To characterize the ongoing evolution of myxoma virus in Australian rabbits, we used experimental infections of laboratory rabbits to determine the virulence and disease phenotypes of recent virus isolates. The viruses, collected between 2012 and 2015, fell into three lineages, one of which, lineage c, experienced a punctuated increase in evolutionary rate. All viruses were capable of causing acute death with aspects of neutropenic septicemia, characterized by minimal signs of myxomatosis, the occurrence of pulmonary edema and bacteria invasions throughout internal organs, but with no inflammatory response. For the viruses of highest virulence all rabbits usually died at this point. In more attenuated viruses, some rabbits died acutely, while others developed an amyxomatous phenotype. Rabbits that survived for longer periods developed greatly swollen cutaneous tissues with very high virus titers. This was particularly true of lineage c viruses. Unexpectedly, we identified a line of laboratory rabbits with some innate resistance to myxomatosis and used these in direct comparisons with the fully susceptible rabbit line. Importantly, the same disease phenotype occurred in both susceptible and resistant rabbits, although virulence was shifted toward more attenuated grades in resistant animals. We propose that selection against inflammation at cutaneous sites prolongs virus replication and enhances transmission, leading to the amyxomatous phenotype. In some virus backgrounds this creates an immunosuppressive state that predisposes to high virulence and acute death. The alterations in disease pathogenesis, particularly the overwhelming bacterial invasions that characterize the modern viruses, suggest that their virulence grades are not directly comparable with earlier studies.IMPORTANCE The evolution of the myxoma virus (MYXV) following its release as a biological control for European rabbits in Australia is the textbook example of the coevolution of virus virulence and host resistance. However, most of our knowledge of MYXV evolution only covers the first few decades of its spread in Australia and often with little direct connection between how changes in virus phenotype relate to those in the underlying virus genotype. By conducting detailed experimental infections of recent isolates of MYXV in different lines of laboratory rabbits, we examined the ongoing evolution of MYXV disease phenotypes. Our results reveal a wide range of phenotypes, including an amyxomatous type, as well as the impact of invasive bacteria, that in part depended on the level of rabbit host resistance. These results provide a unique insight into the complex virus and host factors that combine to shape disease phenotype and viral evolution. Oryctolagus cuniculus) in Australia is the canonical example of viral and host adaptation after a pathogen spillover . The virus likely originated in the Americas in rabbit-like Sylvilagus species of lagomorphs. Although apparently largely innocuous in its natural hosts  experiencing a rapid burst of evolution between 1996 and 2012 . We therViruses tested in the experiments reported here are listed in Five viruses isolated in 2012, three from lineage c and two from lineage b, were tested in the same manner and rabbit line (Oak) as previously described , 11. SurThree of the viruses tested—Hattah (lineage c), Euchareena (lineage b), and Acton-12 (lineage b)—had highly lethal grade I virulence, amyxomatous phenotypes  characterized by 100% case fatality with clinical signs and pathology typical of the highly immunosuppressive phenotype previously described , 11.Clinically, rabbits infected with these three grade I viruses developed moderately swollen heads, together with weight gain, between days 7 and 10, suggesting tissue edema. Eyelids tended to be moderately swollen with eyes still open at time of death. Ears were generally only swollen at the base, and there was usually some degree of anogenital swelling. Primary lesions were flat, diffuse, and poorly defined from the surrounding skin with no scabbing. Rectal temperatures transiently spiked around day 4 to 5 and then returned to normal, but most rabbits had a second high spike to as high as 42°C in the 24 h prior to death. All rabbits died or were euthanized between days 9 and 13 after infection. In some cases, rabbits rapidly became moribund having been apparently normal several hours earlier. Others were found with respiratory distress or trembling and recumbent. At necropsy, there was typically massive pulmonary edema; histopathology often revealed