Outcome prediction by immunophenotypic minimal residual disease detection in adult T-cell acute lymphoblastic leukaemia

Flow-cytometric detection of minimal residual disease (MRD) identifies patients with high relapse risk in childhood acute lymphoblastic leukaemia (ALL). We studied the efficacy of this method in adult T-ALL treated with the Italian co-operative GIMEMA (Gruppo Italiano Malattie Ematologiche dell'Adulto) LAL0496 protocol. Bone marrow samples from 53 patients were taken at fixed treatment time points and MRD was analysed using a leukaemia-specific immunophenotype (cytoplasmic-CD3/nuclear-terminal desoxynucleotidyl transferase). The median follow-up was 17 months (range 3-61) and a median of 4.5 analyses/patient was performed (range 3-12). Six out of 53 (11.3%) patients were refractory to treatment, 30/53 (56.6%) relapsed and 17/53 (32.1%) remain in continuous complete remission. The probability of relapse at 2 years for MRD-positive patients at preconsolidation was 81.5%vs 38.9% for MRD-negative patients (P = 0.00078). This risk was still 54.5% for MRD-positive vs 15.8% for MRD-negative patients pre-third reinduction (P = 0.0098) and 50.0% for MRD-positive vs 16.4% for MRD-negative patients pre-sixth reinduction (P = 0.032). The relapse-predicting value of MRD did not depend on features at diagnosis such as age, sex and leucocyte count. Our data suggest that immunophenotypic MRD monitoring in the first year of treatment is a useful outcome predictor for adult T-ALL patients.     

Simultaneous detection and quantification of minimal residual disease in childhood acute lymphoblastic leukaemia using real-time polymerase chain reaction

A number of prospective studies have indicated the clinical utility of measuring minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia (ALL) and have highlighted the need for improved methodology for quantification of residual disease. We describe a novel real-time polymerase chain reaction (PCR) strategy for MRD analysis based on the exonuclease activity of Taq polymerase to cleave a fluorescently labelled probe. Using a consensus probe designed to the framework 2 region of the IgH gene, together with leukaemia-specific primers, the utility of this technique for simultaneous detection and quantification of MRD was demonstrated in samples from six ALL patients. This technique provides a rapid quantitative assay for determining MRD levels which lends itself to the routine monitoring of minimal residual leukaemia.     

Interphase FISH to detect PBX1/E2A fusion resulting from the der(19)t(1;19)(q23;p13.3) or t(1;19)(q23;p13.3) in paediatric patients with acute lymphoblastic leukaemia

Approximately 6% of paediatric patients with precursor B-cell acute lymphoblastic leukaemia (B-ALL) harbour a rearrangement involving the gene regions of PBX1 (1q23) and E2A (19p13.3) which is visualized cytogenetically either as a der(19)t(1;19)(q23;p13.3) or the less common balanced t(1;19)(q23;p13.3). Unfortunately, no commercial dual-colour, double fusion fluorescence in situ hybridization (D-FISH) strategies are available to detect this recurrent anomaly. Therefore, we have created a D-FISH assay to detect these translocations and monitor minimal residual disease. This probe set was created using four bacterial artificial chromosomes (BACs) corresponding to the PBX1 gene region at 1q23 and four BACs corresponding to the E2A gene region at 19p13.3. We analysed 30 negative bone marrow controls and 20 diagnostic and post-treatment specimens from 13 paediatric B-ALL patients with a cytogenetically defined 1;19 translocation. Once unblinded, the results demonstrated that our D-FISH method effectively identified all diagnostic samples as abnormal and identified disease in four post-treatment samples that were previously considered to be normal by conventional cytogenetic analysis. The development of this FISH strategy for the detection of der(19)t(1;19)(q23;p13.3) and t(1;19)(q23;p13.3) proved to be an effective technique, allowing both the detection of disease in diagnostic samples and in post-treatment samples.     

Assessment of minimal residual disease in acute promyelocytic leukaemia with t(15;17) by chromosome painting

Abstract: To detect the minimal residual disease (MRD) in acute promyelocytic leukaemia patients treated with all-trans retinoic acid, we compared the sensitivity of metaphase fluorescence in situ hybridization (FISH) with conventional analysis of G-banded metaphases. 5 out of 6 patients studied at diagnosis showed the t(15;17) translocation. 4 out of 5 patients carrying t(15;17) achieved complete remission and conventional cytogenetic conversion. In 3 cases the whole chromosome painting (WCP) probe 17 discovered one normal chromosome 17 and two fragments indicative of t(15;17) persistence. The FISH-WCP technique seems to be highly sensitive and recommendable in monitoring leukaemias with specific chromosome rearrangements.     

Low frequency of clonotypic Ig and T-cell receptor gene rearrangements in t(4;11) infant acute lymphoblastic leukaemia and its implication for the detection of minimal residual disease

Infant t(4;11) acute lymphoblastic leukaemia (ALL) is a rare but cytogenetically well defined subgroup of immature B-cell precursor (BCP) ALL. To date, the configuration of their antigen receptor genes has not been studied in a large group of patients so far. In this study on 27 t(4;11) infant ALL, we have used standardized primer sets for the detection of all incomplete and complete immunoglobulin (Ig) heavy chain (IGH) rearrangements, as well as for the Ig light chain kappa (IGK), T-cell receptor delta (TCRD) and gamma (TCRG) rearrangements that are most common in childhood BCP ALL. Only 52% of cases displayed clonotypic antigen receptor gene rearrangements (IGH in 48%, IGK, TCRD and TCRG in 12%, 41% and 6% respectively). This low frequency suggests, together with the findings of predominantly incomplete DJh joins and monoallelic IGH rearrangements, that they are derived from an immature progenitor cell. As 48% of the t(4;11) infant ALL cases had no detectable antigen receptor gene rearrangements that could be used for minimal residual disease (MRD) analysis, we established an expression-independent, leukaemia-specific polymerase chain reaction (PCR) using the genomic sequence of the MLL-AF4 fusion genes. This method had high sensitivity and specificity and resulted in identical estimations of tumour loads when compared with IGH targets. Thus, genomic MLL-AF4 fusion genes are a good alternative target for the analysis of MRD in patients with t(4;11) leukaemias.     

Clinical value of immunological monitoring of minimal residual disease in acute lymphoblastic leukaemia after allogeneic transplantation

In this study, we used multiparameter flow cytometry to quantify minimal residual disease (MRD) in 165 serial bone marrow samples from 40 patients diagnosed with acute lymphoblastic leukaemia (ALL) who underwent allogeneic stem cell transplantation (allo-SCT) from siblings (n = 34) or unrelated donors (n = 6). Samples were prospectively taken from 24 patients before starting the conditioning regimen, at days +30, +60 and +90 and subsequently every 2-3 months. Samples from 16 patients in complete remission (CR) after allo-SCT were taken at least twice. Six of 24 patients harboured MRD (0.2-10% of mononuclear cells) at transplant and 18 were negative. Estimated disease-free survival for the MRD+ and MRD- groups at transplant was 33.3% and 73.5% respectively (P = 0.03). During follow-up, increasing MRD levels were detected in nine patients, a finding that preceded marrow relapse by 1-6 months. Two patients with stable low MRD levels remained in CR. When we used flow cytometry to test the effect of donor leucocyte infusions (DLI) in six patients, we observed that the only sustained remission was achieved when DLI was applied prior to overt relapse. We conclude that MRD by flow cytometry can rapidly assess tumoral burden before transplant to predict outcome, and can be clinically useful for the timing of DLI for increasing levels of leukaemia after transplant.