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Primer and probe designs. Schematic presentation of primer and hybridization probe design for quanti fi cation of (A) AML1-ETO , (B) PML-RARA , and (C) CBFB-MYH11 fusion transcript types. Arrows to the right are forward primers; arrows to the left are reverse primers.
Source publication
To evaluate the prognostic significance of quantitative PML-RARA, AML1-ETO, and CBFB-MYH11 fusion transcript expression, real-time polymerase chain reaction was used to analyze bone marrow samples of 349 such patients at diagnosis and 522 samples of 142 patients also during therapy (total analyses, n = 859; median number of follow-up samples, 4/pat...
Contexts in source publication
Context 1
... quantification of the respective fusion gene in the individual sample, real-time PCR using the LightCycler technology was performed (Roche Diagnos- tics). All primers and hybridization probes used for this study are given in Table 1 and Figure 1. For AML1-ETO the forward primer was positioned to AML1 exon 4, whereas the reverse primer and the hybridization probes were positioned to exon 3 of the ETO gene ( Figure 1A). ...
Context 2
... primers and hybridization probes used for this study are given in Table 1 and Figure 1. For AML1-ETO the forward primer was positioned to AML1 exon 4, whereas the reverse primer and the hybridization probes were positioned to exon 3 of the ETO gene ( Figure 1A). PML-RARA transcript quantification was performed with forward primers in PML exon 6 for bcr1 (L-transcript) or in exon 3 for bcr3 (S-transcript) and reverse primers and the hybridization probes in RARA exon 3 ( Figure 1B). ...
Context 3
... AML1-ETO the forward primer was positioned to AML1 exon 4, whereas the reverse primer and the hybridization probes were positioned to exon 3 of the ETO gene ( Figure 1A). PML-RARA transcript quantification was performed with forward primers in PML exon 6 for bcr1 (L-transcript) or in exon 3 for bcr3 (S-transcript) and reverse primers and the hybridization probes in RARA exon 3 ( Figure 1B). Patients with bcr2 breakpoints were excluded from this study. ...
Context 4
... all CBFB-MYH11-specific sets, the forward primers, the 5FL, and the 3LCred probe were positioned in exon 4 of the CBFB gene. Only the reverse primers were variably positioned to MYH11 exons 7, 8, 9, 10, 11, and 12 to produce PCR fragments of similar sizes and to obtain similar PCR sensitivities for each fusion transcript type ( Figure 1C). ...
Context 5
... bone marrow cells were obtained by Ficoll Hypaque density gradient centrifugation. Total RNA was extracted from 10 7 cells with RNeasy (Qiagen; 1997-2000) or mRNA with the MagnaPureLC mRNA Kit I (Roche Diagnostics, Mannheim, Germany; since January 2001). The cDNA-synthesis of 1 to 2 g total RNA or mRNA from an equivalent of 10 7 cells was performed using 300 U Superscript II (Gibco BRL/Invitrogen, Karlsruhe, Germany) and random hexamer primers (Pharmacia, Freiburg, Germany). PCRs for AML1-ETO, CBFB-MYH11, or PML-RARA fusion transcripts were performed as previously described. 13,51 For each sample a cABL speci fi c RT-PCR was performed to control the integrity of RNA using primers abl5 Ј : 5 Ј -GGCCAGTAGCATCTGACTTTG-3 Ј and abl3 Ј 5 Ј - ATGGTACCAGGAGTGTTTCTCC-3 Ј . Strict precautions were taken to prevent contamination. Water instead of cDNA was included as a blank sample in each experiment. Ampli fi cation products were analyzed on 1.5% agarose gels stained with ethidium bromide. For quanti fi cation of the respective fusion gene in the individual sample, real-time PCR using the LightCycler technology was performed (Roche Diagnostics). All primers and hybridization probes used for this study are given in Table 1 and Figure 1. For AML1-ETO the forward primer was positioned to AML1 exon 4, whereas the reverse primer and the hybridization probes were positioned to exon 3 of the ETO gene (Figure 1A). PML-RARA transcript quanti fi cation ...
Citations
... In recent years, many studies have developed risk-scoring systems to assess the prognosis of AML. One example is the score established by Schnittger et al. (13), which is based on the expression level of fusion genes. Their findings indicate that fusion transcript levels at diagnosis have a significant impact on the overall survival (OS) and event-free survival (EFS) of AML. ...
Background
Fusion genes are considered to be one of the major drivers behind cancer initiation and progression. Meanwhile, non-acute promyelocytic leukemia (APL) pediatric patients with acute myeloid leukemia (AML) in children had limited treatment efficacy. Hence, we developed and validated a simple clinical scoring system for predicting outcomes in non-APL pediatric patients with AML.
