In light of evidence suggesting that Ca2+ affects energetics through “push” (activating metabolite flux and enzymes in the Krebs cycle to push the redox flux) and “pull” (acting directly on ATP synthase and driving the redox flux through the electron transport chain and increasing ATP production) pathways, we investigated whether both pathways are necessary to maintain atrial ATP supply-to-demand matching. Rabbit right atrial cells were electrically stimulated at different rates, and oxygen consumption and flavoprotein fluorescence were measured. To gain mechanistic insight into the regulators of ATP supply-to-demand matching in atrial cells, models of atrial electrophysiology, Ca2+ cycling and force were integrated with a model of mitochondrial Ca2+ and a modified model of mitochondrial energy metabolism. The experimental results showed that oxygen consumption increased in response to increases in the electrical stimulation rate. The model reproduced these findings and predicted that the increase in oxygen consumption is associated with metabolic homeostasis. The model predicted that Ca2+ must act both in “push” and “pull” pathways to increase oxygen consumption. In contrast to ventricular trabeculae, no rapid time-dependent changes in mitochondrial flavoprotein fluorescence were measured upon an abrupt change in workload. The model reproduced these findings and predicted that the maintenance of metabolic homeostasis is due to the effects of Ca2+ on ATP production. Taken together, this work provides evidence of Ca2+ “push” and “pull” activity to maintain metabolic homeostasis in atrial cells.To maintain atrial function, ATP supply-to-demand matching must be tightly controlled. Ca The atria conduct the electrical activity from the sinoatrial node to the heart’s ventricles and both passively and actively enhance ventricular diastolic filling. High and rapidly fluctuating metabolic rates are necessary to perform these electrical and mechanical tasks , thus AT2+ and ADP have been suggested as key regulators of matching respiratory to energy supply to varying demands  Ca2+ both “pulls” and “pushes” ATP production and iii) metabolic homeostasis is maintained in atrial cells during increased electrical stimulation or abrupt changes in workload.We hypothesized that: i) Ca2+ both “pulled” and “pushed” ATP. Moreover, no rapid time-dependent changes in mitochondrial flavoprotein fluorescence level were observed in response to an abrupt change in workload, due to Ca2+ ion regulation of cell bioenergetics.By combining experimental and computational results, we found ATP supply only matched the demand when CaAnimals were treated in accordance with the Technion Ethics Committee. The experimental protocols were approved by the Animal Care and Use Committee of the Technion (Ethics numbers: IL-118-10-13 and IL-001-01-19).Hearts were isolated from healthy male New Zealand white rabbits weighing 2.3–2.7 kg. Each rabbit was weighed and then sedated with an intramuscular injection of ketamine (0.1 ml/kg) and xylazine (0.1 ml/kg). An intravenous cannula was inserted in the rabbit’s ear for delivery of 200 mg/ml sodium pentobarbital diluted with heparin. The adequacy of the anesthesia was evaluated by observing the loss of reflexes in the eye and foot. The atrial cell isolation procedure is described in .2+ cycling into and out of the atrial cell cytosol was measured with Fluo-4 AM (ThermoFisher Scientific), as previously described  glued to a custom-made chamber top.Cytosolic CaThe autofluorescence of mitochondrial flavoprotein of atrial cells was imaged, at different electrical stimulation frequencies , at 37°C ± 0.5°C, under an inverted fluorescence microscope (Zeiss Observer Z1) with a 40x/1.4 N.A oil immersion lens and a 445 nm light-emitting diode (LED). Images were captured at 2 frames per second.2 2, and CaCl2 1 (pH 7.4 with NaOH). The cell suspension was divided into two equal aliquots, one of which was subjected to electrical stimulation and the second served as a control. The atrial cell suspensions were stirred gently under quiescent conditions in HEPES buffer for 2 min at 36°C, and then electrically stimulated at 1, 2 and 3 Hz for 1 min and again maintained under quiescent conditions for 1 min. Following measurement of oxygen consumption, total protein concentration (BCA™ Protein Assay) and the number of viable cells in the cell suspension were determined. Oxygen consumption of the atrial cells was normalized to the protein concentration.Oxygen consumption by either quiescent or electrically stimulated  atrial cell suspensions was measured using Clark-type electrodes . For the oxygen chamber, a custom-sealed plunger that included platinum wires was designed, as previously described . Atrial p < 0.05 taken to indicate statistical significance.All experiments were performed on cells from at least three rabbits. All data are presented as mean ± SD. Comparisons were made using a one-way repeated measurement ANOVA test with 2+ cycling, force generation and energetics  The ATP consumption by Naribed in .(ii) The force generation can be described by a four-state rather than a two-state kinetics  model.2 concentration is not constant ((iii) Similar to NADH, FADHconstant .2 and NADH are created in the TCA cycle in a 1:4 ratio (FADH2:NADH), and we assume that their consumption by the electron transfer chain follows the same ratio ((iv) As the reducing reagents, FADHme ratio .(v) Regulation of oxidative phosphorylation by the cellular energy demands can be modeled using a push and pull mechanism .2+ regulates the activity of F1Fo-ATPase ((vi) Cao-ATPase .2+ into the sarcoplasmic reticulum (SR) equation in , as suggested by  and we assume that their consumption by the electron transfer chain follows the same ratio. This means that 1/5 of oxygen consumption is attributed to FADH2-fueled and 4/5 to NADH-fueled electron transfer cycles:As the reducing reagents, FADH2 to the respiratory chain:In a modification of the original formulation in , it is a2 oxidation (VHe(F)) has the same form as VHe, except for the adjustment of the redox potential and the H+ stoichiometry. 