Results of the Dana-Farber Cancer Institute ALL Consortium Protocol 95-01 for children with acute lymphoblastic leukemia

The Dana-Farber Cancer Institute (DFCI) Childhood ALL Consortium Protocol 95-01 was designed to minimize therapy-related morbidity for children with newly diagnosed ALL without compromising efficacy. Patients participated in randomized comparisons of (1) doxorubicin given with or without dexrazoxane, a cardioprotectant (high-risk patients), (2) intensive intrathecal chemotherapy and cranial radiation (standard-risk patients), and (3) Erwinia and Escherichia coli asparaginase (all patients). Between 1996 and 2000, 491 patients (aged 0-18 years) were enrolled (272 standard risk and 219 high risk). With a median of 5.7 years of follow-up, the estimated 5-year event-free survival (EFS) for all patients was 82%+/-2%. Dexrazoxane did not have a significant impact on the 5-year EFS of high-risk patients (P=.99), and there was no significant difference in outcome of standard-risk patients based on type of central nervous system (CNS) treatment (P=.26). Compared with E coli asparaginase, Erwinia asparaginase was associated with a lower incidence of toxicity (10% versus 24%), but also an inferior 5-year EFS (78%+/-4% versus 89%+/-3%, P=.01). We conclude that (1) dexrazoxane does not interfere with the antileukemic effect of doxorubicin, (2) intensive intrathecal chemotherapy is as effective as cranial radiation in preventing CNS relapse in standard-risk patients, and (3) once-weekly Erwinia is less toxic than E coli asparaginase, but also less efficacious.     

Improved outcome for children with acute lymphoblastic leukemia: results of Dana-Farber Consortium Protocol 91-01

The Dana-Farber Cancer Institute (DFCI) acute lymphoblastic leukemia (ALL) Consortium Protocol 91-01 was designed to improve the outcome of children with newly diagnosed ALL while minimizing toxicity. Compared with prior protocols, post-remission therapy was intensified by substituting dexamethasone for prednisone and prolonging the asparaginase intensification from 20 to 30 weeks. Between 1991 and 1995, 377 patients (age, 0-18 years) were enrolled; 137 patients were considered standard risk (SR), and 240 patients were high risk (HR). Following a 5.0-year median follow-up, the estimated 5-year event-free survival (EFS) +/- SE for all patients was 83% +/- 2%, which is superior to prior DFCI ALL Consortium protocols conducted between 1981 and 1991 (P =.03). There was no significant difference in 5-year EFS based upon risk group (87% +/- 3% for SR and 81% +/- 3% for HR, P =.24). Age at diagnosis was a statistically significant prognostic factor (P =.03), with inferior outcomes observed in infants and children 9 years or older. Patients who tolerated 25 or fewer weeks of asparaginase had a significantly worse outcome than those who received at least 26 weeks of asparaginase (P <.01, both univariate and multivariate). Older children (at least 9 years of age) were significantly more likely to have tolerated 25 or fewer weeks of asparaginase (P <.01). Treatment on Protocol 91-01 significantly improved the outcome of children with ALL, perhaps due to the prolonged asparaginase intensification and/or the use of dexamethasone. The inferior outcome of older children may be due, in part, to increased intolerance of intensive therapy.     

Detection of minimal residual disease in T-cell acute lymphoblastic leukemia using polymerase chain reaction predicts impending relapse.

After achieving remission, approximately one-third of patients with T-cell acute lymphoblastic leukemia (T-ALL) relapse due to the resurgence of residual leukemic cells that cannot be detected in remission by morphologic methods. Thus, the early detection of residual disease is highly desirable to monitor the efficacy of therapy, or to institute an alternative mode of therapy. Toward this aim, we have examined the applicability of polymerase chain reaction (PCR) amplification in the detection of minimal residual disease (MRD) in bone marrow samples from patients with T-ALL in morphologic remission. Two different approaches were taken to identify leukemic clone-specific sequences that could be used as targets for PCR amplification. The first technique used T-cell receptor-delta (TCR-delta) gene rearrangements that were sequenced directly after PCR amplification of leukemic DNA. This method was successful in generating clone-specific probes for 76% of T-ALL patients screened. An alternative method was used to clone and sequence a TCR-beta chain gene from leukemic cells to generate a specific probe. The PCR assays that we used were specific for each patient's leukemic clone, and were capable of routinely detecting one leukemic cell in 10(4) normal cells. Using these sensitive PCR-based assays, we found no evidence for persistence of the leukemic clone in any of the bone marrow samples from four T-ALL patients who are in long-term (3.9 + to 8.1 + years) remission. In contrast, we detected residual disease in clinical remission samples from two patients who subsequently relapsed. In one patient, where we had appropriate samples, we observed a dramatic expansion of the leukemic clone 3 months before clinical relapse. These results suggest that PCR-based assays for detection of MRD in T-ALL patients have great potential in predicting impending relapse, and in determining the efficacy of the anti-leukemic therapy. These methods may also allow the identification of long-term survivors.     