Method
A total of 184 non-APL pediatric patients with AML who were admitted to our hospital and an independent dataset (318 patients) from the TARGET database were included. Least absolute shrinkage and selection operation (LASSO) and Cox regression analysis were used to identify prognostic factors. Then, a nomogram score was developed to predict the 1, 3, and 5 years overall survival (OS) based on their clinical characteristics and fusion genes. The accuracy of the nomogram score was determined by calibration curves and receiver operating characteristic (ROC) curves. Additionally, an internal verification cohort was used to assess its applicability.
Results
Based on Cox and LASSO regression analyses, a nomogram score was constructed using clinical characteristics and OS-related fusion genes ( CBFβ::MYH11 , RUNX1::RUNX1T1 , KMT2A::ELL , and KMT2A::MLLT10 ), yielded good calibration and concordance for predicting OS of non-APL pediatric patients with AML. Furthermore, patients with higher scores exhibited worse outcomes. The nomogram score also demonstrated good discrimination and calibration in the whole cohort and internal validation. Furthermore, artificial neural networks demonstrated that this nomogram score exhibits good predictive performance.
Conclusion
Our model based on the fusion gene is a prognostic biomarker for non-APL pediatric patients with AML. The nomogram score can provide personalized prognosis prediction, thereby benefiting clinical decision-making.
... Long-standing evidence suggests that early detection of measurable residual disease (MRD) by identifying leukemia-associated immunophenotypes (LAIP) by flow cytometry, and fusion transcripts as well as gene expression (e.g., WT1) by quantitative RT-PCR, are able to predict relapse in patients with acute myeloid leukemia (AML) [4][5][6]. Recent technological advances to detect MRD after allo-HSCT such as multicolor flow cytometry, the introduction of droplet digital PCR (ddPCR) and next-generation sequencing (NGS), have resulted in improved MRD detection [7][8][9][10]. As recently reported by an expert panel, MRD detection in AML is based either on PCR amplification of leukemia-associated targets or on flow cytometric detection of LAIPs. ...
Relapse of the underlying disease is a frequent complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we describe the clinical utility of measurable residual disease (MRD) and mixed chimerism (MC) assessment in circulating cell-free DNA (cfDNA) analysis to detect earlier relapse in patients with hematological malignancies after allo-HSCT. A total of 326 plasma and peripheral blood mononuclear cell (PBMCs) samples obtained from 62 patients with myeloid malignancies were analyzed by droplet-digital PCR (median follow-up: 827 days). Comparison of MC in patients at relapse and in complete remission identified an optimal discriminating threshold of 18% of recipient-derived cfDNA. After performing a targeted next-generation sequencing (NGS) panel, 136 mutations in 58 patients were detected. In a total of 119 paired samples, the putative mutations were detected in both cfDNA and PBMCs in 73 samples (61.3%). In 45 samples (37.8%) they were detected only in cfDNA, and in only one patient (0.9%) were they detected solely in DNA from PBMCs. Hence, in 6 out of 23 patients (26%) with relapse after allo-HSCT, MRD positivity was detected earlier in cfDNA (mean 397 days) than in DNA derived from PBMCs (mean 451 days). In summary, monitoring of MRD and MC in cfDNA might be useful for earlier relapse detection in patients with myeloid malignancies after allo-HSCT.
... In previous studies, molecular genetic aberrations have become important approaches for minimal residual disease (MRD) detection for AML and MDS. Especially, the polymerase chain reaction (PCR)-based gene detection has been proven to be an effective MRD monitoring method for AML patients [5][6][7]. However, more than half of AML cases still lack effective specific MRD molecular markers [5]. ...
Background
The mixed-lineage leukemia ( MLL ) gene is located on chromosome 11q23. The MLL gene can be rearranged to generate partial tandem duplications (MLL-PTD), which occurs in about 5-10% of acute myeloid leukemia (AML) with a normal karyotype and in 5-6% of myelodysplastic syndrome (MDS) patients. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently one of the curative therapies available for AML and MDS with excess blasts (MDS-EB). However, how the prognosis of patients with high levels of MLL -PTD after allo-HSCT, and whether MLL -PTD could be used as a reliable indicator for minimal residual disease (MRD) monitoring in transplant patients remains unknown. Our study purposed to analyze the dynamic changes of MLL -PTD peri-transplantation and the best threshold for predicting relapse after transplantation.
