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.     

The detection of minimal residual disease in acute lymphoblastic leukaemia.

The detection of minimal residual disease in acute lymphoblastic leukaemia (ALL) can be achieved by assessing leukaemia-specific features at a cellular, chromosomal or molecular level. The application of the polymerase chain reaction to the amplification of leukaemia-specific chromosomal translocations and clone-specific immunoglobulin and T-cell receptor gene rearrangements allows assessment of the majority of cases of ALL. The sensitivity of detection of this technique is around one leukaemia cell in 10(5) normal marrow cells. A comparative review of the advantages and pitfalls of the different methods of detecting minimal disease is presented. The clinical relevance of such detection is discussed, with early results suggesting that this may have predictive value for future disease relapse.     

Monitoring minimal residual disease in peripheral blood in B-lineage acute lymphoblastic leukaemia

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.1 × 10⁻² to < 9.4 × 10⁻⁷ leukaemic cells/total cells, corresponding to 1.3 × 10⁷ to < 2 × 10³ leukaemic cells/l. In 15 paired samples, leukaemia could be quantified in both tissues and in 20 paired samples, leukaemia was not detected in one or both tissues so that only upper level limits could be set. In the former 15 pairs, the level of leukaemia in peripheral blood was directly proportional to that in marrow but was a mean of 11.7-fold lower. Leukaemia in blood was detected in 10/12 pairs in which the level in marrow was > 10⁻⁴, but in only two of 13 pairs in which the level in marrow was < 10⁻⁵. Patients studied at multiple time-points showed parallel declines in the number of leukaemic cells in both tissues. The results showed that leukaemia could be monitored in peripheral blood during induction therapy, and quantitative considerations based on the results suggest that monitoring of blood during post-induction therapy may be of value in detecting molecular relapse.     

Clinical significance of minimal residual disease in adult acute lymphoblastic leukemia

Monitoring minimal residual disease (MRD) in patients with acute lymphoblastic leukemia (ALL) is a useful way for assessing treatment response and relapse. We studied the value of MRD and showed a correlation with relapse for 34 adult patients with ALL. MRD was evaluated by real-time quantitative polymerase chain reaction (RQ-PCR) with probes derived from fusion chimeric genes (BCR/ABL) (n = 12) or PCR-based detection of clonal immunoglobulin and T cell receptor gene rearrangements (n = 16), or both (n = 6). We analyzed 27 of the 34 patients who could be examined for MRD on day 100 after induction therapy. The overall survival (OS) rate (45.0%) and relapse-free survival (RFS) rate (40.0%) at 2 years in complete remission (CR) patients with MRD level ≥10^?3 (n = 12) were significantly lower than those in CR patients with MRD level <10^?3 (n = 15) (OS rate 79.0%, RFS rate 79.4%) (log-rank test, P = 0.017 and 0.0007). We also applied multicolor flow cytometry for comparison with MRD results analyzed by PCR methods. The comparison of results obtained in 27 follow-up samples showed consistency in 17 samples (63.0%) (P = 0.057). MRD analysis on day 100 is important for treatment decision in adult ALL.     

The significance of detection of minimal residual disease in childhood acute lymphoblastic leukaemia

Summary. In acute lymphoblastic leukaemia (ALL), minimal residual disease (MRD) can be defined as disease occurring at a subclinical level and beyond detection by conventional methods of assessment. Application of the polymerase chain reaction (PCR) to the hypervariable segment of the immunoglobulin heavy chain (IgH) gene, allows detection of MRD at a level of one leukaemic cell in 10⁴-10⁵ normal marrow cells. We have performed a retrospective study using this technique in the assessment of children with precursor B-cell ALL in whom the clinical outcome is known. In the early treatment period MRD is commonly detected in children who remain in complete remission on subsequent follow up. Thus, the detection of MRD at this time may have little value in the prediction of future relapse. However, at the end of treatment, children who remain in complete remission have no evidence of MRD. Conversely, detectable MRD at this time would seem to predict for future relapse, though this can be a delayed event. Remarkably, in two children who suffered a bone marrow relapse 8.5 and 9 years after completing therapy for their initial disease, MRD was detected, in their end of initial treatment marrow samples. Clearly PCR technology is changing the definition of the remission state in childhood ALL. and may have predictive value in the assessment of children who are at a high risk of future relapse. Large prospective studies of molecular monitoring are now required to confirm these preliminary results.     

