Assessment of Minimal Residual Disease in Standard-Risk AML.

BACKGROUND: Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission. METHODS: We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction. RESULTS: Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status. CONCLUSIONS: The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.).

Loss of imprinting at the 14q32 domain is associated with microRNA overexpression in acute promyelocytic leukemia.

Distinct patterns of DNA methylation characterize the epigenetic landscape of promyelocytic leukemia/retinoic acid receptor-α (PML-RARα)-associated acute promyelocytic leukemia (APL). We previously reported that the microRNAs (miRNAs) clustered on chromosome 14q32 are overexpressed only in APL. Here, using high-throughput bisulfite sequencing, we identified an APL-associated hypermethylation at the upstream differentially methylated region (DMR), which also included the site motifs for the enhancer blocking protein CCCTC-binding factor (CTCF). Comparing the profiles of diagnostic/remission paired patient samples, we show that hypermethylation was acquired in APL in a monoallelic manner. The cytosine guanine dinucleotide status of the DMR correlated with expression of the miRNAs following a characteristic position-dependent pattern. Moreover, a signature of hypermethylation was also detected in leukemic cells from an established transgenic PML-RARA APL mouse model at the orthologous region on chromosome 12, including the CTCF binding site located upstream from the mouse miRNA cluster. These results, together with the demonstration that the region does not show DNA methylation changes during myeloid differentiation, provide evidence that 14q32 hypermethylation is implicated in the pathogenesis of APL. We propose a model in which loss of imprinting at the 14q32 domain leads to overexpression of the miRNAs in APL.

Neighbor of Brca1 gene (Nbr1) functions as a negative regulator of postnatal osteoblastic bone formation and p38 MAPK activity.

The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1(tr/tr)) have approximately 50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1(tr/tr) osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1(tr/tr) cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.

Strikingly different molecular relapse kinetics in NPM1c, PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 acute myeloid leukemias.

Early relapse detection in acute myeloid leukemia is possible using standardized real-time quantitative polymerase chain reaction (RQ-PCR) protocols. However, optimal sampling intervals have not been defined and are likely to vary according to the underlying molecular lesion. In 74 patients experiencing hematologic relapse and harboring aberrations amenable to RQ-PCR (mutated NPM1 [designated NPM1c], PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11), we observed strikingly different relapse kinetics. The median doubling time of the CBFB-MYH11 leukemic clone was significantly longer (36 days) than that of clones harboring other markers (RUNX1-RUNX1T1, 14 days; PML-RARA, 12 days; and NPM1c, 11 days; P < .001). Furthermore, we used a mathematical model to determine frequency of relapse detection and median time from detection of minimal residual disease to hematologic relapse as a function of sampling interval length. For example, to obtain a relapse detection fraction of 90% and a median time of 60 days, blood sampling every sixth month should be performed for CBFB-MYH11 leukemias. By contrast, in NPM1c(+)/FLT3-ITD(-), NPM1c(+)/FLT3-ITD(+), RUNX1-RUNX1T1, and PML-RARA leukemias, bone marrow sampling is necessary every sixth, fourth, and fourth and second month, respectively. These data carry important implications for the development of optimal RQ-PCR monitoring schedules suitable for evaluation of minimal residual disease-directed therapies in future clinical trials.

RARalpha-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia.

Leukemia-associated chimeric oncoproteins often act as transcriptional repressors, targeting promoters of master genes involved in hematopoiesis. We show that CRABPI (encoding cellular retinoic acid binding protein I) is a target of PLZF, which is fused to RARalpha by the t(11;17)(q23;q21) translocation associated with retinoic acid (RA)-resistant acute promyelocytic leukemia (APL). PLZF represses the CRABPI locus through propagation of chromatin condensation from a remote intronic binding element culminating in silencing of the promoter. Although the canonical, PLZF-RARalpha oncoprotein has no impact on PLZF-mediated repression, the reciprocal translocation product RARalpha-PLZF binds to this remote binding site, recruiting p300, inducing promoter hypomethylation and CRABPI gene up-regulation. In line with these observations, RA-resistant murine PLZF/RARalpha+RARalpha/PLZF APL blasts express much higher levels of CRABPI than standard RA-sensitive PML/RARalpha APL. RARalpha-PLZF confers RA resistance to a retinoid-sensitive acute myeloid leukemia (AML) cell line in a CRABPI-dependent fashion. This study supports an active role for PLZF and RARalpha-PLZF in leukemogenesis, identifies up-regulation of CRABPI as a mechanism contributing to retinoid resistance, and reveals the ability of the reciprocal fusion gene products to mediate distinct epigenetic effects contributing to the leukemic phenotype.