Methods
We retrospectively collected the clinical data of 48 patients with MLL -PTD AML or MDS-EB who underwent allo-HSCT in Peking University People’s Hospital. The MLL -PTD was examined by real-time quantitative polymerase chain reaction (RQ-PCR) at the diagnosis, before transplantation and the fixed time points after transplantation. Detectable MLL -PTD/ABL > 0.08% was defined as MLL -PTD positive in this study.
Results
The 48 patients included 33 AML patients and 15 MDS-EB patients. The median follow-up time was 26(0.7-56) months after HSCT. In AML patients, 7 patients (21.2%) died of treatment-related mortality (TRM), 6 patients (18.2%) underwent hematological relapse and died ultimately. Of the 15 patients with MDS-EB, 2 patients (13.3%) died of infection. The 3-year cumulative incidence of relapse (CIR), overall survival (OS), disease-free survival (DFS) and TRM were 13.7 ± 5.2, 67.8 ± 6.9, 68.1 ± 6.8 and 20.3% ± 6.1%, respectively. ROC curve showed that post-transplant MLL -PTD ≥ 1.0% was the optimal cut-off value for predicting hematological relapse after allo-HSCT. There was statistical difference between post-transplant MLL -PTD ≥ 1.0% and MLL -PTD < 1.0% groups (3-year CIR: 75% ± 15.3% vs. 0%, P < 0.001; 3-year OS: 25.0 ± 15.3% vs. 80.7% ± 6.6%, P < 0.001; 3-year DFS: 25.0 ± 15.3% vs. 80.7 ± 6.6%, P < 0.001; 3-year TRM: 0 vs. 19.3 ± 6.6%, P = 0.277). However, whether MLL -PTD ≥ 1% or MLL -PTD < 1% before transplantation has no significant difference on the prognosis.
Conclusions
Our study indicated that MLL -PTD had a certain stability and could effectively reflect the change of tumor burden. The expression level of MLL -PTD after transplantation can serve as an effective indicator for predicting relapse.
... Marcucci et al. [74], as well as Corbacioglu et al. [81], described only a correlation between copy number and high percentage of BM blasts at disease onset. No correlation was also found with the type of fusion transcript, the type A mutation being the most frequently observed [77,81]. Conversely, Schnittger et al. [77] brought out a strong prognostic impact of the level of transcript on both OS and EFS. ...
... No correlation was also found with the type of fusion transcript, the type A mutation being the most frequently observed [77,81]. Conversely, Schnittger et al. [77] brought out a strong prognostic impact of the level of transcript on both OS and EFS. A score based on the median expression ratio, after consolidation therapy, and the 75th percentile of the expression ratio at diagnosis was formulated, although it was not possible to identify an absolute threshold to define an early molecular response. ...
... Some studies proposed a threshold of copy number for discriminating subgroups with shorter remission (i.e., 100 copies in BM [17,73,75], 10 copies in PB [82]). Others, instead, concluded that early assessment of MRD did not predict the disease course [74,77,81], contrary to what is generally thought for early morphologic response, possibly due to the well-known good responsiveness of CBF AML to induction treatment, in addition to a rather slow decline in the disease burden. Interestingly, the prognostic impact of MRD after induction is mostly referred to relapse risk rather than to survival, emphasizing the efficacy of salvage treatment in this category of AML. ...
Acute myeloid leukemia (AML) carrying inv(16)/t(16;16), resulting in fusion transcript CBFB-MYH11, belongs to the favorable-risk category. However, even if most patients obtain morphological complete remission after induction, approximately 30% of cases eventually relapse. While well-established clinical features and concomitant cytogenetic/molecular lesions have been recognized to be relevant to predict prognosis at disease onset, the independent prognostic impact of measurable residual disease (MRD) monitoring by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), mainly in predicting relapse, actually supersedes other prognostic factors. Although the ELN Working Party recently indicated that patients affected with CBFB-MYH11 AML should have MRD assessment at informative clinical timepoints, at least after two cycles of intensive chemotherapy and after the end of treatment, several controversies could be raised, especially on the frequency of subsequent serial monitoring, the most significant MRD thresholds (most commonly 0.1%) and on the best source to be analyzed, namely, bone marrow or peripheral blood samples. Moreover, persisting low-level MRD positivity at the end of treatment is relatively common and not predictive of relapse, provided that transcript levels remain stably below specific thresholds. Rising MRD levels suggestive of molecular relapse/progression should thus be confirmed in subsequent samples. Further prospective studies would be required to optimize post-remission monitoring and to define effective MRD-based therapeutic strategies.