2 oxidation.The flux of protons driven by FADHATPase is defined as the coupling coefficient representing the pull effect of the ATP synthase on the activity of the electron transfer chain.The regulation of oxidative phosphorylation by the cellular energy demands is modeled using a push and pull mechanism. ADP activates enzymes in the Krebs cycle, which “pushes” the respiratory flux toward ATP generation . In parai. The chemiosmotic hypothesis dictates that ΔΨm is lowered by an H+ influx, which drives the production of ATP by F1Fo-ATPase.1F0-ATPase concentration, According to the concept of respiratory control, mitochondrial function is governed by the availability of ADP and P2+ transients, oxygen consumption and flavoprotein at a 1 Hz stimulus frequency were used as input for the computational model, and the values predicted at 2 Hz and 3 Hz stimulus frequencies were compared to the experimental results.Experimental data (see results section) of Ca2+ on ATP synthase was withheld by fixing the mitochondrial Ca2+ to its initial condition in the 2+ was fixed at a constant value  throughout the model, which canceled the Ca2+ “push” effect on the TCA cycle and ATP synthase. In addition, the coupling variable between ATP synthase activity and the electron transfer chain oxygen consumption was fixed to its initial value, which eliminated “pull” entirely.To eliminate the “pull” mechanism, the effect of Ca2+ intake by the SR and sarcomere contractile elements. For the membrane component, the Na+-K+ ATPase and the membrane Ca2+ pump were considered:ATP consumption was calculated as the sum of the three main energy-consuming processes in the cell: active membrane transporters, Ca2+ uptake by the SR:ATP consumption by the SR is correlative to CaThe sarcomere energy consumption is calculated as part of the sarcomere model, in Eq. 112 in the supplement:The total energy consumption is the sum of the three:http://bioelectric-bioenergetic-lab.net.technion.ac.il and on https://github.com/yyLabPhysAI/energetics-atrium.The software was run in MATLAB . Numerical integration was performed using the MATLAB ode23tb stiff solver. Impulse stimulations were modeled as boundary conditions and the ODE was solved on the intervals between impulses. A 1-s quiescent window was added between stimulation rates to ensure the completion of the last beat before initiation of a new one. Computations were performed on an Intel(R) Core(TM) i7-8550U CPU @ 1.80 GHz machine with 16 GB RAM. Upon publication, the source code of the numerical model will be available at Fundamental experiments were first performed to verify that an increase in electrical stimulus frequency increases ATP supply. In a fully coupled system, oxygen consumption is an indicator of ATP production, i.e., supply. Increases in stimulus frequency increased oxygen consumption and were statistically significant at 3 Hz compared to 1 Hz . Only th2+ pump) consumed only 0.05% of the ATP budget. By calculating the relative consumption of ATP by the above consumers we found that increasing the stimulus frequency to 2 Hz and 3 Hz increased the ATP consumption rate by the SERCA and sarcomeres, due to an increased uptake rate (2+ pump currents slightly decreased with increased stimulus frequency. On change of the stimulus frequency (2 Hz and 3 Hz), there was no change in the relative ATP consumption by the consumers compared to 1 Hz. Thus, the rate-dependent changes in contractility and Ca2+ handling are the same, likely because the majority of Ca2+ is bound to troponin.Using the computational model, we found that at 1 Hz st0069mulus frequency , the SER2+  was 166.8 ± 168 ms and t90  was 407 ± 204 ms (n = 36 cells). At 3 Hz, t50 was 115 ± 48 ms and t90 was 187 ± 48 ms, n = 36  and pull  mechanisms were suggested . The comparison was enabled by novel oxygen consumption measurements, performed for the first time in isolated and electrically stimulated atrial cells. Ca2+ was regulated by both ATP supply and by the mitochondria that produce ATP. We simulated the two main ATP-consuming processes, both of which are Ca2+-dependent, i.e., pumping Ca2+ by the SERCA and force generation by myofilaments, to which the majority of Ca2+ is attached  diminished the correlation between mitochondrial Ca2+ and oxygen consumption. Moreover, without Ca2+ signaling, the metabolic homeostasis that we observed experimentally could not be maintained in the simulation. Thus, our results and simulation indicate that within the simulated workload range, Ca2+ is an important regulator of ATP supply to demand in both “push” and “pull” modes in atrial cells, as has been previously shown in ventricular cells  in the rate of tangible Ca2+-dependent effects on the Krebs cycle.It was postulated that “push” and “pull” modules lead to temporal changes in NADH levels in response to changes in workload . The difabeculae  were perechanism . It shouechanism  or in isechanism . Both he2+ data measured under a 1 Hz stimulation rate, reproduced Ca2+ kinetics and oxygen consumption under a 3 Hz stimulation rate. The model also reproduced FADH2 homeostasis and predicted that metabolic homeostasis existed at all levels of bioenergetics, from the Krebs cycle enzymes to respiratory fluxes , such as CaMKII signaling  and the xchanger . Of noten demand .2+ handling have been correlated with AF  and electrophysiological and structural remodeling of the atria has been described , recent  with AF  and can 2+ and ADP in both “push” and “pull” modes. However, this work did not rule-out the possibility that other mechanisms and/or mediators are involved, such as posttranslational modification signaling, like PKA  concentration is constant and does not model the CK kinetics in two compartments as was done in . In miceOur computational model represented only the control mechanisms that operate on a time scale of seconds. Electrical stimulation for several minutes may activate additional energy balance maintenance mechanisms. Of note, even for longer periods of electrical stimulation (1 minute) metabolic homeostasis was maintained experimentally .2+, work on a beat-to-beat basis. However, the tools we used to quantify bioenergetics do not support such a level of resolution.One should also take into consideration that some control mechanisms, such as Ca2+ is an important regulator of ATP supply-to-demand matching and that Ca2+ works in both “push” and “pull” directions.This work measured oxygen consumption and metabolic balance indicators in rabbit atrial cells and developed an integrated model of the electrophysiology, excitation-contraction coupling and mitochondrial bioenergetics of atrial cells. The model reproduced the increase in oxygen consumption in response to electrical stimulation and maintained metabolic homeostasis under abrupt changes in workload. The model predicted that Ca


