Leukemia-associated Aberrant Immunophenotype Research Articles

Use of 5-Fold Staining for Multiparameter Flow Cytometry-Based Quantification of Minimal Residual Disease in Acute Myeloid Leukemia Results in Improved Sensitivity.

The quantification of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) using triple staining has been shown to yield prognostic information independent of other parameters in patients with acute myeloid leukemia (AML). Due to the immunophenotypic heterogeneity of AML the application of 5-fold staining may result in a better characterization of the leukemia-associated aberrant immunophenotype (LAIP) and thus in an improved sensitivity of the method as compared to triple staining. We analyzed bone marrow samples from 114 patients with newly diagnosed and untreated AML by MFC using a comprehensive antibody panel with 5-fold combinations. Sensitivity was estimated by quantification of LAIP-positive cells for each LAIP in 18 normal bone marrow samples. In each patient at least one LAIP was identified (total, 203 LAIPs). The LAIPs were present on a median of 15.88% of the bone marrow cells at diagnosis (range, 2.11% to 79.64%). The median number of normal bone marrow cells displaying the LAIPs ranged from 0.001% to 0.065% (median, 0.010%). As a result, the logarithmic difference (LD) in LAIP-positive cells between leukemic and normal bone marrow amounted to a median of 3.33 (range, 1.96 to 4.88). Similarly, if only the most sensitive LAIP was considered for each patient the median frequencies of LAIP-positive cells were 14.07% (range, 2.11% to 77.57%) in leukemic bone marrow and 0.010% (range, 0.001% to 0.065%) in normal bone marrow. Importantly, however, in this setting the resulting LD amounted to a median of 3.45 (range, 1.96 to 4.88). In order to estimate the impact of applying 5-fold staining on the sensitivity the information of each of the applied colors was skipped once while the results of the other four colors, respectively, were used. Skipping one color resulted in an increase of LAIP-positive normal bone marrow cells (median, 0.050%; range, 0.001% to 3.6%) while the percentages of LAIP-positive leukemic cells changed only marginally (median, 22.65%; range, 2.25% to 90.06%). The gain in LD by applying 5-fold staining in comparison to 4-fold staining amounted to a median of 0.58 (maximum gain, 3.14). In 32 patients a total of 120 follow-up samples have been analyzed appyling the combination of antibodies that allowed the best LAIP definition. The LD from diagnosis to follow-up amounted to a median of 2.82 (range, 0.77 to 4.82). Clinical follow-up data is available in 26 of these 32 patients. MRD assessment after completion of consolidation therapy has been performed in 15 patients. The median LD between diagnosis and follow-up assessment is 2.84 (range, 1.07 to 4.33). Separating patients according to this median LD identified a group of patients with no relapses yet (LD >2.84) while patients with an LD <2.84 had an event-free survival of only 50% at one year (p=0.075). These data confirm that flow cytometrically-based assessment of MRD is feasible in AML and results in prognostic information. It is suggested that the application of 5-fold staining significantly improves the sensitivity and thereby the overall accuracy of the method.

Different Minimal Residual Disease Levels between Childhood Acute Lymphoblastic Leukemia and Acute Myeloid Leukemia Detected with Multi-Parameter Flow Cytometry.