bacterial invasion of multiple tissues such as lung, lymph nodes, spleen, heart, liver, and kidney. The primary lesions ranged in thickness on cut section from 1 to 5 mm, with most being 1 to 2 mm. Often, there was subcutaneous edema fluid over the rump and down the hind legs.Interestingly, although the Acton-12 isolate showed an amyxomatous acute death phenotype in laboratory rabbits, the wild rabbit from which it was isolated clearly had multiple flat lesions of the typical myxomatous phenotype .TF1213 (lineage c) was still highly lethal (grade II virulence), with four of the rabbits exhibiting pulmonary edema/toxemia around days 12 to 13, and three of these having a temperature spike in the 24 h prior to death. The remaining two rabbits were euthanized on day 15, but one had pulmonary edema on histology, and the other had bacteria in tissues, indicating a pathogenesis similar to the more acute deaths. Compared to the lineage b viruses, rabbits infected with TF1213 had an obviously more proliferative disease with more swelling around head, eyelids, and ears and edematous ridging of the skin of the anterior surface of the forelegs. There was some hair loss over the head area. Eyelids were fully closed at time of death. Anogenital swelling was much more severe, and there was usually some scrotal edema. Primary lesions were more defined, slightly raised and 3 to 10 mm thick on cut sections. Subcutaneous edema of rump and hind legs was also a feature.Wonga Park (lineage c) was quite different, with only one rabbit moribund and euthanized at 15 days with hemorrhage and pulmonary edema. Three of five of the remaining rabbits developed extreme swelling of the head and ears with marked increase in body weight, while the other two had a less-severe clinical progression and also developed multiple coalescing secondary lesions. A feature of the Oak rabbits infected with this virus was grossly swollen, drooping ears and extreme folding of the thickened skin at the base of the ears and over the face . Swollen drooping ears are often seen with MYXV isolates from the 1950s but were rarely seen with the more recently isolated viruses we have tested. All five remaining rabbits were euthanized on day 19 and scored as category C on the disease appearance scale, but these had much more cutaneous swelling than we had previously seen. It was difficult to predict the outcomes for these rabbits and so define an endpoint for euthanasia, but the virus was obviously considerably attenuated and so continuing the trial could not be justified; at day 19, when the trial was terminated, the surviving rabbits were all still eating and drinking.At this point in our investigation, a change in rabbit supplier occurred. This meant that NZ White rabbits from an independent genetic line were supplied—here named CR rabbits. To control for any variation between the rabbit lines, we tested a previously well characterized, highly virulent lineage a virus, BRK isolated in 1993, along with nine viruses isolated between 2012 and 2015 from lineages a, b, and c not previously tested. Based on experience with multiple rabbit lines, we were not anticipating any differences between the lines of NZ White rabbits. However, it quickly became obvious that this CR line of rabbits had some innate resistance to myxomatosis compared to previously tested laboratory rabbits in the United States and in Australia.The control virus, BRK, had previously been shown to have grade I virulence in Australia and in multiple trials in the United States , 35. InfThe nine new viruses tested were mostly of grade II or III virulence in the CR rabbits with only ABC Range being grade I. Prolonged survival of one or more rabbits was a feature of all the viruses tested in the CR rabbits . HoweverWith the exception of seven rabbits (see below) all of the viruses generally caused an amyxomatous phenotype with minimal primary lesions and a lack of secondary lesions. All viruses caused some acute deaths due to pulmonary edema/toxemia, where these occurred on or before day 15 they were classed as category B. Those rabbits that survived longer, including many scored as recovered or likely to recover, were scored as category C. A few of these rabbits developed secondary lesions, mostly around the head and ears. Mt Gambier caused death with pulmonary edema and bacteria in tissues, but these deaths occurred after day 15 and so were somewhat arbitrarily classed as category C. It was difficult to categorize four rabbits infected with TF2930, that developed secondary lesions on the ears