Methodological approach to minimal residual disease detection by flow cytometry in adult B-lineage acute lymphoblastic leukemia

A flow cytometric approach to minimal residual disease (MRD) monitoring useful in childhood B-lineage acute lymphoblastic leukemia (ALL) is discussed here in the context of ALL in adults. Of 64 leukemia samples analyzed, 95.3% had at least one abnormal phenotype (57.3% had two or more) as compared to physiologic B-cell precursors in adult bone marrow. The method was sensitive enough to detect one leukemic cell among 10,000 normal cells in 16/19 experiments (84.2%). Blast phenotypes were stable in culture and at relapse, and were useful for MRD monitoring in patients. Marker combinations for childhood ALL are also applicable to adult cases.     

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.     

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.     

Pre‐ and post‐transplant minimal residual disease predicts relapse occurrence in children with acute lymphoblastic leukaemia

Relapse remains the leading cause of treatment failure in children with acute lymphoblastic leukaemia (ALL) undergoing allogeneic haematopoietic stem cell transplantation (HSCT). We retrospectively investigated the prognostic role of minimal residual disease (MRD) before and after HSCT in 119 children transplanted in complete remission (CR). MRD was measured by polymerase chain reaction in bone marrow samples collected pre‐HSCT and during the first and third trimesters after HSCT (post‐HSCT1 and post‐HSCT3). The overall event‐free survival (EFS) was 50%. The cumulative incidence of relapse and non‐relapse mortality was 41% and 9%. Any degree of detectable pre‐HSCT MRD was associated with poor outcome: EFS was 39% and 18% in patients with MRD positivity <1 × 10⁻³ and ≥1 × 10⁻³, respectively, versus 73% in MRD‐negative patients (P < 0·001). This effect was maintained in different disease remissions, but low‐level MRD had a very strong negative impact only in patients transplanted in second or further CR. Also, MRD after HSCT enabled patients to be stratified, with increasing MRD between post‐HSCT1 and post‐HSCT3 clearly defining cohorts with a different outcome. MRD is an important prognostic factor both before and after transplantation. Given that MRD persistence after HSCT is associated with dismal outcome, these patients could benefit from early discontinuation of immunosuppression, or pre‐emptive immuno‐therapy.     

Kinetics of minimal residual disease during induction/consolidation therapy in standard-risk adult B-lineage acute lymphoblastic leukemia

We have compared the kinetics of minimal residual disease (MRD) by simultaneous polymerase chain reaction (PCR) monitoring with oligonucleotides for the immunoglobulin heavy chain (IgH) complementarity-determining region 3 (CDR3) and the T-cell receptor chain gene (TCR), as well as clone-specific CDR3 sequences in adult patients (aged 17–51 years) with acute lymphoblastic leukemia (ALL) who entered a complete hematological remission (CR) after chemotherapy with the German multicenter ALL (GMALL) protocol. The sensitivities were one in 10^2–3 for the CDR3- and TCR-PCR and one in 10^5–6 for a two-step, seminested CDR3/clone-specific PCR. At diagnosis, 7/7 patients were CDR3 positive and four were TCR positive in their bone marrow (BM). At the end of induction therapy (after 2 months) 4/6 tested positive for CDR3, 2/6 for TCR, and 5/6 for clone-specific rearrangements. At the end of consolidation treatment (after 7 months) only 1/7 remained positive for CDR3, 2/7 for TCR, and 5/7 for clone-specific rearrangements. After an observation period of 18–36 months, 4/7 patients were still in CR and all were PCR negative by the clone-specific method during or after maintenance therapy. Two patients died in leukemic relapse; one patient relapsed but is still alive. All three of these patients remained PCR positive throughout the course of their disease. Clonal evolution in the IgH locus was found in one of these patients. We conclude that the molecular response to chemotherapy in adult B-lineage ALL is slow, even in patients without risk factors other than age. As in childhood ALL, most patients with long-term CR convert to PCR negativity approximately 18 months after the start of chemotherapy. The data also suggest the existence of early clone-specific PCR negativity in a small proportion of long-term survivors. The predictive value of this observation will now have to be confirmed in a larger study.     