Simplified strategies for minimal residual disease detection in B-cell precursor acute lymphoblastic leukaemia

We have developed a simplified fluorescent run-off (FluRO) based IgH PCR strategy in order to facilitate follow-up of large numbers of B-cell precursor (BCP) acute lymphoblastic leukaemias (ALL) in a routine molecular diagnostic laboratory. DNA samples from 26 BCP-ALL and one B-cell line were amplified using IgH FR1 and FR2 consensus primers and analysed in parallel either by ethidium bromide non-denaturing PAGE or, after rendering the PCR products fluorescent with an internal JH consensus primer, by high-resolution analysis on an automated fragment analyser. The latter led to a minimum of one log increase in sensitivity of detection in 62% of alleles from 19 samples (16/28 in FR1; 11/15 in FR2) tested in parallel on log DNA dilutions, and to at least a 10⁻² level of sensitivity of detection in 15/19. The improved resolution allowed an approximate 20% increase in the number of clonal alleles detected, and consequently doubled the incidence of oligoclonality (6/26; 23%). Using these strategies, 6/17 (35%) of children analysed prospectively showed residual IgH positivity in the post induction complete remission bone marrow sample. Both early deaths occurred within this subgroup of patients and of the three of four surviving patients tested, two remained positive 2–3 months later. Although this simplified strategy is, as expected, less sensitive than anti-V-D-J junction specific strategies, it enables detection of a category of 'slow-remitters' which may have prognostic significance at a stage where therapeutic decisions are taken.     

Molecular detection of minimal residual disease is associated with early relapse in adult acute lymphoblastic leukemia

Several studies in childhood acute lymphoblastic leukemia (ALL) have documented that molecular detection of minimal residual disease (MRD) based on screening for T-cell receptor and immunoglobulin gene rearrangements can identify patients at a high risk of relapse. In our experience, evaluation of MRD in adult ALL can help to identify high risk patients.     

Relationship between minimal residual disease and outcome in adult acute lymphoblastic leukemia

In children with acute lymphoblastic leukemia (ALL), the level of minimal residual disease (MRD) at the end of induction strongly predicts outcome, presumably because it measures both drug sensitivity and the number of leukemic cells requiring elimination. Children with high levels (> 10(-3) leukemic cells per marrow cell) nearly always relapse, whereas those with low levels (<2 x 10(-5)) seldom do. However, the importance of MRD in adult ALL is unclear. We studied 27 patients aged 14 to 74 who were treated with a standard protocol and who attained morphological remission. MRD in the marrow at first remission was quantified by using the polymerase chain reaction (PCR), with the rearranged immunoglobulin heavy chain gene as a molecular marker. Levels of MRD varied from 3 x 10(-1) to <7 x 10(-7). The probability of long-term relapse-free survival was significantly related to the level of MRD and only one of nine patients with MRD >10(- 3) did not relapse. For patients who did relapse, there was an inverse relationship between MRD level and the length of remission. Overall, MRD in adults in whom a translocation had not been identified was significantly higher than in comparably-treated children, suggesting that ALL in adults is more drug-resistant than in children.     

Detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia by quantitative flow cytometry

The clinical significance of detecting minimal residual disease (MRD) in B-lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B-cell precursor ALL were followed during and after completion of treatment (median follow-up 23 months). Nine patients relapsed and MRD had been detected in six of them, 5–15 weeks before relapse despite morphological complete remission. 43 patients remain in clinical remission and in none of these was MRD detected. Disease-free survival based on the detection of MRD by flow cytometry showed a statistically significant difference between both groups ( P  < 0.0001). The absence of MRD correlates with a low relapse rate, whereas the presence of MRD predicted early relapse. This study has shown that flow cytometry can improve the morphologic assessment of bone marrow (BM) remission status in B-lineage ALL. The finding of < 5% blasts in BM aspirates did not correlate with 'true' remission in a proportion of cases as residual leukaemic blasts were detected by flow cytometry in nine samples from six patients. On the other hand, the presence of > 5% blasts assessed by morphology was not necessarily a feature of relapse in five patients as these cells were shown to have a phenotype identical to normal TdT-negative B-cell precursors. Quantitative flow cytometry was more informative than conventional morphology to assess remission status and showed a strong correlation with clinical outcome. This methodology is useful to define MRD in the majority of patients with B-lineage ALL and should be tested in prospective clinical trials.     

Detection of minimal residual disease in childhood acute lymphoblastic leukaemia using fluorescence in-situ hybridization

Summary. Using centromere-specific probes and a fluorescence in-situ hybridization (FISH) technique in cases of childhood hyperdiploid acute lymphoblastic leukaemia (ALL), cells with extra copies of chromosomes can be differentiated from normal cells by their extra signals in both metaphase and interphase nuclei. In this way the entire cell population, not only those cells in division, can be analysed, thereby providing a valuable technique not only for determining leukaemia cell karyotype at diagnosis but also for the detection of minimal residual disease (MRD). We have conducted 161 analyses of remission bone marrow aspirates (BMs) in 13 children with hyperdiploid ALL. Slides were analysed blind and in parallel to 35 control samples. Control BMs showed very low numbers of trisomic cells (mean ± 2 × SD = 013 ± 0.34%). MRD was detected in 5/13 cases of ALL investigated while on chemotherapy. One out of five newly diagnosed cases and all three relapse cases of ALL had significantly raised levels of hyperdiploid cells in day 28 BMs. The presence of detectable disease in day 28 BMs suggests the need for larger studies to find whether this data is of prognostic value.     