between days 10 and 12, two of which developed more generalized secondary lesions. In addition, three rabbits (one from the Mulligans Flat group and two from the Acton-14 group) developed secondary lesions early and had a mild disease course with rapid recovery. These seven rabbits have been classed as category D (attenuated myxomatous disease) based on the secondary lesion development, but the disease course could be considered atypical since the primary lesion was clearly of the amyxomatous type.Rabbits in category C infected with lineage c viruses that survived for longer tended to develop the extreme folding of the thickened skin of the base of the ears and anterior skin of the forelegs induced by Wonga Park in the previous trial. This was particularly the case with the attenuated Mt Gambier and Coomandook viruses. However, one rabbit infected with the Monarto virus (lineage a) that was euthanized at day 26 developed very similar lesions. The virulence grade for Acton 8-14 is somewhat arbitrary. With 2 survivors, both of which had an atypical disease course, it could be considered a grade IIIB or IV virus, but an AST of 19.1 days is typical of a grade IIIA virus. Similarly, TF2930 on survival times was grade IIIA virulence virus, but this was skewed by the two acute deaths, with the remaining rabbits having much more prolonged survival more like a grade IIIB or IV virus.To further investigate this observed resistance in the CR rabbits, we obtained Oak rabbits from the previous supplier  and CR rabbits to undertake a simultaneous comparison of infections between the two lines of rabbits. For this we selected eight viruses across a range of virulence grades: six used in trial 2 in CR rabbits, Wonga Park from trial 1 as a highly attenuated virus, and the Lausanne strain as a known grade 1 virulent virus with the classic myxomatous nodular phenotype.We have published Lu in the susceptible (Oak) rabbits on the survival time and pathogenesis previously , and in P = 0.018)  . SubsequP = 0.001; No clinical differences were seen between the two groups of rabbits infected with ABC Range; both groups had an amyxomatous acute death phenotype category B see  below. AP = 0.007; At the other end of the virulence spectrum, the highly attenuated Wonga Park also showed clear differences in virulence, survival curves, and clinical signs between the two groups of rabbits. In Oak rabbits, Wonga Park was estimated as grade IIIB virulence, whereas it was assigned grade V virulence in the CR rabbits . ClinicaRabbits infected with the Hoppers Crossing, Mt Gambier, and Coomandook viruses showed similar trends between the groups with lower estimated virulence in the CR rabbits  but no sResults from trial 2 with CR rabbits were not always duplicated in this comparative trial. This was particularly the case for Acton 8-14, which was a grade IIIA virulence in the previous trial but a grade I virulence in the comparative trial with no difference between the Oak and CR rabbits and an amyxomatous phenotype . The wil9 PFU/g in both groups of rabbits. These are extremely high titers; 107 rabbit ID50/g is regarded as the threshold for high mosquito transmissibility  the titers for each virus at each time point have been pooled. Despite these limitations, some interesting trends can be seen. Titers in cutaneous lesions are of interest because these correlate with transmissibility . Primarysibility  and so asibility . The treIn general, necropsy findings in all three trials recapitulated our findings reported for Australian viruses from the 1990s and some recent UK viruses , 11. TwoFor the Australian viruses, rapid collapse and death between days 8 and 15 occurred unpredictably with relatively minor clinical signs of myxomatosis and was most often due to acute pulmonary edema with massively swollen lungs and fluid and froth filling the bronchi and trachea . SometimEarly primary lesions were poorly differentiated from the surrounding skin. Histologically, there was some hyperplasia and hypertrophy of epidermal cells, including the cells of the hair follicles, and minor disruption of the upper dermis evident as disruption of the collagen fibers, together with deposition of gray-blue-staining mucin-like material. Occasional macrophages were present, but there was a general absence of inflammatory cells such as neutrophils or lymphocytes. Fibroblasts were often prominent in the upper dermis (see below).Despite the presence of bacteria in multiple tissues, neutrophils were absent from the tissues of rabbits that died acutely from infection