... 8,19,29 Relationship between MRD in PB and BM in patients with imminent relapse Several studies have investigated the ability of longitudinal qPCR MRD monitoring in both PB and BM during follow-up after completion of therapy to identify an imminent relapse in CBF-AML. 8,11,19,27,29,31,40,[42][43][44] These studies have uniformly shown that both persistently high MRD levels and rising transcript levels in PB and BM are highly predictive of relapse, often using exceedance of a critical MRD threshold followed by a confirmatory test to determine molecular relapse. 8,19,29,40,44 In the largest prospective study to date, the UK Medical Research Council (MRC) assessed the clinical value of serial qPCR MRD monitoring in PB and BM in 278 patients with CBF-AML in the UK MRC AML-15 trial. ...
Longitudinal molecular measurable residual disease (MRD) sampling after completion of therapy serves as a refined tool for identification of imminent relapse of acute myeloid leukaemia (AML) among patients in long‐term haematological complete remission. Tracking of increasing quantitative polymerase chain reaction MRD before cytomorphological reappearance of blasts may instigate individual management decisions and has paved the way for development of pre‐emptive treatment strategies to substantially delay or perhaps even revert leukaemic regrowth. Traditionally, MRD monitoring is performed using repeated bone marrow aspirations, albeit the current European LeukemiaNet MRD recommendations acknowledge the use of peripheral blood as an alternative source for MRD assessment. Persistent MRD positivity in the bone marrow despite continuous morphological remission is frequent in both core binding factor leukaemias and nucleophosmin 1‐mutated AML. In contrast, monthly assessment of MRD in peripheral blood superiorly separates patients with imminent haematological relapse from long‐term remitters and may allow pre‐emptive therapy of AML relapse.
... Kaplan-Meier method was applied to estimate the probabilities of OS, and the log-rank test was utilized to compare the P values. Major molecular remission (MMR) was based on the RUNX1-RUNX1T1 transcript level as previously described [12][13][14]. Statistical analyses were performed with SPSS 25.0 (IBM) and GraphPad Prism 6.0. ...
t(8;21)(q22;q22) acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy with a high relapse rate in China. Two leukemic myeloblast populations (CD34+CD117dim and CD34+CD117bri) were previously identified in t(8;21) AML, and CD34+CD117dim cell proportion was determined as an independent factor for this disease outcome. Here, we examined the impact of CD34+CD117dim/CD34+CD117bri myeloblast-associated gene expression on t(8;21) AML clinical prognosis. In this study, 85 patients with t(8;21) AML were enrolled. The mRNA expression levels of CD34+CD117dim-associated genes (LGALS1, EMP3, and CRIP1) and CD34+CD117bri-associated genes (TRH, PLAC8, and IGLL1) were measured using quantitative reverse transcription PCR. Associations between gene expression and clinical outcomes were determined using Cox regression models. Results showed that patients with high LGALS1, EMP3, or CRIP1 expression had significantly inferior overall survival (OS), whereas those with high TRH or PLAC8 expression showed relatively favorable prognosis. Univariate analysis revealed that CD19, CD34+CD117dim proportion, KIT mutation, minimal residual disease (MRD), and expression levels of LGALS1, EMP3, CRIP1, TRH and PLAC8 were associated with OS. Multivariate analysis indicated that KIT mutation, MRD and CRIP1 and TRH expression levels were independent prognostic variables for OS. Identifying the clinical relevance of CD34+CD117dim/CD34+CD117bri myeloblast-associated gene expression may provide new clinically prognostic markers for t(8;21) AML.
... The most sensitive method for molecular MRD evaluation involves the detection of different genes. [7] However, more than 50% of AML cases lack known genetic lesions or clonality markers suitable for MRD monitoring. [8] Therefore, a proposed alternative marker is the expression of Wilms' tumour 1 (WT1) gene. ...
... Pathogenic fusion transcript detection in inherited disease is particularly notable as it has been traditionally associated with oncology. Initially believed to be isolated to blood-based neoplasia (Daley and Ben-Neriah, 1991) and later shown to be common in solid tumors (Barr, 1998;Aman, 1999), fusion transcripts received significant attention due to their diagnostic, prognostic and sometimes remarkable therapeutic implications (Burchill, 2003;Schnittger et al., 2003;An et al., 2010). Discussion of fusion transcripts detected in normal tissues centered on apparently benign events resulting from co-transcription of neighboring genes or more controversially from trans-splicing (Akiva et al., 2006;Peng et al., 2015;Babiceanu et al., 2016;Yuan et al., 2017;He et al., 2018). ...