Minimal residual diseases (MRD) are the problem of disease recurrence for childhood acute leukemia. Different levels of MRD determined with multi-parameter flow cytometry (MP-FCM) have been revealed prognostic for both adult and childhood acute leukemia in several recent studies. However, the MRD levels at the time of complete remission and the various check point during further treatment have not been reported.Methods: 122 cases (Male 73, Female 49, aged 2 month to 15yrs with a median of 6.5yrs) with childhood acute leukemia treated in our hospital from 2001.3 ~ 2004.7 were enrolled into this study. A total of 610 BM samples were detected with three or four color MP-FCM based on individualized leukemia-associated aberrant immunophenotypes (LAIP) in each patient according to their initial complete immunophenotypic profiles (B lineage ALL: 79 cases, T lineage ALL: 11 cases; AML: 32 cases). A total of 99 LAIPs (B lineage ALL: 48, T lineage ALL: 16, AML: 31, HAL: 4) were identified and single LAIP was used in most patients, but some patients needed more than one (2 or 3) LAIPs to detect their MRDs. At least 100,000 events were collected for each analysis. ALL and AML patients were treated with Chinese Childhood Cancer Study Group (CCCSG) ALL-protocol-1998 and AML-protocol-1998, respectively.Results: The CR rates for B lineage ALL, T lineage ALL and AML were 99.3%, 94.7% (overall ALL 98.8%) and 89.1%, respectively. The 1 yr/2 yrs of event free survival (EFS) for the three types of leukemia was 92.5%/85.2%, 78.3%/68.9% and 68.3%/56.4%, respectively. At the time of first CR after induction therapy, MRD level of B lineage ALL (79 cases) was 0.166%±0.057% (mean ± SE), significantly lower than those of T lineage ALL (11 cases, 0.783%±0.328%, t = 3.1926, P = 0.002) and AML (32 cases, 1.191%±0.257, t = 5.5177, P < 0.00001), respectively. No significance was identified between the MRD levels of T lineage ALL and AML (t = 0.8488, P = 0.40). The MRD level of the overall ALL (T + B lineage: 90 cases) was also significantly lower than that of AML (32 cases) (t = 5.0249, P < 0.00001). With further detection of MRD following the continuous consolidation and maintenance therapy, some patients relapsed and were excluded the study, and some had not reached the time for detection. At the time of 6 months after first CR, the MRD level of B lineage ALL (53 cases, 0.224%±0.065%) was not statistically different from that of T lineage ALL (7 cases, 0.627% ± 0.276%, t = 1.767, P = 0.083), while it was significantly higher than that of AML (16 cases, 0.966%±0.347%, t =3.334, P = 0.0014). At the time of 1 yr after CR, the MRD level in ALL (50 cases, 0.347%±0.090%) was not significantly different from that of AML (7 cases, 0.094%±0.072, t = 1.043, P = 0.31). At the time of 2 yrs after CR, the MRD level in ALL (49 cases, 0.302%±0.105%) was also not significantly different from that of AML (8 cases, 0.485%±0.246%, t = 0.6599, P = 0.51).Conclusions: MRD level of B lineage ALL is significantly lower than those of T lineage ALL and AML at the time of first CR, being true at the time of 6 months after CR. The differences are lost at 12 and 24 months after CR indicating that MRD detection with MP-FCM are well associated with the prognosis of childhood acute leukemia. Continuous follow-up to accumulate more cases is now underway.

Minimal Residual Disease in Patients with Acute Myeloid Leukemia Quantified by Multiparameter Flow Cytomety and Quantitative RT-PCR Is an Independent Prognostic Parameter.

