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.     

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)     

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.     

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.     

Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.     

Rituximab can be useful as treatment for minimal residual disease in bcr-abl-positive acute lymphoblastic leukemia

We report the results of administering CD20 monoclonal antibody (MoAb) in a 32-year-old man with bcr-abl-positive acute lymphoblastic leukemia. Morphological complete remission was achieved after two lines of chemotherapy with persistence of blast cells (2%) in flow cytometric analysis of marrow cells. Since no HLA-matched donor for allogeneic bone marrow transplantation (BMT) was found, anti-CD20 MoAb therapy was administered for in vivo marrow purging, prior to autologous peripheral blood stem cell (PBSC) harvest and transplantation. After MoAb therapy <0.1% of blast cells were observed and the molecular abnormality (bcr-abl gene rearrangement) disappeared.     

Detection of minimal residual disease in acute leukemia

A significant proportion of patients with acute leukemia who achieve remission subsequently experience frank relapse of their disease, and their ultimate prognosis is typically poor. Although such disease recurrences have been impossible to predict using standard laboratory techniques, new methods have been studied that identify patients destined to relapse. Sensitive polymerase chain reaction analyses of unique breakpoint fusion regions, and, in cases of acute lymphoblastic leukemia, patient-specific gene rearrangements have been used to detect minimal residual disease. Multiparameter flow cytometry has also been used to identify rare leukemia cells among populations of predominantly normal cells. Because these types of studies in pediatric acute lymphoblastic leukemia have convincingly demonstrated that patients with evidence of minimal residual disease during remission have a much higher incidence of relapse, therapeutic protocols have been initiated that intensify therapy for patients with minimal residual disease detected during remission.     

Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia

Adult patients with acute lymphoblastic leukemia (ALL) who are stratified into the standard-risk (SR) group due to the absence of adverse prognostic factors relapse in 40% to 55% of the cases. To identify complementary markers suitable for further treatment stratification in SR ALL, we evaluated the predictive value of minimal residual disease (MRD) and prospectively monitored MRD in 196 strictly defined SR ALL patients at up to 9 time points in the first year of treatment by quantitative polymerase chain reaction (PCR). Frequency of MRD positivity decreased from 88% during early induction to 13% at week 52. MRD was predictive for relapse at various follow-up time points. Combined MRD information from different time points allowed definition of 3 risk groups (P < .001): 10% of patients with a rapid MRD decline to lower than 10(-4) or below detection limit at day 11 and day 24 were classified as low risk and had a 3-year relapse rate (RR) of 0%. A subset of 23% with an MRD of 10(-4) or higher until week 16 formed the high-risk group, with a 3-year RR of 94% (95% confidence interval [CI] 83%-100%). The remaining patients whose RR was 47% (31%-63%) represented the intermediate-risk group. Thus, MRD quantification during treatment identified prognostic subgroups within the otherwise homogeneous SR ALL population who may benefit from individualized treatment.     

Monitoring of minimal residual disease in acute myeloid leukemia

Monitoring minimal residual disease (MRD) becomes increasingly important in the risk-adapted management of patients with acute myeloid leukemia (AML). The two most sensitive and quantitative methods for MRD detection are multiparameter flow cytometry (MFC) and real-time polymerase chain reaction (QRT-PCR). Fusion gene-specific PCR in AML is based on the RNA level, and thus in contrast to MFC expression levels rather than cell counts are assessed. For both methods independent prognostic values have been shown. The strong power of MFC has been shown mainly in the assessment of early clearance of the malignant clone. MRD levels in AML with fusion genes have the strongest prognostic power after the end of consolidation therapy. In addition, with QRT-PCR highly predictive initial expression levels can be assessed. With both methods early detection of relapse is possible. So far, validated PCR-based MRD was done with fusion genes that are detectable in only 20-25% of all AML MFC is superior since it is applicable for most AML. However, QRT-PCR is still more sensitive in most cases. Thus, it is desirable to establish new molecular markers for PCR-based studies. Large clinical trials will determine the role and place of immunologic and PCR-based monitoring in the prognostic stratification of patients with AML.     

Flow cytometric evidence for minimal residual disease and cytological heterogeneities in acute lymphoblastic leukemia with severe hypodiploidy

Two subpopulations of small and large leukemia cells and binucleated cells were present in the bone marrow of a 10-year-old girl with acute lymphoblastic leukemia (ALL). Cytogenetic studies showed some cells with a karyotype of 34,X,-X,-2,-3,-4,-5,-7,-9,-13,-15,-16,-17,-20, and others with a karyotype that was exactly double the chromosome set in the cells with 34 chromosomes. Flow cytometric (FCM) examination of surface common ALL antigen (CALLA) and DNA content of the lymphoblasts led to the identification of the primary hypodiploid DNA stemline (DI = 0.72), which corresponds to the small-sized blasts, and the secondary hyperdiploid DNA stemline (DI = 1.44), which corresponds to the large-sized blasts. Sequential bone marrow examinations with FCM and cytogenetics revealed the persistence of the primary hypodiploid clone during remission and their proliferation with chromosomal evolution at full relapse. These results suggest that more rational inductive therapy should be designed to achieve the favorable outcome of ALL with severe hypodiploidy and that FCM is a useful tool to monitor the minimal residual disease of this subgroup in ALL.     

Detection of minimal residual disease in acute lymphoblastic leukemia by flow cytometry with monoclonal antibodies.

To detect more precisely the minimal residual disease in acute lymphoblastic leukemia (ALL), two-color flow cytometric analysis for the detection of cell-surface antigen (CD10; CALLA) and nuclear terminal deoxynucleotidyl transferase (TdT) was performed in the six patients with CALLA-positive ALL coexpressing TdT. In all patients, the leukemic blasts coexpressed Ia (HLA-DR), CD9, CD19, CD20, CD24, and CD10. Five of six patients achieved complete remission, but one has so far relapsed. No leukemic blasts (CD10+, TdT+) were detected at the time of complete remission. During maintenance chemotherapy, leukemic blasts coexpressed C10 and TdT were found 2.32% in the patient's peripheral blood by two-color analysis, whereas no obvious leukemic cells were recognized morphologically. The patient relapsed leukemia with the same phenotype 4 weeks after the examination. On the basis of our findings, we suggest that two-color flow cytometric analysis with the use of these antibodies is quite valuable to detect the minimal residual leukemic cells in a patient with ALL. The reduction of leukemic cells below the threshold of detection of methods currently available appears to be necessary to achieve a cure in ALL. Hence accurate diagnosis of ALLs with monoclonal antibodies (MAbs) should contribute substantially to the development of an effective form of therapy for their cure.     

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.