Real-time quantitation of minimal residual disease in inv(16)-positive acute myeloid leukemia may indicate risk for clinical relapse and may identify patients in a curable state

Abstract

The inv(16) cytogenetic subtype of acute myeloid leukemia (AML) has a relatively good prognosis. Many patients achieve complete remission (CR). The prognostic uncertainty of negative qualitative reverse transcription-polymerase chain reaction (RT-PCR) assays suggests the need to identify prognostically significant critical thresholds by real-time RT-PCR. A reliable and sensitive (10 ؊5 ) real-time RT-PCR assay was set up for the evaluation of relevant CBF␤-MYH11/ABL transcript ratios and was applied to the 21 patients with inv(16) AML routinely referred for cytogenetic and molecular monitoring in Serà gnoli Institute (Bologna, Italy) since 1990. Among the 18 patients who underwent ablative chemotherapy, all achieved CR with a 3-year disease-free survival probability of 63% (95% CI, 40%-87%) and no recorded events after 26 months. Five patients had relapses; 2 died of disease and 3 entered second CR. Analysis of the 125 bone marrow (or peripheral blood) samples studied by real-time RT-PCR showed that transcript ratios of samples taken during CR at any time before a relapse were always greater than 0.12%, whereas those of samples taken during first or second CR from patients who did not subsequently have relapses were always less than 0.25%. This suggests that transcript ratios greater than 0.25% may correspond to high risk for relapse, whereas ratios below 0.12% might indicate the patient is in a curable state. IntroductionInversion of chromosome 16, inv(16)(p13q22), and its related translocation, t(16;16)(p13;q22), are recurrent rearrangements found in a subset of patients with acute myeloid leukemia (AML), particularly the M4Eo subtype. [1][2][3][4] Inv(16) is associated with relatively good long-term, disease-free survival (DFS). 5,6 It generates a chimeric mRNA transcript, CBF␤-MYH11, by fusing the CBF␤ gene located on band q22 of chromosome 16 7,8 and the MYH11 gene, which encodes a heavy chain of the smooth-muscle myosin protein, located at the p13 breakpoint. 9,10 Messenger RNA analysis by reverse transcription-polymerase chain reaction (RT-PCR) can help define leukemic subsets and provide potentially valuable diagnostic and prognostic information. 11 Qualitative RT-PCR reveals the presence of detectable levels of minimal residual disease (MRD) in patients in complete clinical remission (CR) after induction chemotherapy. 12 Introduction of real-time RT-PCR 13 should allow routine quantitation of the transcript. The 5Ј-3Ј nuclease activity of the Taq polymerase cleaves an internal fluorogenic probe specific for the target sequence, causing the emission of a fluorescent signal that can be detected during amplification. This allows rapid quantitation of specific RT-PCR products with a dynamic detection range of more than 5 orders of magnitude. Several groups have used real-time RT-PCR with TaqMan technology to quantitate MRD with leukemia-specific chromosome aberrations such as t(9;22), t(l5;17), and t(8;21). [14][15][16] Most patients with inv(16) AML treated in our institute achieved long-term CR. 17,18 Ho...

Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement

“…Interestingly, a recent RQ-RT-PCR study on adults positive for CBFB/MYH11 [inv (16)] has shown that most of the patients (4/5) with more than 100 normalized CBFB/MHY11 copies after induction had a relapse, whereas patients below this level never relapse. 41 Similar results were reported from Buonamici et al 42 All four relapse patients of our study were randomized in different therapy trials. In three of them an increase of the AML1/ ETO fusion transcript became obvious 9 weeks (pt.…”

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

“…Quantitative RT-PCR studies using competitive PCR or RQ-PCR enabled monitoring of the decrease in CBFB-MYH11 FG transcripts during early phases of induction and consolidation therapies. 33,45,46,176,177 However, due to the low number of patients so far examined, it was not possible to define a kinetic or a cutoff level for predicting relapse. 178 …”

“…4 Such favorable results with conventional chemotherapy led some authors to consider that allo-BMT is not indicated to consolidate first CR in these patients, even when a suitable donor is available. 46,172,173 Nevertheless, the relapse rate is still high indicating that reliable methods to detect MRD during hematologic CR are needed in order to better adapt the intensity of postremission therapy to specific cohorts of patients. So far, the use of qualitative RT-PCR-based methods employed to detect CBFB-MYH11 FG transcripts did not allow consistent Table 25 Expression values of the PML-RARA FG transcript in NB-4 cell line and bcr1 patients at diagnosis (phase IV) Comparison of PML-RARA FG transcript expression between BM and PB at diagnosis using either ABL, B2M or GUS as CG.…”

“…35,36 There have been numerous manuscripts from individual laboratories demonstrating the reliability of this technology and its potential clinical value for MRD studies using FG transcripts as PCR targets such as BCR-ABL in CML [37][38][39] or ALL, 40 PML-RARA, 41 AML1-ETO [42][43][44] and CBFB-MYH11 45,46 in AML and TEL-AML1 [47][48][49] in ALL patients. However, standardized RQ-PCR procedures are warranted in order to apply this innovative technology for large-scale MRD studies within multicenter therapeutic trials.…”

Prognostic impact of RT-PCR-based quantification of WT1 gene expression during MRD monitoring of acute myeloid leukemia

“…Recently, molecular monitoring of minimal residual disease was shown to early identify patient groups at high risk of relapse and therefore provide a window for therapeutic intervention. [1][2][3] Monitoring of MRD is mostly based on quantitative PCR. Common targets include specific fusion transcripts, for example, AML1-ETO, CBFB-MYH11, PML-RARA, and MLL gene fusions.…”