Quantification of minimal residual disease (MRD) is becoming increasingly important to guide therapy in patients with acute myeloid leukemia (AML). While MFC can be applied to more patients with AML than QPCR, the latter has the advantage of a higher sensitivity in many cases. We compared data obtained by both methods in parallel in bone marrow samples in 160 patients at diagnosis and at 469 follow-up checkpoints. MFC was applied at diagnosis with a comprehensive panel of antibodies to identify leukemia-associated aberrant immunophenotypes (LAIP) useful for MRD monitoring. QPCR targeted on the leukemia-specific fusion transcripts AML1-ETO, AML1-EVI1, CBFB-MYH11, MLL-AF10, MLL-AF6, MLL-AF9, MLL-ELL, MLL-ENL, and PML-RARA as well as overexpression of EVI1, length mutations of FLT3, and partial tandem duplications of MLL. In order to adjust for differences in the percentages of bone marrow cells covered by the respective LAIP by MFC at diagnosis and for the heterogeneity of transcript levels detected by QPCR at diagnosis, the logarithmic difference (LD) was calculated for each follow-up sample in comparison to the diagnostic sample. There was a significant correlation between MFC and QPCR with regard to the LD from diagnosis to follow-up checkpoint (r=0.645, p=0.000001). Concordant results with regard to negativity between QPCR (no signal) and MFC (&lt;0.01% positive cells) was found in 301/469 (64.2%) samples (both methods positive, 270 (57.6%); both methods negative, 31 (6.6%)). In 44 samples (9.4%) QPCR detected positivity and MFC negativity while in 124 samples (26.4%) MFC detected positivity and QPCR negativity (sensitivity of QPCR was lower than 1:100,000 in some cases). In 133 patients clinical follow-up data was available allowing the analysis of the prognostic impact of MRD levels. Cytogenetics were favorable, intermediate, and unfavorable in 86, 30, and 17 cases, respectively. Median age was 46 years (range, 17–83). Median event-free survival (EFS) was 22.1 months, overall survival (OS) at three years was 77%. The median LDs for MFC and QPCR at the checkpoint 1 (up to day 21), 2 (day 22–60), 3 (day 61–120), 4 (day 121–365), and 5 (after day 365) were 2.40 and 0.62, 2.05 and 1.55, 2.51 and 3.34, 2.71 and 3.70, and 2.60 and 3.45, respectively. Separating patients according to these median LDs resulted in a better EFS and OS for cases with higher LDs at all five checkpoints for each method. Significant differences in EFS were observed at checkpoints 2 (MFC, 22.1 vs. 12.6 months, p=0.0379; QPCR, median not reached vs. 9.9, p=0.0081), 3 (QPCR, 30.9 vs. 14.1 months, p=0.0011), 4 (MFC, median not reached vs. 16.9 months, p=0.0007; QPCR, median not reached vs. 15.1 months, p=0.0102), and 5 (QPCR, median not reached vs. 17.2, p=0.0008). Cox regression analysis taking into consideration cytogenetics, age, WBC count, and bone marrow blast count at diagnosis identified the LD at checkpoint 4 determined by MFC and the LD at checkpoints 2 and 5 determined by QPCR as independent prognostic factors. The results of our analyses confirm that both MFC and QPCR are highly sensitive methods capable of quantifying MRD in AML. While data are concordant for both methods in many cases, either of the two has advantages in distinct cases depending on the individual MRD marker. Clinical trials should consider MRD monitoring by both methods in order to prove their respective roles in risk prediction and treatment stratification.

Determination of Minimal Residual Disease in Patients with Acute Myeloid Leukemia by Multiparameter Flow Cytometry: Prognostic Impact at Different Checkpoints.

Guiding antileukemic treatment in patients with acute myeloid leukemia (AML) is increasingly based on levels of minimal residual disease (MRD) which can be quantified with high sensitivity by multiparameter flow cytometry (MFC). The optimum checkpoint for determination of MRD during the course of therapy, however, has not yet been determined. We applied MFC using a comprehensive panel of antibodies to identify leukemia-associated aberrant immunophenotypes (LAIPs) at diagnosis and to quantify MRD by individually selected antibody combinations. The prognostic impact of MRD levels was assessed in comparison to cytogenetics and age. Patients received double induction, consolidation, and maintenance therapies and underwent allogeneic stem cell transplantation if they were younger than 60 years and had a matched related donor. In 286 patients with newly diagnosed and untreated AML MFC-based assessment for the presence of LAIP has been performed. The median percentage of LAIP-positive bone marrow cells at diagnosis was 16.04% (range, 2.54%–76.14%). All individual LAIPs were applied to 26 normal bone marrow samples to estimate sensitivity based on the median percentages of LAIP-positive normal bone marrow cells which ranged from 0.00% to 1.01% (median, 0.02%). A total of 550 follow-up samples has been analyzed in these patients at different checkpoints (CP1, up to day 21 after start of therapy, n=85; CP2, day 22–60, n=122; CP3, day 61–120, n=158; CP4, day 121–365, n=137; CP5, after day 365, n=48). In order to adjust for differences in the percentages of LAIP-positive bone marrow cells at diagnosis the logarithmic difference (LD) between diagnosis and follow-up was calculated for each follow-up sample. The median LDs at the respective checkpoints were: CP1, 2.02; CP2, 2.29; CP3, 2.39; CP4, 2.53; and CP5, 2.81. Separation of patients according to the respective median LDs resulted in differences in event-free survival (EFS; CP1: 21.1 vs. 9.1 months, p=0.0711; CP2: 14.2 vs. 9.3 months, p=0.0095; CP3: 30.9 vs. 13.5 months, p=0.0055; CP4: median not reached vs. 14.1 months, p<0.0001; CP5: median not reached vs. 22.5 months, p=0.0001) and overall survival (OS; CP3: median not reached vs. 21.6 months, p=0.0332; CP4: 90% vs. 53% at 2 years, p=0.0058). Cox analysis using the LDs at the different checkpoints as continuous variables confirmed the prognostic impact on EFS (CP2, p=0.002; CP3, p=0.0003; CP4, p<0.0001; CP5, p<0.0001) and revealed an impact also on OS (CP3, p=0.003; CP4, p=0.001; CP5, p=0.029). Cox regression analysis taking into consideration cytogenetics and age as covariates proved the independent prognostic impact of LD at checkpoints 2 to 5 on both EFS and OS with the exception of LD at checkpoint 2 and OS. In fact, LD at checkpoint 5 was the only parameter independently related to EFS and OS. These data suggest that quantification of MRD by MFC in AML results in powerful and independent prognostic parameters. In particular during the first year of treatment MRD levels provide important prognostic information. Clincal trials should use MRD-based stratification in order to assess the efficacy of early treatment intensification in high-risk AML patients.

Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia

AbstractQuantification of minimal residual disease (MRD) reveals significant prognostic information in patients treated for acute myeloid leukemia (AML). The application of multiparameter flow cytometry (MFC) for MRD assessment has resulted in significant prognostic information in selected cases in previous analyses. We analyzed MRD in unselected patients with AML in complete remission (CR) after induction (n = 58) and consolidation (n = 62) therapies. By using a comprehensive panel of monoclonal antibodies we identified at least one leukemia-associated aberrant immunophenotype (LAIP) in each patient. The degree of reduction between diagnosis and CR in LAIP-positive cells (log difference [LD]) as a continuous variable was significantly related to relapse-free survival (RFS) both after induction (P = .0001) and consolidation (P = .000 08) therapies, respectively. The LD determined after consolidation therapy was the only parameter related to overall survival (OS) (P = .005). Separation of patients based on the 75th percentile of LD after consolidation therapy resulted in groups with highly different RFS (83.3% versus 25.7%, P = .0034) and OS (87.5% versus 51.4%, P = .0507) at 2 years. Multivariate analysis identified LD as an independent prognostic factor for RFS at both checkpoints. MFC-based quantification of MRD reveals important prognostic information in unselected patients with AML in addition to cytogenetics and should be further evaluated and used in clinical trials.

Stability of leukemia-associated aberrant immunophenotypes in patients with acute myeloid leukemia between diagnosis and relapse: comparison with cytomorphologic, cytogenetic, and molecular genetic findings.

Multiparameter flow cytometry is increasingly used to monitor minimal residual disease in patients with acute myeloid leukemia to identify leukemic cells by leukemia-associated aberrant immunophenotypes (LAIPs). Changes in LAIPs during the course of the disease may be a limitation for this approach. We analyzed 49 patients at diagnosis and relapse by flow cytometry, cytomorphology, cytogenetics, and molecular genetics. In 37 patients (76%), at least one LAIP detectable at diagnosis was present at relapse; in 12 patients (24%), none of the original LAIPs were present in at least 1% of bone marrow cells. Three groups were identified: no change in LAIPs, partial changes in LAIPs, and complete change in LAIPs. There were significant differences across these groups with regard to changes in cytomorphology (11%, 40%, and 58% of all cases, respectively; P = 0.007), cytogenetics (15%, 20%, and 25%; not significant), and molecular genetics (18%, 0, and 86%; P = 0.002). These data indicate that, in a subset of patients with acute myeloid leukemia, the disease is biologically different at relapse; therefore, monitoring of minimal residual disease is difficult to accomplish. In most patients with acute myeloid leukemia, multiparameter flow cytometry may be used to monitor minimal residual disease.