Frontiers in Physiology is a collection of ten original research and review papers, showing the state-of-the-art research and future directions in the physiology and pathophysiology of myocardium.Myocardium has evolved to contract in a rhythmic fashion to provide blood from the heart to the body. The mechanical activities of myocardium originate in sarcomeres, composed of three filaments . Cardiac researchers have developed and applied various new technologies to elucidate the in-depth mechanisms of sarcomeric functions in the heart  and tropomyosin (Tm) complex blocks myosin binding to actin (“off” state). Following an increase in the intracellular Ca2+ concentration ([Ca2+]i), the binding of Ca2+ to TnC (one of the three subunits of Tn) causes displacement of Tm on thin filaments (“on” state), allowing myosin to interact with actin, and as a result, active force is generated   in which myosin heads are in closer proximity to thin filaments, and can readily bind to actin  e.g., . SRX is in e.g., . Decreasin e.g., . It is cin e.g.,  or titinin e.g., , shifts Kawana et al. reviewed HCM, from mutations to mechanisms and therapies. Based on rapid advances of biochemical and biophysical techniques, they have studied the molecular effects of HCM mutations on human β-cardiac myosin heavy chain, combining insights from clinical genetics and structural analyses of cardiac myosin. Accordingly, they concluded that HCM-causing mutations in sarcomere proteins cause hypercontractility, and that the increase in the number of myosin molecules available for interacting with actin is the primary driver. While β-blockers and Ca2+ channel blockers have been widely used to alleviate the heart’s hyperdynamic contraction for decades, their effectiveness is often poorly tolerated in the clinical setting, especially for young patients. Recently, two small molecule cardiac myosin inhibitors that shift the SRX-DRX equilibrium to SRX have been developed. One of these, mavacamten, showed improvement in the composite endpoint of exercise capacity and symptom severity in the phase III study of HCM patients, and it has recently been approved by the US Food and Drug Administration. Mavacamten is the first compound that showed a benefit for HCM in a randomized controlled trial. The other small molecule cardiac myosin inhibitor is aficamten; it is currently under evaluation in a phase III trial, and the results are awaited.Hypertrophic cardiomyopathy (HCM) affects more than one in 500 people worldwide with high morbidity due mostly to arrhythmia, heart failure and sudden death. 2+]i. This is a favorable property as an inotropic agent because an increase in [Ca2+]i often causes arrhythmias due to membrane depolarization coupled with Na+ entry via the Na+/Ca2+ exchanger. Nakanishi et al. demonstrated by using skinned porcine venticular and atrial muscles that OM increases Ca2+ sensitivity in a concentration-dependent manner in a clinically relevant range , with the effect more pronounced in ventricular muscle. The Ca2+-sensitizing effect of OM became less following thin filament reconstitution with fast skeletal Tn complex, and the ensuing decrease in the number of “recruitable” cross-bridges. Therefore, they concluded that OM’s Ca2+ sensitizing effect results from the strongly bound cross-bridge-dependent allosteric activation of thin filaments. Also, as pointed out by On another front, omecamtive mecarbil (OM) was developed as a first-in-class myosin activator for the treatment of heart failure in patients with reduced ejection fraction (HFrEF). Several lines of evidence suggest that OM is effective in the treatment of HFrEF by improving cardiac function without increasing i. This refined understanding of thin filament structures will provide us with many opportunities such as designing molecular interventions for genetic heart diseases.Three papers have added to the recent advances in the understanding of thin filament regulations. Accumulating evidence indicates that mutations in thin filament proteins cause genetic heart diseases such as HCM, dilated cardiomyopathy (DCM) and restictive cardiomyopathy (RCM) e.g., . Due to Chalovich et al. reviewed thin filament regulations with a focus on Tn mutants, in particular the HCM-causing mutation of Δ14 of TnT that is missing the last 14C-terminal residues of cardiac TnT. It is important that removal of the basic residues in this region eliminates the inactive Ca2+-free state. Indeed, cardiac muscle fibers containing Δ14-TnT shows increased Ca2+ sensitivity by ∼0.23 pCa units with no change in maximal Ca2+-activated force  on the myocardial relaxation rate when sarcomere lengthening (1%) was applied. Accordingly, they found that Pi enhanced the stretch-dependent increase in the cross-bridge detachment rate, with the magnitude becoming greater with faster stretching. Considering that Pi exists in the mM range in cardiomyocytes under physiological conditions, their finding will promote our understanding of the molecular mechanism of sarcomeric relaxation during the transition from late-systole to early-diastole.Relaxation of myocardium is the key process for effective ventricular filling, and the duration of isovolumic relaxation depends on the rate of the transition from late-systole to early-diastole. There are two important contributors to this transition; i.e., the fall of [Ca2+]i in myocytes under physiological conditions. In 2015, Shintani and colleagues found that when warmed to 37–43°C, sarcomeres exhibit rapid spontaneous sarcomeric auto-oscillations independent of [Ca2+]i changes in neonatal rat cardiomyocytes .” HSOs are similar in characteristics to “Spontaneous Oscillatory Contractions (SPOCs)” that occur at partial activation of pCa ∼6.0 in both neonatal and adult myocardium  for the quantification of cardiac function. STE uses B-mode imaging which records a video clip of the entire heart wall. Therefore, as an average measurement, STE provides data points along the entire heart wall rather than merely two from M-mode imaging. STE allows us to obtain displacement, velocity, strain and strain rate in radial, longitudinal and circumferential axes of the heart. STE is useful in basic research; in particular, further improvement of spatial and temporal resolutions will allow for more accurate analyses of the in vivo assessment of all tiers of cardiovascular performance in small rodents like mice, including left ventricular systolic and diastolic function and contractility.One paper introduces a technological advancement of the non-invasive analysis of In summary, this Research Topic highlights the considerable advancements cardiac researchers have made since the turn of the 21st century. We must move forward to further elucidate the physiology and pathophysiology of myocardium not only by taking advantage of the current technologies but also by developing newer advanced technologies. In doing so, all the contributions made by the authors in the Research Topic will be integrated for the prevention, diagnosis and treatment of disorders of the heart.