Several recent studies have demonstrated the utility of RNA-Seq in the diagnosis of rare inherited disease. Diagnostic rates 35% higher than those previously achievable with DNA-Seq alone have been attained. These studies have primarily profiled gene expression and splicing defects, however, some have also shown that fusion transcripts are diagnostic or phenotypically relevant in patients with constitutional disorders. Fusion transcripts have traditionally been studied as oncogenic phenomena, with relevance only to cancer testing. Consequently, fusion detection algorithms were biased toward the detection of well-known oncogenic fusions, hindering their application to rare Mendelian genetic disease studies. A recent methodology published by the authors successfully tailored a traditional algorithm to the detection of pathogenic fusion events in inherited disease. A key mechanism of decreasing false positive or biologically benign events was comparison to a database of events detected in normal tissues. This approach is akin to population frequency-based filtering of genetic variants. It is predicated on the idea that pathogenic fusion transcripts are absent from normal tissue. We report on an analysis of RNA-Seq data from the genotype-tissue expression (GTEx) project in which known pathogenic fusions are computationally detected at low levels in normal tissues unassociated with the disease phenotype. Examples include archetypal cancer fusion transcripts, as well as fusions responsible for rare inherited disease. We consider potential explanations for the detectability of such transcripts and discuss the bearing such results have on the future profiling of genetic disease patients for pathogenic gene fusions.
... Current approaches for detecting and measuring MRD include multi-parametric flow cytometry (4)(5)(6), FISH (7), PCR detection of fusion transcripts (8,9) and targeted sequencing of common mutations (10)(11)(12)(13)(14)(15)(16). Each of these methods have their utility, but many are limited in their sensitivity, specificity and/or applicability to all patients. ...
Measuring minimal residual disease in cancer has applications for prognosis, monitoring treatment and detection of recurrence. Simple sequence-based methods to detect nucleotide substitution variants have error rates (about 10-3) that limit sensitive detection. We developed and characterized the performance of MASQ (multiplex accurate sensitive quantitation), a method with an error rate below 10-6. MASQ counts variant templates accurately in the presence of millions of host genomes by using tags to identify each template and demanding consensus over multiple reads. Since the MASQ protocol multiplexes 50 target loci, we can both integrate signal from multiple variants and capture subclonal response to treatment. Compared to existing methods for variant detection, MASQ achieves an excellent combination of sensitivity, specificity and yield. We tested MASQ in a pilot study in acute myeloid leukemia (AML) patients who entered complete remission. We detect leukemic variants in the blood and bone marrow samples of all five patients, after induction therapy, at levels ranging from 10-2 to nearly 10-6. We observe evidence of sub-clonal structure and find higher target variant frequencies in patients who go on to relapse, demonstrating the potential for MASQ to quantify residual disease in AML.
... Minimal residual disease (MRD) is a powerful tool for evaluating the response to treatment and for predicting the risk of relapse in patients affected with acute myeloid leukemia (AML). Nowdays, monitoring of specific gene mutations or rearrangements represents the most sensitive method (one target cells per 10 4 -10 6 ) to estimate MRD but its applicability is restricted to 30-50% of AML cases (Krönke et al., 2011;Schnittger et al., 2003;Yin et al., 2012). Of interest, the introduction of assays based on next generation sequencing, or deep-sequencing has not increased this frequency, despite the large number of reported somatic mutations (Morita et al., 2018;Papaemmanuil et al., 2016;Patel et al., 2012). ...
Background:
The assessment of minimal residual disease (MRD) by flow cytometry (FC) has a prognostic impact in acute myeloid leukemia (AML), despite the low sensitivity in predicting relapse. Nonetheless, the role of leukemic-associated immunophenotypes (LAIPs)-related specificity on the sensitivity of MRD has not been clarified yet. In this respect, we accomplished this study.
Methods:
LAIP-frequencies of bone marrow samples from healthy donors and patients after treatment were quantified and subdivided in "categories of specificity" named as: "strong," "good," and "weak." At the following, the diagnostic performance of MRD was investigated in terms of sensitivity, specificity, predictive values, likelihood ratio (LR).
Results:
"Strong" LAIPs were identified by CD7, CD2, CD4, and CD56 markers while "weak" LAIPs, independently of coexpressed markers, were mainly observed in CD33+ cells. MRD identified patients with significantly low DFS and OS but showed a low sensitivity in predicting relapse. Interestingly, majority of recurrences was noticed in patients with two LAIPs and lacking of "strong" LAIPs or only with one "good" LAIP. Thus, only patients showing one "strong" or two "good" LAIPs were considered suitable for MRD monitoring and selected to be further investigated. In this subset, positive MRD predicted a poor prognosis. Moreover, a higher sensitivity, negative predictive value (NPV) and LR- were observed after comparison with the previous series.
Conclusions:
These data highlight the relevant role of LAIP classification in "categories of specificity" in improving the sensitivity of MRD as assessed by FC.
