Monitoring of minimal residual disease in acute myeloid leukemia Wolfgang Kern, Claudia Schoch, Torsten Haferlach, Susanne Schnittger  Critical Reviews in Oncology / Hematology  Volume 56, Issue 2, Pages 283-309 (November 2005) DOI: 10.1016/j.critrevonc.2004.06.004 Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Leukemia treatment outcome. The red line indicates a sequential reduction of the leukemic cell mass with the exception of a slight increase before the second consolidation therapy. Eventually, cure is achieved. The green lines indicate cases with relapses of AML in which increasing MRD levels are present before relapse. The light blue area refers to the cytomorphologic finding of 1–5% bone marrow blasts which is compatible with complete remission. The intermediate blue area refers to a 0% bone marrow blasts count with MRD levels detectable by MFC or QRT-PCR. The dark blue area refers to a 0% bone marrow blasts count with MRD levels below the sensitivity of MFC and QRT-PCR. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Four different classes of leukemia-associated aberrant immunophenotypes. First and second plots are leukemic bone marrow; third and fourth plots are normal bone marrow; second and fourth plots display only cells gated in first and third plots (green cells); green dots represent cells with immunophenotype as defined in AML by first two antigens, red dots represent cells with immunophenotype as defined in AML by third antigen and OLS-signal; A (cross-lineage expression): CD33+CD34+ and CD7+SSClow; B (overexpression): CD45++CD64++ and CD4+SSClow; C (lack of expression): CD34+CD33+ and HLA-DR−SSClow; D (asynchronous expression): CD34+CD117+ and CD11b+SSClow. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Prognostic impact of MRD at day 16 as quantified by multiparameter flow cytometry. Cases with an MRD level at day 16 lower than the median at day 16 have a better event-free survival than patients with an MRD level higher than the median (53% at 2 years vs. a median of 2.8 months, p<0.0001). Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Prognostic impact of MRD after consolidation therapy as quantified by multiparameter flow cytometry. Cases with an MRD level after consolidation therapy lower than the 75%-percentile after consolidation therapy have a better event-free survival than patients with an MRD level higher than the 75%-percentile (event-free survival at 2 years: 83.3% vs. 25.7%, p=0.0034). Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Improvement of separation of populations by CD45 gating. (A) Normal bone marrow (left: FLS-OLS plot, right: CD45-OLS plot) and (B) AML bone marrow (left: FLS-OLS plot, right: CD45-OLS plot). G, granulocytes; M, monocytes; L, lymphocytes; E, erythrocytes; B, blasts. CD45-OLS gating allows to isolate bone marrow blasts from all other populations which is not possible by FLS-OLS gating. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 Exponential amplification during PCR. In a standard or nested PCR reaction the detection is performed after completion of the reaction when PCR has reached the plateau-phase (endpoint analysis). Real-time PCR allows the quantitative detection during the logarithmic amplification phase (log-phase analysis). Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 7 Schematic presentation of the three main types of RQ-PCR techniques. The four most relevant PCR phases (annealing; early, late, and end of extension) for fluorescence detection are depicted. Arrows indicate forward and reverse primers of the reaction. Yellow jagged arrows indicate the time point of fluorescence detection. By use of the hydrolysis and SYBR green format the detection is in the end of the annealing phase. By use of the hybridization probe format detection is performed in the annealing phase. R, reporter dye; Q, quencher dye; D, fluorescence donor dye; A, fluorescence acceptor dye; FRET, fluorescence resonance energy transfer. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 8 Quantification by real-time PCR. (a) Amplification of defined plasmid dilutions containing the target to be quantified. (b) Standard curve that is constructed from the plasmid dilution. (c) Limited dilution series of a fusion gene positive in a fusion gene negative sample, indicating the sensitivity of the assay. (d) Assessment of the ABL control gene in the same samples as depicted in “c”. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 9 Molecular fusion subtypes. Molecular fusion subtypes of (a) PML-RARA and (b) CBFB-MYH11 due to different exon usage. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 10 Prognostic relevance of fusion gene expression levels at diagnosis. Overall survival (OS) and event-free survival (EFS) for patients separated according to the 75%-percentile of the expression level of the respective fusion transcript (PML-RARA, AML1-ETO, CBFB-MYH11) at diagnosis. Patients with a lower level (blue lines) have a better prognosis than those with a higher level (red lines). Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 11 Prognostic impact of MRD after consolidation therapy as quantified by quantitative RT-PCR. Patients with MRD levels after consolidation therapy lower than the median MRD level after consolidation therapy (blue line) have a better prognosis than patients with MRD levels higher than the median (red line). Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 12 Combined prognostic impact of expression levels at diagnosis and MRD levels. Patients with both a low transcript level at diagnosis and a low MRD level after consolidation therapy (blue lines) have a better prognosis than patients not fulfilling both criteria (red lines). Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 13 Semiquantitative and quantitative analysis of FLT3-LM. (a) Semiqualitative conventional agarose gel electrophoresis. The wildtype allele (WT) is the smallest fragment. All that are larger than this fragment indicate FLT3-LM. Patients P1, P5, and P6 are positive for a FLT3-LM. P1 in addition has a loss of the WT-allele. –C, H2O control; M, molecular weight marker. (b) Fragment analysis for quantification ot the FLT3-LM. In red the standard; in blue the FLT3-WT and the FLT3-LM. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 14 Dilution series of MLL-PTD positive AMLs in normal control samples. (a) exon9/exon3 fusion type and (b) exon11/exon3 fusion type. The sensitivity is 1:1000 to 1:10,000. In the highly diluted samples, the MLL-PTD expression is like in normal controls. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 15 Examples of follow-up with MLL-PTD in three patients. In pink, a resistant patient. In blue, a case with bad response to standard chemotherapy that underwent bone marrow transplantation. In yellow, a good responder. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 16 Scattergram of EVI1 expression in different cytogenetic AML subtypes. Scattergram of EVI1 expression in different AML subtypes. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 17 Correlation of MRD quantification as determined by MFC and QRTPCR in AML cases with t(11q23). The logarithmic difference between measurements at diagnosis and at follow-up for both multiparameter flow cytometry (FC log-difference) and quantitative RT-PCR (PCR log-difference) are plotted against each other. Pearson correlation (r=0.719, p=0.003) indicates a very good quantitative agreement between both methods. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions

Fig. 18 Correlation of courses of MRD. MRD monitoring by flow cytometry and PCR. Green lines show measurements by multiparameter flow cytometry, red lines show measurements by quantitative RT-PCR. The respective markers are given. Critical Reviews in Oncology / Hematology 2005 56, 283-309DOI: (10.1016/j.critrevonc.2004.06.004) Copyright © 2005 Elsevier Ireland Ltd Terms and Conditions