RARA gene translocations, which are of high importance in the diagnosis of and proper therapy selection for APL. However, in some cases acute myeloid leukemia (AML) demonstrates APL-like morphological features such as atypical promyelocytes accumulation. This type of AML is characterized by the involvement of other RAR family members or completely different genes. In the present study, we used conventional karyotyping, FISH and high-throughput sequencing in a group of 271 de novo AML with atypical promyelocytes accumulation. Of those, 255 cases were shown to carry a typical chromosomal translocation t with PML::RARA chimeric gene formation (94.1%). Other RARA-positive cases exhibited cryptic PML::RARA fusion without t  and variant t translocation with NPM1::RARA chimeric gene formation . However, 7 RARA-negative AMLs with atypical promyelocytes accumulation were also discovered. These cases exhibited TBL1XR1::RARB and KMT2A::SEPT6 fusions as well as mutations, e.g., NPM1 insertion and non-recurrent chromosomal aberrations. Our findings demonstrate the genetic diversity of AML with APL-like morphological features, which is of high importance for successful therapy implementation.Acute promyelocytic leukemia (APL) pathogenesis is based on  RAR family genes [RAR genes encode the retinoic acid steroid receptors of three isotypes: RARA, RARB and RARG, which differ in their ability to bind to the SMRT corepressor [RARE (retinoid acid responsive element)—thus blocking RNA synthesis in the absence of retinoic acid. Upon activation of the RARA receptor by retinoic acid, its conformation changes, eventually leading to normal myeloid cell differentiation [Acute promyelocytic leukemia (APL) is a subvariant of acute myeloid leukemia (AML). Distinctive features of APL are the accumulation of atypical promyelocytes and chromosomal translocations affecting the ly genes . The RARepressor . The RARntiation ,4.PML::RARA chimeric gene formation, where PML is a tumor suppressor gene on chromosome 15 [PML::RARA were identified with different breakpoints in PML: bcr1 in intron 6, bcr2 in exon 6 and bcr3 in intron 3 , while RARA gene rupture occurs in intron 2 [PML::RARA may also form as a result of cryptic chromosomal material exchange between chromosomes 15 and 17 with no t detectable by karyotyping or FISH [More than 90% of APL carry a chromosomal translocation t15;17) with osome 15 ,5. Such ;17/ZBTB16::RARA, t/NPM1::RARA, t/NuMa::RARA, t/STAT5b::RARA, etc. [RARA rearrangement can be detected. This group of RARA-negative AML with APL-like morphological features is either associated with other RAR family genes rearrangements such as RARB and RARG [RAR rearrangements at all [RARA rearrangements as well as RARB and RARG cases exhibit an unfavorable prognosis. In the current work, we aimed to study tumor-specific genetic lesions in AML with APL-like morphological features, with a focus on RARA-negative cases.In 1–2% of APL, RA, etc. ,13,14. H::RARA, t;17. Further characterization was carried out using immunophenotypic, cytogenetic and molecular genetic methods.From 2010 to 2022, 271 cases of de novo AML with atypical promyelocytes were investigated at the Dmitry Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology  smears corresponding to the M3 or M3v FAB subtype ,7,8. AllThe hypergranular variant is characterized by an excess of cytoplasmic azurophilic granules and numerous Auer rods (faggot cells). The hypogranular type is represented by a low content or absence of granules in the cytoplasm of promyelocytes, as well as single faggot cells containing 1–3 Auer rods . If therIn addition, to accurately differentiate AML from APL and monocytic leukemia, all samples were subjected to cytochemical analysis consisting of 5 reactions: assessment of myeloperoxidase (MPO) activity, Sudan black (SB) staining for lipids, Schiff (PAS) reaction, assessment of nonspecific esterase and suppression of esterase with sodium fluoride. Promyelocytes have strong staining in reactions to MPO and SB, in contrast to undifferentiated blasts. In addition, in APL, the PAS reaction demonstrates the diffuse distribution of the dye, and the excess of nonspecific esterase in myeloid cells is not suppressed by sodium fluoride.Diagnostic immunophenotyping was performed as described previously . The antBM aspirate obtained at diagnosis was cultured overnight without mitogenic stimulation and processed as previously described . G-bandePML::RARA-negative by FISH were further analyzed with Kreatech RARA DCBA probe  for other types of RARA rearrangements. Kreatech KMT2A DCDF probe (Leica) was used for all patients with additional chromosomal abnormalities and patients without metaphase plates. All FISH procedures were performed according to the manufacturer’s instructions and at least 100 interphase nuclei were analyzed.FISH screening for t was performed with Kreatech PML-RARa DCDF probe  in any newly diagnosed AML. Cases with atypic promyelocytes by morphological examination but PML::RARA was used in routine [NPM1::RARA was analyzed with a nested PCR system [Total DNA and RNA were simultaneously extracted from the BM samples using the InnuPrep DNA/RNA Mini Kit . Reverse transcription-polymerase chain reaction (RT-PCR) with the Biomed multiplex system for  routine . NPM1::RR system ,24. The RARA gene rearrangements by either method were subjected to whole transcriptome sequencing with NEBNext Ultra II Directional RNA library preparation kit  and sequenced on Illumina NextSeq . Alignment to NCBI preformatted GRCh38 reference genome [KMT2A-positive sample was also subjected to LDI-PCR [Samples negative for e genome  was perfe genome . Fusion e genome . Additioe genome  and annoe genome . Short ie genome . A KMT2A LDI-PCR . Fusion-Overall survival (OS) was defined as the duration from the date of diagnosis to death or last follow-up. Event-free survival (EFS) was defined as the time from diagnosis to the first event—relapse or death from any cause. Survival rates were estimated with the Kaplan–Meier method , and staAccording to morphological examination, most patients had hypergranular M3 AML . Hypogranular M3v AML was found in 17.3% (47 of 271), and in 36.2%, M3 and M3v could not be differentiated (98 of 271).Standard karyotyping was successful in 69.4% cases (188 of 271). T was found to be a sole abnormality in 64.4% (121 of 188), while it conjoined with additional abnormalities in 23.4% (44 of 188), and had complex structure in 3.7% . Of those, 173 were studied by routine qRT-PCR. It revealed bcr3 to be the most common , followed by bcr1  and bcr2 . In 5.8% of patients, the type of PML::RARA was not determined (10 of 173).The presence of RARA-rearranged PML::RARA-negative cases were uncovered (4 of 271). All of them carried NPM1::RARA transcript detected by nested RT-PCR.No translocation between chromosomes 15 and 17 was found in 5.9% (16 of 271). Additional studies with RARA DCBA were performed and 1.48% RARA-negative