with the Australian viruses. This was in contrast to rabbits that died at similar time points following infection with Lu, where bacteria were not present, but neutrophils were prominent in tissues such as primary and secondary lesions, the lymph nodes, the liver, and the spleen . Proliferation of epithelial cells from glandular elements was a feature of the eyelid pathology. As with the primary lesions, there was a difference between time and virus lineage, with earlier lesions showing much less pathology and lineage c viruses tending toward more proliferative lesions, which could begin to resemble Lu but taking many days longer than the Lu lesions to develop. Even at 23 days, the lineage b Acton 8-14 virus showed much less proliferative change than did the lineage c viruses (data not shown).The aim of this study was to examine the phenotypic consequences of about 15 years of evolution of MYXV by characterizing the disease caused by Australian MYXV isolated between 2012 and 2015. A particular focus was whether any feature of the disease caused by the rapidly evolving lineage c viruses might indicate ongoing adaptive evolution.All of the Australian viruses tested induced acute death mimicking neutropenic septicemia in some rabbits. Most had an amyxomatous phenotype with little or no inflammatory response in the cutaneous lesions. We hypothesize that the amyxomatous phenotype in laboratory rabbits is the result of selection against an inflammatory response in cutaneous lesions to enhance transmissibility in resistant wild rabbits. In some virus genetic backgrounds, neutrophil activity is sufficiently suppressed that generalized bacteremia can occur, causing acute death in virtually all laboratory rabbits and mimicking very high grade I virulence but with a completely different pathogenesis compared to earlier grade I virulent viruses. In other genetic backgrounds, some infected rabbits have a more prolonged disease course, and these viruses were of grade II to V virulence. The amyxomatous phenotype suggests that a neutrophil or other innate inflammatory response is involved in the formation of discrete, circumscribed primary and secondary lesions and the early scabbing that limits vector access in infections with viruses such as Lu and SLS. It is questionable whether the virulence exhibited by these modern viruses should be compared to the earlier viruses since the bacteremia/toxemia seems critical, and we have previously demonstrated that survival time could be extended by treatment with antibacterials, whereas antibacterials had no effect on SLS .Rabbits infected with viruses from each lineage developed the novel disease syndrome that we have previously described for Australian viruses from the 1990s and more recent viruses from Great Britain , 11. ThiImportantly, when viruses were tested in the innately resistant CR line of laboratory rabbits, the same acute disease phenotype was observed in some rabbits in each group except those infected with the highly attenuated Wonga Park virus. This confirmed that the novel disease phenotype was independent of rabbit lineage. Acute pulmonary edema had also been observed with viruses from the 1990s using outbred laboratory rabbits in Australia, but the mechanism and pathology of the disease syndrome had not been characterized . We have not yet been able to identify a specific viral mutation associated with this phenotype, and it is possible that the mutation has a different mechanism of action in resistant wild rabbits.M005L/R and M153R genes, both of which have major virulence functions in the Lu virus , had the amyxomatous phenotype, which our data suggest is strongly linked to a lack of inflammation in the cutaneous lesions. All of the viruses were capable of inducing acute collapse/bacteremia in at least some rabbits. However, some viruses, such as ABC Range, were extremely lethal, causing acute collapse with short ASTs in 6/6 Oak rabbits and 11/12 CR rabbits, while others were much more attenuated with extended ASTs. At this stage, we do not have a molecular explanation for this difference. However, ABC Range is the most virulent Australian virus we have tested and, interestingly, has reading frame disruptions in the Lu virus , 37. It Lineage c MYXVs were isolated from a broad area of southeastern Australia between 2012 and 2016 coincident with viruses from lineages a and b. Phylogenetically, we infer that a rapid, punctuated burst of evolution occurred in this lineage at some point between 1996 and 2012 . In contWe tested eight lineage c viruses in laboratory