The use of DNA probes to monitor minimal residual disease in childhood acute lymphoblastic leukaemia

DNA probes to both the joining region (JH) of the immunoglobulin heavy chain gene (IgH) and to the beta chain of the T-cell antigen receptor complex (TCR) have been used as tumour-specific markers to monitor the rearrangements of the IgH chain gene and the TCR beta gene in the blast cells of children presenting with acute lymphoblastic leukaemia (ALL) of B or T cell origin. Blast cells from 68 children with early B cell ALL and eight with T-ALL were examined at presentation, at day 28 after commencement of therapy and at varying times thereafter. An additional 43 patients (42 with B cell ALL, one with T-ALL) were studied both at presentation, at completion of their 2-year treatment course and 3 months later. Twelve patients, drawn from both these groups, were studied at relapse as were a further eight patients in whom an extramedullary relapse had occurred. Persistence of clonally-derived cells as a predictor of early relapse was seen in the day 28 bone marrows of 11/76 newly-diagnosed children (nine early B and two T-ALL) followed by rapid, overt relapse in four of the early B ALL cases. No minimal residual disease (MRD) was detected in bone marrows from any of the 43 patients completing their 2-year treatment course, but six of these subsequently relapsed at varying time periods thereafter. Identical patterns of rearrangement at both presentation and relapse were seen in most cases. Oligoclonality, or multiple IgH chain gene rearrangements was seen in the blast cells of 15% of patients with early B cell ALL. No correlation between oligoclonality, high white count, unfavourable phenotype or abnormal karyotype could, however, be ascertained.     

Molecular analysis of chromosome 22 breakpoints in adult Philadelphia-positive acute lymphoblastic leukaemia

The Philadelphia (Ph) translocation, t(9:22)(q 34:q11), is found in the majority of patients with chronic myelogenous leukaemia (CML) as well as in approximately 20% of adult acute lymphoblastic leukaemia (ALL) patients. The chromosome 22 breakpoint in CML has been localized within a restricted 5.8 kb segment of DNA known as the breakpoint cluster region (bcr). To investigate the chromosome 22 breakpoint in ALL, we analysed five adult Ph-positive ALL patients for bcr rearrangement. Rearrangement was detected within bcr in two patients. However, in one patient the break occurred 5' to the first exon of bcr and in two patients the bcr region was not involved. We conclude that the identical cytogenetic marker, t(9:22), may yield a different genomic configuration in ALL and CML.     

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.     

Defining the correct role of minimal residual disease tests in the management of acute lymphoblastic leukaemia

Minimal residual disease (MRD) has acquired a prominent role in the management of childhood and adult Acute Lymphoblastic Leukaemia (ALL) for its high prognostic value. Several studies have demonstrated the strong association between MRD and risk of relapse in childhood and adult ALL, irrespective of the methodology used. MRD is now used in clinical trials for risk assignment and to guide clinical management. Negativity at early time points may be considered to decrease treatment burden in patients who are likely to be cured with reduced intensity regimens. On the other hand, high MRD levels at late time points (end of consolidation) define ALL subgroups which deserve investigation of more effective treatments. The predictivity of MRD as a measurement of drug response in vivo opened new perspectives for its use in clinical decision, to deliver risk-based treatments, and possibly as a surrogate for efficacy in the evaluation of novel therapeutic approaches.     

Modelling a minimal residual disease-based treatment strategy in childhood acute lymphoblastic leukaemia

The measurement of minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia offers the promise of individualized, risk-stratified treatment, but an optimal protocol needs establishing. A model was developed to explore certain unanswered questions. The model assumes that all patients have MRD assessed after induction chemotherapy and children above a certain threshold are offered intensive chemotherapy. Using parameter estimates derived from published studies of MRD, the model predicted event-free survival (EFS) rates, relapse rates and treatment-related mortality for a cohort of children in the first presentation who were Philadelphia chromosome negative. Using the level of MRD after induction in order to decide on the use of intensive therapy resulted in an increase in EFS rates of up to 2.9 per 100 children, although if the error of the MRD measurement were too great, the benefit was almost nullified. Taking and analysing more than one marrow sample from the patient for the MRD measurement, in order to reduce sampling and measurement error, improved EFS by a further 1.1 patients per 100 treated, and decreased the number of patients offered intensive therapy by up to 2.6 per 100 treated. The optimal threshold for offering intensive therapy was in the range of 10-3.5- 10-4.5 cells if the intensive treatment-related mortality was 13-18% (allograft options), but 10-5- 10-6 cells if it was less than 8% (intensified chemotherapy). Using MRD to target patients at a high risk of relapse improved EFS rates, but the accuracy of measurements was of critical importance.