by either FISH approach  were first analyzed by routine qRT-PCR. This uncovered 1.9% cases of cryptic 15q25 and 17q21 chromosomal material exchange leading to PML::RARA formation (5 of 271) transcribed as bcr3 and bcr1 subtypes . This left seven cases negative for any type of detectable RARA rearrangement. They were further subjected to RNAseq which revealed only 0.8% of unconventional fusion transcripts .Patients TBL1XR1::RARB-positive sample was found to have conventional hypergranular M3 morphology   d,e. None6 exon 2 g,h. The The remaining five patients did not carry any gene fusions 1.8%; . All thePTPN11, NRAS, IDH2 and a single case of NPM1 insertion and FLT3-ITD co-occurrence. Nonsynonymous deleterious exonic variants are listed in Fusion-negative AML with APL-like morphological features did not carry any recurrent chromosomal aberrations. They included two cases of non-recurrent chromosomal rearrangements and three AMLs with normal karyotype. Additional mutations affected RARA gene rearrangements placed on ATRA combined with cytarabine and daunorubicin therapy was 90.0 ± 2.0%, while EFS was 75.0 ± 5.0% (n = 227). RARA-negative patients were treated with either APL approach  or AML intermediate risk approach . RARA-negativity was associated with lower survival rates ; however, the difference did not reach statistical significance due to the small size of the RARA-negative cohort . Survival curves are depicted in Outcome data were available for 86.3% of the studied cohort (234 of 271). The three-year OS rate in patients with RARA gene rearrangements. In our study, 94.1% of patients demonstrated classic APL with t/PML::RARA, 1.9% carried cryptic PML::RARA and 1.5% had t/NPM1::RARA variant . PML::RARA bcr1:bcr2:bcr3 ratio was reported to be roughly 2:1:2 in both adult and pediatric APL [Atypical promyelocytes accumulation, as defined by morphology, strongly correlates with the tric APL ,33. In otric APL .NPM1 gene at 5q35 encodes the nucleophosmin protein and moves between the nucleus, nucleolus and cytoplasm, participating in the regulation of p53, the tumor suppressor ARF and many other cellular functions [RARA-negative AML with APL-like features were also described featuring RARB or RARG [KMT2A fusions [RARB isotype gene rearrangement TBL1XR1::RARB (0.4%), one case of KMT2A-rearranged AML (0.4%) and five patients without fusions (1.9%).NPM1::RARA fusion transcript was found in four patients and exhibited a conservative exon 4–exon 3 breakpoint junction. unctions . NPM1::R or RARG ,45,46,47 fusions ,48,49 an fusions ,50,51,52TBL1XR1 (transducin β-line 1 X-linked receptor 1) is located at 3q26 and encodes WD-40 protein, which has the LiSH domain [TBL1XR1::RARB transcript discovered in the study had breakpoints in exon 5 and exon 2, respectively, which was previously described in APL [PML::RARA in its action and reduces the transcriptional activity of the retinoic acid pathway [H domain . Transcrd in APL ,43. The  pathway .KMT2A::SEPT6-positive AML resembling APL at diagnosis deserves special attention. KMT2A, also known as MLL, located at 11q23, encodes a lysin-specific histone methyltransferase critical for the HOX genes expression. As a result of KMT2A rearrangements, upregulated HOX expression in hematopoietic stem cells leads to disruption of their differentiation and development of leukemogenesis [KMT2A, and SEPT6 is the most widespread among them [An unusual case of ogenesis . Septin ong them . They enong them .KMT2A rearrangements were diagnosed with acute lymphoblastic leukemia, it was suggested that the KMT2A::SEPT6 fusion gene tends to induce differentiation of cells into the myeloid lineage [Although most pediatric patients with  lineage . The desKMT2A-rearranged AML with morphological features resembling APL but without RARA rearrangement [KMT2A::SEPT6 fusion.There is a limited number of reports on angement ,48,49. NKMT2A-rearranged AML, such as very bright expression of CD33 and HLA-DR and expression of CD4 and CD56, which have been noted [KMT2A [KMT2A-rearrangement antigen profiles as well.Despite the cytomorphological characteristics typical of APL, leukemia cells in the reported case lacked the signature characteristic immunophenotypic signs of malignant promyelocytes observed in APL, i.e., they were positive for HLA-DR, CD34 and brightly positive for CD117. On the other hand, they exhibited typical features of en noted . NG2 expd [KMT2A ,60, was PML::RARA-negative AML cases with APL-like morphological features, except the KMT2A-positive patient (due to the specific tumor biology), had more or less a typical myeloid immunophenotype, with strong expression of pan-myeloid antigens . Moreover, all of them were HLA-DR-negative as can be expected for APL cases [PML::RARA-negative AML cases with APL-like morphological features displayed relatively heterogeneous antigen expression profiles with a mix of typical and atypical APL immunophenotypic features.The leukemic cells of the other six PL cases . On the PL cases . The expPL cases , was fouPML::RARA is considered to be a combination of ATRA and ATO which both lead to disruption of the fusion protein and restore the activity of the retinoic acid receptor pathway, unblock myeloid differentiation and decrease the proliferative capacity of abnormal cells [Modern therapy for classical APL with al cells ,64,65,66al cells . We haveX::RARA-associated APLs are considered to have the same pathogenetic mechanism and were shown to respond to ATRA therapy. However, NPM1::RARA cases require a higher dosage of ATRA due to the high affinity for corepressor molecules and are at higher risk of relapse [PLZF::RARA, STAT3::RARA, STAT5B::RARA, or CPSF6::RARA were shown to have poor response to ATRA [TBL1XR1::RARA, remission was achieved after prolonged treatment with ATRA, ATO and chemotherapy [NPM1::RARA-positive patients who received ATRA and chemotherapy, who reached the first CR confirmed by RT-PCR and were alive in CR for 28 and 44 months.Variant  relapse ,66,68,69 to ATRA . On the otherapy . As for RARB and RARG demonstrated ATRA resistance and frequent relapses [NPM1::RARG::NPM1 fusion and M3 morphology who had clinical resistance to ATO and ATRA drugs due to the lack of binding sites for these substances [TBL1XR1::RARB-positive patient received APL induction therapy, achieved morphological remission, was set to HSCT but was lost for follow-up 2 months after diagnosis.The limited data on ATRA therapy in APL-like AML with retinoic receptors B and G isotypes involvement show their adverse clinical effects. relapses ,37,41,70bstances . Thus, iKMT2A::SEPT6-positive AMLs meet adverse risk stratification criteria, and intensive induction therapy followed by HCST is a generally recommended treatment option [KMT2A::SEPT6 fusion with KMT2A intron 11 involvement which was reported to attribute to the lowest survival rate [KMT2A::SEPT6-positive patient started treatment with ATRA combined with cytarabine