rabbits using viruses from different branches of the tree and from a range of geographic locations. Virulence grades ranged from I  to IIIB/V . Most were moderately attenuated grade II or III, with a tendency to be more attenuated in the resistant CR rabbits. While most viruses tested had an amyxomatous phenotype, compared to the lineage a and b viruses tested, in the lineage c infections there appeared to be much more proliferative swelling at the base of the ears, around the eyelids and the skin of the anterior forelegs. In rabbits that survived for longer, this swelling became folded into firm ridges that could become distorted especially around the eyelids and base of the ears and eventually morph into more discrete lesions. This is a distinctly different pathogenesis from the secondary lesions that occur with the progenitor SLS, where widespread discrete secondary lesions develop from around 6 days after infection , and froInsect transmissibility is dependent on high titers of virus in cutaneous sites accessible to the vector. Studies on transmissibility have usually focused on the primary lesion as a proxy for cutaneous titers. However, the virus-rich tissues of eyelids, face, and ears are probably essential for transmission since mosquitoes are less likely to feed on the more densely haired parts of the body . This maFrom the results obtained here, we hypothesize that lineage c viruses are capable of enhanced cutaneous dissemination to sites around the head where mosquitoes are more likely to feed and that they are able to suppress inflammatory responses at these sites, allowing persistent virus replication to high titers. This does not seem to be necessarily linked to virulence, at least in laboratory rabbits. In contrast, the lineage b viruses were generally more virulent, causing more limited cutaneous swelling around the head and ears, although still with an amyxomatous phenotype. Although we have not tested enough recent lineage a viruses to draw any conclusions about these viruses, one CR rabbit infected with each lineage a virus (Monarto and BRK) developed the folded ears and face seen with the lineage c viruses, suggesting that prolonged survival in resistant rabbits could enhance transmissibility. This indicates selection at multiple sites affecting both virulence and cutaneous immune suppression in the lineage c viruses. However, it is difficult to point to a genetic signature of this across the genetically diverged lineage c viruses. It seems likely that there has been selection against inflammatory responses and that in some virus genetic backgrounds this causes overwhelming immunosuppression with bacteremia and acute collapse.M005L/R gene , which reverses a single nucleotide deletion present in the progenitor SLS virus and all subsequent Australian virus isolates sequenced. This deletion disrupted the M005 reading frame, with predicted loss of activity of any protein translated from this gene  viruses in nonresistant rabbits both here and in earlier work . Based oFenner and Woodroofe noted in 1965  that wheWe also saw occasional accelerated recoveries in the resistant rabbit trials. Of particular interest, these rabbits tended to develop widespread discrete secondary cutaneous lesions early in the disease course. Although the overall disease could be quite mild and was classified as category D, the disease was quite different from that caused by attenuated viruses from the 1950s . The remThe main measure of MYXV evolution in Australia from 1950 through to 1981 was testing virus isolates for virulence using the Fenner grading system in laboratory rabbits. Through most of this period, the virulence of field isolates was relatively stable, with most isolates being of intermediate grade III or grade IV virulence and small numbers showing higher or lower virulence . There mSomething appears to have changed in this evolutionary dynamic between 1980 and the early 1990s. Detailed genetic and phenotypic studies of viruses from the 1990s in laboratory rabbits showed that the disease caused in laboratory rabbits was quite different to earlier myxomatosis . This suThe continued isolation of highly virulent, attenuated, and moderately attenuated viruses (as measured in laboratory rabbits) suggests that under different ecological conditions or in different rabbit populations viruses with quite different virulence are able to establish and transmit. Does this mean that the nexus between virulence and transmission is breaking down? Earlier studies on flea transmission demonstrated that rabbits that