Development of minimal residual disease-directed therapy in acute myeloid leukemia.

The last three decades have seen major advances in understanding the genetic basis of acute myeloid leukemia (AML). Comprehensive molecular and cytogenetic analysis can distinguish biologically and prognostically distinct disease subsets that demand differing treatment approaches. Definition of these pretreatment characteristics coupled with morphological response to induction chemotherapy provides the framework for current risk-stratification schemes, aimed at identifying subgroups most (and least) likely to benefit from allogeneic transplant. However, since such parameters lack the precision to distinguish the individual patient likely to be cured with conventional therapy from those destined to relapse, there has been considerable interest in development of multiparameter flow cytometry, identifying leukemia-associated aberrant phenotypes, and real-time quantitative polymerase chain reaction (RQ-PCR) detecting leukemia-specific targets (eg, fusion gene transcripts, NPM1 mutation) or genes overexpressed in AML (eg, WT1), to provide a more precise measure of disease response. Minimal residual disease (MRD) monitoring has been shown to be a powerful independent prognostic factor and is now routinely used to guide therapy in patients with the acute promyelocytic leukemia (APL) subtype. We consider the challenges involved in extending this concept, to develop a more tailored personalized medicine approach to improve the management and outcome of other forms of AML.

Can we say farewell to monitoring minimal residual disease in acute promyelocytic leukaemia?

Molecularly targeted therapies have transformed the management of PML-RARA+ acute promyelocytic leukaemia (APL), with survival rates now exceeding 80% in clinical trials. This raises questions about the relevance of post-remission monitoring for PML-RARA transcripts, which has been widely used to predict relapse, guiding early intervention to prevent disease progression and the inherent risk of fatal bleeding. Given the treatability of haematological relapse, survival benefits would only be seen if monitoring could identify patients who could be salvaged if treated early but not later on, although it could be argued that early deployment of arsenic trioxide (ATO) can avoid inducing hyperleucocytosis and the associated differentiation syndrome, which frequently complicate treatment of frank relapse. However, given the low rates of relapse now observed in patients presenting with standard risk disease (i.e. presenting WBC<10×10(9)/l) who achieve early molecular remission, subsequent sequential minimal residual disease (MRD) monitoring confers only a marginal benefit, so could be avoided in this group. However, sequential MRD monitoring may still be of value in patients with high risk APL, although evidence tends to come from historically controlled studies. Therefore, there may remain a role for MRD monitoring in the most clinically challenging subsets of APL, but the continuing debate highlights the need for robust evidence in developing a more individualized approach to management of other subtypes of acute leukaemia.

Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia.

Molecular detection of minimal residual disease (MRD) has become established to assess remission status and guide therapy in patients with ProMyelocytic Leukemia-RARA+ acute promyelocytic leukemia (APL). However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF-RARA and STAT5b-RARA. Despite their rarity (<1% of APL) we identified 6 cases (PLZF-RARA, n = 5; STAT5b-RARA, n = 1), established the respective breakpoint junction regions and designed reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17) - associated APL, affording assay sensitivities of ∼1 in 10(4)-10(5). Serial samples were available from two PLZF-RARA APL patients. One showed persistent polymerase chain reaction positivity, predicting subsequent relapse, and remains in CR2, ∼11 years post-autograft. The other, achieved molecular remission (CRm) with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b-RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RT-qPCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly defined subsets of acute leukemia.

Prospective minimal residual disease monitoring to predict relapse of acute promyelocytic leukemia and to direct pre-emptive arsenic trioxide therapy.