Next-generation sequencing-defined minimal residual disease before stem cell transplantation predicts acute myeloid leukemia relapse

In acute myeloid leukemia (AML), the assessment of post-treatment minimal residual disease (MRD) may inform a more effective management approach. We investigated the prognostic utility of next-generation sequencing (NGS)-based MRD detection undertaken before hematopoietic stem cell transplantation (HSCT). Forty-two AML subjects underwent serial disease monitoring both by standard methods, and a targeted 42-gene NGS assay, able to detect leukemia-specific mutant alleles (with >0.5% VAF) (mean 5.1 samples per subject). The prognostic relevance of any persisting diagnostic mutation before transplant (≤27 days) was assessed during 22.1 months (median) of post-transplant follow-up. The sensitivity of the NGS assay (27 MRD-positive subjects) exceeded that of the non-molecular methods (morphology, FISH, and flow cytometry) (11 positive subjects). Only one of the 13 subjects who relapsed after HSCT was NGS MRD-negative (92% assay sensitivity). The cumulative incidence of post-transplant leukemic relapse was significantly higher in the pre-transplant NGS MRD-positive (vs MRD-negative) subjects (P = .014). After adjusting for TP53 mutation and transplant conditioning regimen, NGS MRD-positivity retained independent prognostic significance for leukemic relapse (subdistribution hazard ratio = 7.3; P = .05). The pre-transplant NGS MRD-positive subjects also had significantly shortened progression-free survival (P = .038), and marginally shortened overall survival (P = .068). In patients with AML undergoing HSCT, the pre-transplant persistence of NGS-defined MRD imparts a significant, sensitive, strong, and independent increased risk for subsequent leukemic relapse and death. Given that NGS can simultaneously detect multiple leukemia-associated mutations, it can be used in the majority of AML patients to monitor disease burdens and inform treatment decisions.     

Monitoring minimal residual disease in pediatric patients with acute lymphoblastic leukemia

The level of minimal residual disease is an important prognostic factor in childhood acute lymphoblastic leukaemia. The end of induction therapy is the most significant time-point for prediction of treatment outcome. Within a pilot study covered by the Paediatric Haematology Working Group in the Czech Republic 51 childhood patients were analysed at diagnosis of acute lymphoblastic leukaemia and at the end of induction using method based on detection of clonal rearrangements of immuno-receptor genes. The majority of tested patients (32/51, 63%) had a low or non-detectable levels of residual disease, a group of patients with the highest levels and thus the highest risk of relapse included 10% of patients (5/51). Within each of three risk groups one patient has relapsed so far. Therefore, the relapse rate in particular subgroups is 3% (1/32), 7% (1/14) and 20% (1/5) to date, respectively. The results are compared with these published by the BFM group (van Dongen et al., Lancet 1998). The pilot phase of a new BFM treatment protocols includes examination of residual disease for stratification of patients into the different risk groups.     

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.     

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.     

Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia

By using rapid flow cytometric techniques capable of detecting one leukemic cell in 10(4) normal cells, we prospectively studied minimal residual disease (MRD) in 195 children with newly diagnosed acute lymphoblastic leukemia (ALL) in clinical remission. Bone marrow aspirates (n = 629) were collected at the end of remission induction therapy and at 3 intervals thereafter. Detectable MRD (ie, > or = 0.01% leukemic mononuclear cells) at each time point was associated with a higher relapse rate (P < .001); patients with high levels of MRD at the end of the induction phase (> or = 1%) or at week 14 of continuation therapy (> or = 0.1%) had a particularly poor outcome. The predictive strength of MRD remained significant even after adjusting for adverse presenting features, excluding patients at very high or very low risk of relapse from the analysis, and considering levels of peripheral blood lymphoblasts at day 7 and day 10 of induction therapy. The incidence of relapse among patients with MRD at the end of the induction phase was 68% +/- 16% (SE) if they remained with MRD through week 14 of continuation therapy, compared with 7% +/- 7% if MRD became undetectable (P = .035). The persistence of MRD until week 32 was highly predictive of relapse (all 4 MRD(+) patients relapsed vs 2 of the 8 who converted to undetectable MRD status; P = .021). Sequential monitoring of MRD by the method described here provides highly significant, independent prognostic information in children with ALL. Recent improvements in this flow cytometric assay have made it applicable to more than 90% of all new patients. (Blood. 2000;96:2691-2696)     