and daunorubicin. Complete remission was achieved and confirmed by BM morphology and qRT-PCR for KMT2A::SEPT6 fusion transcript. The patient received chemotherapy consolidation consisting of two additional courses of cytarabine and daunorubicin and was placed on maintenance therapy with daily mercaptopurine, weekly methotrexate and ATRA for 2 weeks every 3 months. Due to initial CNS involvement  the patient was also placed on CNS therapy consisting of five intrathecal injections of cytarabine, methotrexate and dexamethasone after the onset of the disease. Six months after remission, the patient presented with left-side face paralysis and was hospitalized. Combined BM and CNS relapse was diagnosed confirmed by BM morphology, qRT-PCR for KMT2A::SEPT6 fusion transcript and direct genomic PCR for KMT2A::SEPT6 fusion gene in cerebrospinal fluid. The patient received high-dose cytarabine and anthracycline without ATO; however, the leukemia proved to be resistant and the patient died because of the progression of the disease.ATRA therapy is even more questionable in AML with APL-like morphological features but without any trans-retinoic acid targets. t option . As for val rate . Our repNPM1 exon 12 insertion accompanied by FLT3-ITD in 60% [NPM1 and FLT3-ITD-positive [NPM1 and FLT3-ITD-positive case [RARA-negative AML with APL-like features described in the literature are summarized in NPM1 and FLT3-ITD-positive and the rest carried various other mutations.Cases mimicking various features of acute promyelocytic leukemia that do not carry any fusion genes were also reported ,33,51. AD in 60% . A case PML::RARA-positive APL [RARA rearrangement, Zhao et al. show a possible approach to transition to AML mode (not M3) starting from the second induction course after the first APL induction course. This resulted in an improved survival of patients who switched to an AML regimen, compared to patients treated with an APL regimen, although the difference was not statistically significant [RARA-positive group) and one patient received cytarabine, mitoxantrone and etoposide (as intermediate risk AML). All patients achieved CR. Remission status was confirmed by flow cytometry, as a unified molecular assessment could not be used in this group. Later, two patients relapsed (9 months after diagnosis each) and were subjected to hematopoietic stem cell transplant. Three patients stayed in the first CR for 14, 25 and 32 months.Regarding therapeutic options, these AML should not be classified as APL, but as intermediate-risk AML according to the ELN recommendations . APL-tartive APL . Howevertive APL , it is hnificant . In our PML::RARA gene formation, and are a heterogeneous AML subgroup. Thus, confirmatory molecular methods are obligatory for chromosomal translocations verification in patients with APL-like features. Our study adds some important traits to characteristics of AML with APL-like features which might help to select appropriate therapy.The literature data, as well as our own data, suggest that AML mimicking the morphological, cytochemical and immunophenotypic features of APL may arise from different mechanisms other than


TP53 allelic state and variant allele frequency (VAF) in AML‐MRC remains poorly defined.Acute myeloid leukemia with myelodysplasia‐related changes (AML‐MRC) generally confers poor prognosis, however, patient outcomes are heterogeneous. The impact of We retrospectively evaluated 266 AML‐MRC patients who had NGS testing at our institution from 2014 to 2020 and analyzed their clinical outcomes based on clinicopathological features.TP53 mutations were associated with cytogenetic abnormalities in 5q, 7q, 17p, and complex karyotype. Prognostic evaluation of TP53MUT AML‐MRC revealed no difference in outcome between TP53 double/multi‐hit state and single‐hit state. Patients with high TP53MUT variant allele frequency (VAF) had inferior outcomes compared to patients with low TP53MUT VAF. When compared to TP53WT patients, TP53MUT patients had inferior outcomes regardless of MRC‐defining criteria, TP53 allelic state, or VAF. TP53 mutations and elevated serum LDH were independent predictors for inferior OS and EFS, while PHF6 mutations and transplantation were independent predictors for favorable OS and EFS. NRAS mutation was an independent predictor for favorable EFS.TP53MUT AML‐MRC defines a very‐high‐risk subentity of AML in which novel therapies should be explored.Our study suggests that  TP53‐mutated AML‐MRC patients have inferior outcomes compared to TP53‐wildtype patients irrespective of MRC‐defining criteria, TP53 allelic state or TP53 variant allele frequency. TP53‐mutated AML‐MRC should be classified as a very‐high‐risk AML subentity for which novel therapeutic strategies should be explored. The diagnosis of AML‐MRC requires at least one of the following: (a) a prior history of myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN), (b) presence of a MDS‐defining cytogenetic abnormality, and (c) morphologic detection of multi‐lineage dysplasia (MLD). AML‐MRC is associated with advanced age, low remission rates, and poor prognosis, with a median overall survival of approximately 12 months.CEBPA, NPM1, FLT3, RUNX1, ASXL1, and TP53 have shown prognostic relevance and have been incorporated into the 2017 ELN risk stratification.In recent years, next‐generation sequencing (NGS) has become a standard tool to risk stratify patients with myeloid malignancies. In AML, mutations in TP53, which is located on the short arm of chromosome 17, encodes the tumor suppressor protein p53 that mediates critical anti‐tumor activity by inducing apoptosis in response to DNA damage.TP53 mutations are detected in up to 10% of de novo AML patients, however, its incidence has been shown to increase to approximately 30% in AML‐MRC.TP53 mutations and abnormalities in chromosome 17p have been identified as poor prognostic factors by the 2017 ELN risk stratification and are associated with advanced age, therapy resistance, and poor prognosis.TP53 mutations were recently identified to have an adverse prognostic impact in AML‐MRC.TP53 allelic state and variant allele frequency (VAF) can further resolve the heterogeneity in AML‐MRC patient outcomes.TP53‐mutated (TP53MUT) AML‐MRC to evaluate whether TP53 allelic state and clonal burden could resolve prognostic heterogeneity in AML‐MRC.In the present study, we performed an extensive molecular evaluation of 266 AML‐MRC patients and retrospectively assessed whether individual mutations were associated with patient outcomes. We further characterized 22.1We screened all the University Health Network patients diagnosed with AML‐MRC as per the 2016 WHO definition between April 2014 and November 2020. The study was approved by the University Health Network Research Ethics Board. Peripheral blood and bone marrow aspirate smears were reviewed by at least two independent hematopathologists and consensus on diagnosis was achieved. Patients were