survived infection were always poor sources of infection, as were those that died early . This suIn Europe, a recent recombination event between MYXV and an unknown poxvirus has led to a virus that can transmit and cause disease in both rabbits and hares , which mAll animal experiments were approved by The Pennsylvania State University Institutional Animal Care and Usage Committee (permits 33615 and 42748).Pasteurella multocida and Bordetella bronchiseptica. Rabbits were housed as previously described  or Charles River, Canada (“CR rabbits”) and were free of multiple pathogens, including escribed  in indivRabbit trials were conducted as previously described . For eac10(ST-8) as described previously (Unless there was clear evidence that death had occurred in the previous 1 to 2 h (warm body and lack of rigor mortis), animals that died unobserved were assigned survival times that were the midpoint between observations. For the purpose of estimating AST and Kaplan-Meier survival analysis, animals that were euthanized when moribund or becoming moribund were assigned a survival time based on estimated likely time to death. These survival times were likely accurate to within hours. Animals euthanized on humanitarian grounds were assigned an additional survival time of 24 to 72 h based on estimated likely time to death. These survival times may be underestimates. To estimate the AST, rabbits classified as likely survivors were assigned a survival time of 60 days . For asseviously  and thenFor virus titers in tissues and for disease category assignments (see below) the actual time of death or euthanasia was used without adjustment, other than for unobserved deaths, where the midpoint time was used as described above and is referred to as the actual survival time.In earlier work , where wSample collection and processing for histology were as previously described . For virThe viruses tested are listed in 10.26207/ya4f-rg91).Accession numbers for the viruses listed in Fig. 3 to


Acute myeloid leukemia with plasmacytoid dendritic cell differentiation (pDC-AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN) are two leukemias characterized by neoplastic pDC proliferation. In this study, we aimed to explore the immunophenotypic and molecular profiles of pDC-AML and compare them with BPDCN. We found that pDCs in pDC-AML and BPDCN possessed different immunophenotype. The mutation profiles of these two were also different. These findings demonstrate that pDC-AML and BPDCN are two distinct entities and pDCs in these two entities derive from different subsets of pDC precursors.RUNX1 mutations. In this study, we investigated a cohort of 53 pDC-AML cases representing about 3% of all AML cases. We characterized their immunophenotype and genetic profiles and compared these findings with blastic plasmacytoid dendritic cell neoplasm (BPDCN). pDC-differentiation/expansion was preferentially observed in AML with an immature myeloid or myelomonocytic immunophenotype, where myeloblasts were frequently positive for CD34 (98%), CD117 (94%), HLA-DR (100%) and TdT (79%), with increased CD123 (89%) expression. The median number of pDCs in pDC-AML was 6.6%  and their immunophenotype reminiscent of pDCs in early or intermediate stages of differentiation. The immunophenotype of pDCs in pDC-AML was different from BPDCN (n = 39), with major disparities in CD34 (96% vs. 0%), CD56 (8% vs. 97%) and TCL1 (12% vs. 98%) and significant differences in frequency of CD4, CD13, CD22, CD25, CD36, CD38, CD117 and CD303 expression. At the molecular level, the genetic landscapes of pDC-AML and BPDCN also differ, with RUNX1 mutations detected in 64% of pDC-AML versus 2% of BPDCN. Disparities in TET2 (21% vs. 56%), FLT3 (23% vs. 0%), DNMT3A (32% vs. 10%) and ZRSR2 (2% vs. 16%)  were also detected. The distinct immunophenotypic and mutation profiles of pDC-AML and BPDCN indicate that the neoplastic pDCs in pDC-AML and BPDCN derived from different subsets of pDC precursors.Acute myeloid leukemia (AML) with ≥2% plasmacytoid dendritic cells (pDC) has been recently described as AML with pDC differentiation (pDC-AML) characterized by pDC expansion with frequent  Plasmacytoid dendritic cells (pDCs) are produced in the bone marrow and traffic to the peripheral tissues and organs when they mature . There aRUNX1 mutations are common in pDC-AML, and the same gene mutations and cytogenetic abnormalities have been shown in pDC and myeloblasts, indicating their shared cell of origin. The immunophenotype of pDCs in pDC-AML was explored by others in a few studies [Mirroring the heterogeneity of normal pDCs, neoplastic proliferations of pDCs can manifest with different disease types such as blastic plasmacytoid dendritic cell neoplasm (BPDCN), mature pDC proliferations associated with myeloid neoplasms and recently described AML with pDC expansion/differentiation (pDC-AML) ,7,8,9. P studies ,7,8,10 w studies  comparedIn this study, we presented 53 cases of pDC-AML from our cancer center, the largest cohort to date. We comprehensively explored the immunophenotypic and molecular features of pDC-AML and discussed the differential diagnosis with BPDCN.We retrospectively screened cases with a diagnosis of AML and mixed phenotype acute leukemia (MPAL) including myeloid/T or myeloid/B, from 7 January 2017 through 31 December 2020 that underwent flow cytometric immunophenotypic analysis; 1768 cases were identified. Among this group, 53 cases showed increased pDCs defined as ≥2% of total nucleated cells by flow cytometry ,12. ThisFlow cytometry immunophenotyping was performed on fresh bone marrow aspirate specimens using a standard staining and lyse/wash technique  as part of routine clinical work-up. Samples were examined with antibody panels designed to assess leukemic cells including: CD2, surface and cytoplasmic CD3, CD4, CD5, CD7, CD13, CD14, CD15, CD19, CD25, CD33, CD34, CD36, CD38, CD41, CD45, CD56, CD64, CD117, CD123, HLA-DR, MPO and TdT. An add-on panel for BPDCN was also performed in some cases consisting of CD2, CD4, CD7, CD34, CD38, CD45, CD56, CD64, CD123, CD303 and HLA-DR. For each analysis, a minimum of 200,000 events was acquired on CANTO II instruments (BD Biosciences), in which standardization was maintained using CS&T Beads with emphasis on comparable cross-platform performance. Data were analyzed using FCS Express .For the patterns and intensity assessed by flow cytometry, positivity for most markers was defined as expression in ≥20% of cells using the cutoff set by comparison to background fluorescence, with 20%–80% of cells being partially positive and >80% of cells being uniformly positive. A small subset was defined as expression in <20% of cells, but expression was deemed as clearly separated from the negative population.CD123/TCF4 and TCL1 immunohistochemistry was performed following a protocol described previously .ANKRD26, ASXL1, ASXL2,  BCOR,  BCORL1,  BRAF,  BRINP3,  CALR,  CBL,  CBLB,  CBLC,  CEBPA,  CREBBP,  CRLF2,  CSF3R,  CUX1,  DDX41,  DNMT3A,  EED,  ELANE,  ETNK1,  ETV6,  EZH2,  FBXW7,  FLT3,  GATA1,  GATA2,  GFI1,  GNAS,  HNRNPK,  HRAS,  IDH1,  IDH2,  IKZF1,  IL2RG,  IL7R,  JAK1,  JAK2,  JAK3,  KDM6A,  KIT,  KMT2A,  KRAS,  MAP2K1,  MPL,  NF1,  NOTCH1,  NPM1,  NRAS,  PAX5,  PHF6,  PIGA,  PML,  PRPF40B,  PTEN,  PTPN11,  RAD21,  RARA,  RUNX1,  SETBP1,  SF1,  SF3A1,  SF3B1,  SH2B3,  SMC1A,  SMC3,  SRSF2,  STAG1,  STAG2,  STAT3,  STAT5A,  STAT5B,  SUZ12,  TERC,  TERT,  TET2,  TP53,  U2AF1,  U2AF2, WT1 and ZRSR2). The remaining 2 cases were tested using a panel including 28 genes  [Targeted next-generation sequencing methods were used to detect genes commonly mutated in myeloid neoplasms. Among 53 pDC-AML cases, 51 were tested using a panel including 81 genes (and WT1) .p-value of <0.05 was considered statistically significant. Descriptive analyses and other statistical analyses were performed using GraphPad Prism 9 .Chi-square was used to assess categorical variables. A CBFB rearrangement and 10 acute myelomonocytic leukemia. There were 3 cases of MPAL including 2 mixed myeloid/B and 1 mixed myeloid/T. In both cases of MPAL-myeloid/B, myeloblasts were predominant and B lymphoblasts were a minor component, representing 5% and 18% of total blasts. In the case of MPAL-myeloid/T, blasts were positive for multiple myeloid markers  as well as CD7 and cytoplasmic CD3.Our cohort was composed of 53 cases of pDC-AML. There were 36 men and 17 women with a median age of 69 years . Fourteen (26%) cases of pDC-AML showed monocytic differentiation including 4 AML with By flow cytometry, the percentages of pDCs in total nucleated cells ranged from 2% to 26.3% with a median of 6.6% in these 53 cases.Morphology and bone marrow distribution of pDC were assessed in conjunction with CD123/TCF4 immunohistochemistry in 18 cases in which a minimum of 10% pDCs was detected by flow cytometry. All cases showed an interstitial distribution of pDCs intermixed with leukemic blasts A,C on thOn bone marrow aspirate smears, cells with pDC morphology were of medium size with oval to irregular nuclei, dispersed chromatin with frequent small distinct nucleoli, and scant to moderate