3 were negative. Both patients in Group 2 showed higher levels of MRD positivity at MRD1 (1% of total bone marrow cells), the first one of them also being positive at MRD2 and the second one becoming MRD+ at MRD4 time point. Although turning to MRD- at MRD3 time point both Group 2 patients relapsed 2.5 and 4.5 months, respectively, after the end of consolidation treatment. Additionally, Group 1 patients had a significantly longer RFS than Group 2 (median 58 months RFS vs. 8.5 months; P <0.001).
Conclusions: Reliable detection of MRD in T-ALL is possible by 3C FCM using a combination of TdT and a T cell marker (cytCD3 or mCD3) as such a combination is normally found exclusively in the thymus. The higher MRD-positive levels in Group 2 reflect the more resistant disease in this group and higher probability of early relapse and shortened overall survival. Early T-cell precursor phenotype in these patients appeared to be a subtype at very high risk for treatment failure irrespective of the lack or the presence of genetic lesions. Based on MRD positivity above 0.18% at time points MRD1 or both MRD1 and MRD2 these patients need reassessment of treatment options and more intensive therapy has to be considered for relapse prevention. Finally, the results of our retrospective study suggest the usefulness of implementation of MRD testing by FCM for taking clinical decisions in the prospective clinical trials for novel therapies for T-ALL.
The study was supported by the Union for International Cancer Control, Geneva, Switzerland (Grant ICRETT-080–2011)
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.