PURPOSE: Molecular diagnostics and early assessment of treatment response that use methodologies capable of detecting submicroscopic disease can distinguish subgroups of patients with leukemia at differing relapse risk. Such information is being incorporated into risk-stratified protocols; however, there are few data concerning prospective use of sequential minimal residual disease (MRD) monitoring to identify more precisely those patients destined to experience relapse, which would allow more tailored therapies. METHODS: Real-time quantitative polymerase chain reaction (RQ-PCR) assays to detect leukemia-specific transcripts (ie, PML-RARA, RARA-PML) were used to prospectively analyze 6,727 serial blood and marrow samples from 406 patients with newly diagnosed acute promyelocytic leukemia (APL) who were receiving all-trans-retinoic acid and anthracycline-based chemotherapy. RESULTS: MRD monitoring according to the recommended schedule successfully identified the majority of patients subject to relapse and provided the most powerful predictor of relapse-free survival (RFS) in multivariable analysis (hazard ratio, 17.87; 95% CI, 6.88 to 46.41; P < .0001); MRD monitoring was far superior to presenting WBC (hazard ratio, 1.02; 95% CI, 1.00 to 1.03; P = .02), which is currently widely used to guide therapy. In patients who were predicted to experience relapse on the basis of MRD monitoring, early treatment intervention with arsenic trioxide prevented progression to overt relapse in the majority, and the RFS rate at 1 year from molecular relapse was 73%. By using this strategy, 3-year cumulative incidence of clinical relapse was only 5% in the Medical Research Council AML15 trial. CONCLUSION: Rigorous sequential RQ-PCR monitoring provides the strongest predictor of RFS in APL and, when coupled with pre-emptive therapy, provides a valid strategy to reduce rates of clinical relapse. This provides a model for development of a more individualized approach to management of other molecularly defined subtypes of acute leukemia.

Real-time quantitative polymerase chain reaction detection of minimal residual disease by standardized WT1 assay to enhance risk stratification in acute myeloid leukemia: a European LeukemiaNet study.

PURPOSE: Risk stratification in acute myeloid leukemia (AML) is currently based on pretreatment characteristics. It remains to be established whether relapse risk can be better predicted through assessment of minimal residual disease (MRD). One proposed marker is the Wilms tumor gene WT1, which is overexpressed in most patients with AML, thus providing a putative target for immunotherapy, although in the absence of a standardized assay, its utility for MRD monitoring remains controversial. PATIENTS AND METHODS: Nine published and in-house real-time quantitative polymerase chain reaction WT1 assays were systematically evaluated within the European LeukemiaNet; the best-performing assay was applied to diagnostic AML samples (n = 620), follow-up samples from 129 patients treated with intensive combination chemotherapy, and 204 normal peripheral blood (PB) and bone marrow (BM) controls. RESULTS: Considering relative levels of expression detected in normal PB and BM, WT1 was sufficiently overexpressed to discriminate > or = 2-log reduction in transcripts in 46% and 13% of AML patients, according to the respective follow-up sample source. In this informative group, greater WT1 transcript reduction after induction predicted reduced relapse risk (hazard ratio, 0.54 per log reduction; 95% CI, 0.36 to 0.83; P = .004) that remained significant when adjusted for age, WBC count, and cytogenetics. Failure to reduce WT1 transcripts below the threshold limits defined in normal controls by the end of consolidation also predicted increased relapse risk (P = .004). CONCLUSION: Application of a standardized WT1 assay provides independent prognostic information in AML, lending support to incorporation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification, and to identify patients who may benefit from allogeneic transplantation.

Low-dose imatinib mesylate leads to rapid induction of major molecular responses and achievement of complete molecular remission in FIP1L1-PDGFRA-positive chronic eosinophilic leukemia.

The FIP1L1-PDGFRA fusion gene is a recurrent molecular lesion in eosinophilia-associated myeloproliferative disorders, predicting a favorable response to imatinib mesylate. To investigate its prevalence, 376 patients with persistent unexplained hypereosinophilia were screened by the United Kingdom reference laboratory, revealing 40 positive cases (11%). To determine response kinetics following imatinib, real-time quantitative-polymerase chain reaction (RQ-PCR) assays were developed and evaluated in samples accrued from across the European LeukemiaNet. The FIP1L1-PDGFRA fusion transcript was detected at a sensitivity of 1 in 10(5) in serial dilution of the EOL-1 cell line. Normalized FIP1L1-PDGFRA transcript levels in patient samples prior to imatinib varied by almost 3 logs. Serial monitoring was undertaken in patients with a high level of FIP1L1-PDGFRA expression prior to initiation of imatinib (100 mg/d-400 mg/d). Overall, 11 of 11 evaluable patients achieved at least a 3-log reduction in FIP1L1-PDGFRA fusion transcripts relative to the pretreatment level within 12 months, with achievement of molecular remission in 9 of 11 (assay sensitivities 1 in 10(3)-10(5)). In 2 patients, withdrawal of imatinib was followed by a rapid rise in FIP1L1-PDGFRA transcript levels. Overall, these data are consistent with the exquisite sensitivity of the FIP1L1-PDGFRalpha fusion to imatinib, as compared with BCR-ABL, and underline the importance of RQ-PCR monitoring to guide management using molecularly targeted therapies.