classified into three groups: (a) AML‐MRC patients with a history of MDS or of MDS/MPN, irrespective of the presence of MDS‐related cytogenetic changes (AML‐MRC‐H); (b) AML‐MRC patients with MDS‐defining cytogenetic abnormalities (AML‐MRC‐C); and (c) AML‐MRC patients with MLD alone, defined as the presence of >50% dysplasia in at least two lineages (AML‐MRC‐M). Patients with therapy‐related AML were excluded from this study. The majority of patients were treated with induction regimen consisting of cytarabine and daunorubicin/idarubicin, or non‐intensive regimens consisting of hypomethylating agent (HMA) or low‐dose cytarabine.2.2TP53 was determined as previously described.In accordance with the International System for Human Cytogenetic Nomenclature guidelines, karyotypes were obtained from diagnostic bone marrow samples and described as appropriate. The cytogenetic loss of 2.3TP53 mutations were assessed by NGS which spanned TP53 exons 2–11 (Table Next‐generation sequencing (NGS) was performed for 105 patients using a custom myeloid panel for 49 genes implicated in myeloid malignancies (Oxford Gene Technologies) and run on the MiSeq platform (Illumina), as previously described.11 Table , variantTP53 state when (A) at least two TP53 variants were detected by NGS, (B) one TP53 variant detected by NGS co‐occurred with cytogenetic loss of TP53 and (C) one TP53 variant was detected by NGS with a VAF of ≥55%, as previously described.Patients were considered to be double/multi‐hit 2.4p‐value was less than 0.05.Evaluation of patient outcomes including overall survival (OS) and event‐free survival (EFS) was carried out from retrospective analysis of patient records. OS was calculated as the time from the date of diagnosis to last follow‐up or death. EFS was defined as the time from the date of diagnosis to last follow‐up, relapse, or death. Univariate survival analysis and comparison of outcome were performed using the Kaplan–Meier product‐limit method and the log‐rank test. The chi‐square or Fisher's exact test, as appropriate, was used to assess associations between categorical parameters. The Kruskal–Wallis and Wilcoxon rank‐sum tests were used to assess whether there were differences in numerical variables between groups, as appropriate. Multivariable Cox proportional hazards regression was performed with variables that were significant prognostic factors by univariable analysis and variables that are known prognostic factors in AML. All statistical tests were performed using R software version 4.0.5  and were interpreted as significant if the two‐sided 33.1Two hundred and sixty‐six AML‐MRC patients were identified, including 142 (53.4%) AML‐MRC‐C patients, 99 (37.2%) AML‐MRC‐H patients, and 25 (9.4%) AML‐MRC‐M patients. The median age of our cohort was 70 years (range 18–91). The median follow‐up time was 6.2 months (range 0–75.7 months) and 160 (60%) patients died at the time of last follow‐up.p = 0.032 and p = 0.018, respectively). The number of mutated genes was higher in AML‐MRC‐H and AML‐MRC‐M compared to AML‐MRC‐C .Patient characteristics as stratified by AML‐MRC subtype are summarized and compared in Table TP53 mutation status are summarized and compared in Table TP53MUT and TP53WT patients. However, the TP53MUT group had a greater female representation and had fewer mutated genes other than TP53 compared to the TP53WT group. Fewer TP53MUT patients had bone marrow transplantation.Patient characteristics as stratified by 3.2TP53, mutated in 96 (36%) patients, followed by DNMT3A (26%), ASXL1 (21%), TET2 (21%), RUNX1 (19%), SRSF2 (15%), IDH2 (14%), U2AF1 (11%), and STAG2 (11%). 140 (53%) patients had at least one secondary‐type mutation involving ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2, as defined by Lindsley et al.The mutational landscape of AML‐MRC is presented in Figure TP53 mutations were significantly enriched in AML‐MRC‐C compared to both AML‐MRC‐H and AML‐MRC‐M . STAG2 mutations were significantly enriched in AML‐MRC‐M compared to both AML‐MRC‐C and AML‐MRC‐H , and were significantly enriched in AML‐MRC‐H compared to AML‐MRC‐C (p < 0.0001). Mutations significantly enriched in both AML‐MRC‐H and AML‐MRC‐M relative to AML‐MRC‐C were in ASXL1 , SRSF2  and IDH2 . Mutations in U2AF1, RUNX1 and TET2 were significantly enriched only in AML‐MRC‐H compared to AML‐MRC‐C . Overall, AML‐MRC‐C was characterized by TP53 mutations, while AML‐MRC‐H and AML‐MRC‐M were characterized by secondary‐type mutations. Indeed, secondary‐type mutations were more frequently harbored in AML‐MRC‐M  and AML‐MRC‐H  compared to AML‐MRC‐C  .The frequency of mutations in each AML‐MRC subtype is presented in Figure 3.3ASXL1, RUNX1, SRSF2, and IDH2 were significantly underrepresented in the TP53MUT group. ASXL1 mutations significantly co‐occurred with mutations in RUNX1, SRSF2, IDH2, and STAG2. STAG2 mutations also significantly co‐occurred with mutations in SRSF2, IDH2, and NRAS. Other significantly co‐occurring mutation pairs included IDH2 and SRSF2, IDH2 and FLT3, and U2AF1 and KRAS.Molecular characterization of AML‐MRC allowed us to identify frequently co‐occurring and mutually exclusive mutations in AML‐MRC. Our correlation analysis of recurrently (≥5) mutated genes is presented in Figure 3.4TP53MUT and 156 TP53WT patients. TP53MUT patients were more likely to have complex karyotype than TP53WT patients . TP53MUT patients were also more likely to have cytogenetic abnormalities in chromosome 17p .Karyotype analysis was available in 248 (93%) AML‐MRC patients, including 92 TP53MUT patients were more likely to have chromosomal abnormalities in 5q  and 7q .Among the 18 patients without available karyotype, 12 patients had interphase FISH for del(5q) and del(7q). TP53 mutations, and complex karyotype had the highest sensitivity (97%) for TP53 mutations.Overall, chromosomal aberrations in 17p had the highest specificity (95%) for 3.5p = 0.058) and had significantly better EFS  compared to AML‐MRC‐C patients. AML‐MRC‐H patients had borderline significantly better OS  and EFS  compared to AML‐MRC‐C patients. AML‐MRC‐M patients had no significant differences in OS  and EFS  compared to AML‐MRC‐H patients.The median OS and EFS of our cohort were 10.7 months and 7.7 months, respectively. Kaplan–Meier curves for patients as stratified by subtype are presented in Figure p‐values for all mutations are presented in Figure TP53 mutations were associated with inferior OS and EFS, while mutations in IDH1, NRAS, and PHF6 were associated with favorable OS and EFS. SF3B1 mutations were associated with favorable OS but not EFS. The respective Kaplan–Meier curves are provided in Figures Given the paucity of data on the prognostic impact of mutations in AML‐MRC, we evaluated the associations between individual gene mutations and patient outcomes. The OS and EFS hazard ratios, median and 2‐year OS and EFS, and log‐rank TP53 mutation status are presented in Figure TP53 mutations were detected