New markers for minimal residual disease detection in acute lymphoblastic leukemia

To identify new markers for minimal residual disease (MRD) detection in acute lymphoblastic leukemia (ALL), we compared genome-wide gene expression of lymphoblasts from 270 patients with newly diagnosed childhood ALL to that of normal CD19?CD10? B-cell progenitors (n = 4). Expression of 30 genes differentially expressed by ≥ 3-fold in at least 25% of cases of ALL (or 40% of ALL subtypes) was tested by flow cytometry in 200 B-lineage ALL and 61 nonleukemic BM samples, including samples containing hematogones. Of the 30 markers, 22 (CD44, BCL2, HSPB1, CD73, CD24, CD123, CD72, CD86, CD200, CD79b, CD164, CD304, CD97, CD102, CD99, CD300a, CD130, PBX1, CTNNA1, ITGB7, CD69, CD49f) were differentially expressed in up to 81.4% of ALL cases; expression of some markers was associated with the presence of genetic abnormalities. Results of MRD detection by flow cytometry with these markers correlated well with those of molecular testing (52 follow-up samples from 18 patients); sequential studies during treatment and diagnosis-relapse comparisons documented their stability. When incorporated in 6-marker combinations, the new markers afforded the detection of 1 leukemic cell among 10(5) BM cells. These new markers should allow MRD studies in all B-lineage ALL patients, and substantially improve their sensitivity.     

Allogeneic bone marrow transplantation (BMT) for adults with acute lymphoblastic leukemia (ALL): predictive role of minimal residual disease monitoring on relapse

We developed a PCR-based method to monitor clonogenic IgH VDJ rearrangement as a possible predictor of relapse in patients with acute B-ALL after allogeneic bone marrow transplantation (BMT). We studied 23 patients at diagnosis, before and after BMT. At the time of BMT, 13 patients were in first complete remission, eight in second complete remission and two in relapse. Four patients were PCR negative before BMT and remained PCR negative also after BMT (-/- pattern). They are still in remission after a median follow-up of 41 months. Nineteen patients were MRD-positive before BMT: three were PCR negative at first determination after BMT (+/- pattern) and maintain remission. Sixteen patients were PCR-positive at first determination after BMT (+/+ pattern): five became PCR negative (+/+/- pattern) (four with chronic graft-versus-host disease (GVHD) and two after donor lymphocyte infusions (DLI)). Nine patients remained PCR-positive (+/+/+ pattern) (four remain in remission, and six relapsed); two patients died before transplant. In conclusion, PCR negative patients before BMT remained negative post-BMT; many pre-BMT positive patients had initial MRD positivity after BMT: 37% of them achieved a molecular remission with cGVHD or DLI.     

Minimal residual disease analysis for the prediction of relapse in children with standard-risk acute lymphoblastic leukaemia

We report a largely retrospective analysis of minimal residual disease (MRD) in a cohort of 66 children suffering from acute lymphoblastic leukaemia (ALL). All patients lacked high-risk features at diagnosis, i.e. the presenting white cell count was <50 × 10⁹/l, age 1–16 years and translocations t(9;22) and t(4;11) were not present. All were treated according to either the MRC protocols UKALL X or XI. PCR of IgH, TCRδ and TCRγ gene rearrangements and allele-specific oligoprobing were employed for the detection of MRD. Sensitivity was at least 10⁻⁴ in 78/82 (93%) probes examined.
A total of 33 patients relapsed (seven on therapy and 26 off) and 33 remain in continuing complete remission (CCR) (median follow-up 69 months from diagnosis). Of those who remain in CCR, MRD was present in the bone marrow in 32%, 10% and 0% at 1, 3 and 5 months into therapy respectively. This is in marked contrast to the presence of MRD at these times in 82%, 60% and 41% of patients who relapsed ( P <0.001, P <0.005 and P <0.005). These results provide further evidence of a strong correlation between clearance of MRD early in therapy and clinical outcome in childhood ALL.     

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.