in 80 (56%) AML‐MRC‐C patients, 15 (15%) AML‐MRC‐H patients, and one (4%) AML‐MRC‐M patient. TP53 mutation had an adverse impact on OS and EFS in AML‐MRC‐C . TP53 mutation was associated with borderline significantly inferior OS  and significantly inferior EFS  in AML‐MRC‐H. For both TP53MUT and TP53WT patients, AML‐MRC subtype had no significant impact on OS  or EFS .Kaplan–Meier curves for patients as stratified by both subtype and TP53 was associated with inferior OS  and EFS   were detected in 26 patients and did not significantly impact OS  or EFS   and EFS , and TP53MUT patients with multiple TP53 mutations had inferior OS  and borderline significantly inferior EFS   or EFS   and EFS   Figure . Among p) Figure . Compare) Figure . Among p) Figure . Compare) Figure .TP53 allelic state in our cohort is presented in Figure TP53 double/multi‐hit had no significant differences in OS  and EFS  compared to patients with TP53 single‐hit  and EFS   Figure .TP53MUT VAF (52.1%), patients with high VAF had inferior OS  and EFS  compared to low VAF patients  and EFS   and EFS  compared to low VAF patients  and EFS   Figure . Similars Figure . Compare) Figure .TP53MUT AML‐MRC patients, concurrent DNMT3A mutation was associated with inferior OS  and EFS , while in TP53WT patients, DNMT3A mutation did not significantly impact OS  or EFS   Figure .p = 0.02) and EFS  compared to patients with <6 mutated genes  and EFS   Figure .TP53WT and TP53MUT patients who went onto transplant had significantly longer OS  and EFS  than those who did not, however, TP53WT patients had dramatically improved and plateauing long‐term survival rates  the overall adverse prognosis in AML‐MRC is largely driven by TP53 mutation status and not AML‐MRC‐defining criteria, and (B) TP53 mutation confers an adverse prognostic impact in AML‐MRC irrespective of its allelic state or VAF.In this study, we report, to the best of our knowledge, the largest molecular profiling of AML‐MRC by detailed NGS. We provide a comprehensive evaluation of the prognostic impact of commonly occurring myeloid gene mutations in AML‐MRC. We identified NPM1, CEBPA, and secondary‐type mutations in ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2 owing to their association with distinct clinical features and outcomes.TP53 mutations to define a standalone AML subtype.TP53” as a distinct disease category owing to its association with distinctly aggressive disease, complex cytogenetic abnormalities and very poor outcome.TP53 mutations were independently associated with adverse outcomes in AML‐MRC. Strikingly, when stratifying patients by both AML‐MRC subtype and TP53 mutation status, patient outcomes largely grouped together by TP53 mutation status, not AML‐MRC subtype  stratifying patients by number of TP53 mutations, (B) stratifying TP53MUT patients by the presence/absence of cytogenetic loss of TP53, or (C) stratifying patients by the number of TP53 hits. Our data suggest that AML‐MRC with any number of TP53 hits should be considered as very‐high‐risk AML.The prognostic impact of TP53 clonal burden and AML patient outcomes remains controversial. Prior studies have reported conflicting data on the prognostic impact of TP53MUT VAF in both non‐intensive and induction chemotherapy patients.TP53MUT VAF had inferior OS and EFS compared to patients with low VAF. Therefore, AML‐MRC patients with high TP53MUT VAF may benefit most from novel TP53MUT therapies. However, low TP53MUT VAF was also associated with poor outcomes and should still be considered as a high‐risk factor for AML‐MRC.The association between TP53 mutations, which independently predicted for poor outcomes in AML‐MRC, were infrequently accompanied by co‐occurring mutations, which was consistent with a prior report.TP53 mutations in AML‐MRC.It is conventionally thought that greater mutational complexity predicts for inferior outcomes due to clonal heterogeneity. Prior studies have reported associations between higher number of mutations and inferior outcomes in AML.TP53 mutations as having independent prognostic value, we also showed that NRAS and PHF6 mutations were independent predictors for improved outcomes. Even after exclusion of AML‐MRC‐M cases, PHF6 mutations retained their association with improved OS  and EFS , while NRAS mutations retained their association with improved EFS  in multivariable analysis (data not shown). NRAS mutations were reported to have a favorable prognostic effect in NPM1MUT/DNMT3AMUT AML.NRAS mutations have not shown a significant prognostic impact in AML overall, studies have shown that NRAS‐mutated clones are chemosensitive and do not persist after treatment.PHF6 mutations in AML, likely due to its low frequency (2%–3%) in AML.PHF6 mutations were associated with adverse outcomes in AML overall and intermediate‐risk AML. Another study in CK‐AML patients revealed that when stratifying patients into “typical CK”, defined as CK containing high‐risk abnormalities in 5q, 7q, and/or 17p, and “atypical CK”, defined as CK without such abnormalities, CK‐AML patients with atypical CK had a significantly higher rate of PHF6 mutation and favorable outcomes.NRAS and PHF6 mutations have an independently favorable prognostic impact in AML‐MRC. Future studies are needed to evaluate whether NRASMUT or PHF6MUT AML‐MRC should still be classified as high‐risk AML.In addition to identifying TP53MUT VAF threshold of the median may not reflect a biologically meaningful threshold, however, this also applies to other prognostic biomarkers such as WBC count. Future investigations are needed to optimize the TP53MUT VAF threshold for risk stratification.We acknowledge that our study has several limitations. First, our study is limited by its retrospective nature and its relatively small subsets for analysis. Future prospective studies with larger cohorts are needed to confirm our data. Second, NGS was performed with two different gene panels which may have resulted in variability in the reported VAFs due to technical differences. However, all NGS experiments were performed on the same MiSeq platform. Third, our 5TP53MUT AML‐MRC patients had inferior outcomes compared to TP53WT patients irrespective of MRC‐defining criteria, TP53 allelic state, or TP53 VAF. Our study suggests that TP53MUT AML‐MRC should be classified as a very‐high‐risk AML subentity and highlights the great need for novel therapies for TP53MUT AML.In conclusion, DZ analyzed data, performed statistical analysis, and wrote the manuscript. DZ, EE, MZ, JM, AS and MM collected data for analysis. EA performed statistical analysis. HC designed the study, analyzed data, supervised the project, and wrote the manuscript.This research was supported by grants from the Leukemia and Lymphoma Research Society of Canada (LLSC) and the Cancer Research Society (CRS).The authors declare no conflicts of interest.The study was approved by the UHN Research Ethics Board.Data S1Click here for additional data file.