basophilic cytoplasm polarized at one side with the appearance of pseudopodia B. The immunophenotype of pDCs was assessed in reference to normal/reactive pDCs ,11,15 inRUNX1 mutated; cases # 35–53, RUNX1 wild type) and a representative case of AML-pDC is shown in n = 53), CD15 (n = 53), CD19 (n = 53) and MPO (n = 52).The detailed immunophenotype of pDCs in pDC-AML is illustrated in As noted above, except for uniformly bright CD123 A and briRUNX1 mutated  of the RUNX1 wild type group (p = 0.19). However, CD2 expression by pDCs was conspicuously more frequent in RUNX1 wild type cases   . The expp < 0.0001). In contrast, CD56 was positive in only 8% of pDCs in pDC-AMLs compared to 97% of BPDCN cases positive for CD56 (p < 0.0001). CD303, a specific marker for pDCs, was positive in all pDC-AML cases and only 44% of BPDCN cases were positive (p = 0.046). Of note, the number of pDC-AML cases tested for CD303 in our study was rather small (n = 6). Two myeloid markers CD13 and CD117 were significantly higher in pDC-AML cases, 33% and 47%, respectively, compared to to 0% (p = 0.0001) and 9% (p = 0.0001) in BPDCN. CD22 and CD25 expression was seen in 60% and 51% of pDC-AML cases, respectively, whereas none of BPDCN cases expressed these two markers (p < 0.0001). CD36 and CD38 expression was more frequently positive in pDC-AML than BPDCN (p = 0.04).We compared the immunophenotype of pDCs in pDC-AML to neoplastic pDCs in 39 BPDCN cases in which comprehensive flow cytometric analysis was performed. As shown in an BPDCN  and on tThe myeloblasts in pDC-AML were positive for CD34 in 52 (98%) of 53 cases. CD117 was positive in 50 (94%) of 53 cases. All 53 cases were positive for HLA-DR. Similarly, all 53 cases were positive for CD123 and 47 (89%) showed an increased  and uniform CD123 expression. In total, the immunophenotype of CD34+/CD117+/HLA-DR+/CD123+inc of myeloblasts was observed in 43 (81%) of 53 pDC-AML cases. TdT was positive in 42 (79%) of 53 cases. MPO was positive in 26 (49%) of 53 cases. Of note, although myeloblasts in most cases showed increased CD123 expression, their expression level was still lower than pDCs from the same sample .RUNX1 mutated and wild type cases revealed that cases with mutated RUNX1 cases had more frequent increased CD123 expression than cases with wild type RUNX1 .Immunophenotypic comparison of myeloblasts between Immunostains were performed in 17 cases; CD123 and TCF4 as well as CD123/TCF4 double stain highlighted increased pDCs in all cases and the expression of both CD123 and TCF4 was strong in pDCs. Interestingly, in addition to pDCs, a subset of myeloblasts also showed CD123 and TCF4 expression but their expression level was much weaker than pDCs . TCL1 waRUNX1 was the most frequently mutated gene, detected in 34 (64%) cases, followed by ASXL1 in 19 (36%), DNMT3A in 17 (32%), SRSF2 and BCOR each in 12/51 (24%). NRAS and FLT3 mutations were detected in 12 (23%) patients each. For FLT3, internal tandem duplication (ITD) was detected in 4 cases, D835 mutation in 4 cases, Y842C mutation in 2 cases, V592A mutation in 1 case and an in-frame deletion-insertion mutation (Y591_V592delinsH) in 1 case.The detailed mutation profile is illustrated in RUNX1 mutated cases had other co-mutations detected; ASXL1 and DNMT3A were most frequent, seen in 15 (44%) cases each. Six (18%) showed RUNX1, ASXL1 and DNMT3A mutations simultaneously.All 34 RUNX1 mutated (n = 34) versus RUNX1 wild type (n = 19) subgroups, DNMT3A mutations were more common in the RUNX1 mutated subgroup , whereas TP53 mutations were only detected in the RUNX1 wild type subgroup . There was no significant differences in the frequency of other mutated genes between these two groups.When we divided cases into RUNX1 mutations, only 1 (2%) case of BPDCN showed RUNX1 mutation (p < 0.0001). TET2 mutations were the most common mutations in BPDCN, seen in 28/50 (56%) of cases. In contrast, only 11 (21%) pDC-AML cases showed TET2 mutations (p = 0.0003). FLT3 mutations were detected in 12 (23%) pDC-AML cases whereas no BPDCN case showed this mutation (p = 0.0003). DNMT3A mutations were detected more frequently in pDC-AML than BPDCN , and ZRSR2 mutations occurred less frequently in pDC-AML than BPDCN .We compared the mutation profile of pDC-AML with 50 cases of BPDCN  in whichIn this study, we explored the immunophenotypic and molecular features of pDC-AML and compared these findings with those of blastic plasmacytoid dendritic neoplasm (BPDCN). Our data provide new insights into understanding the cell origin and characteristics of the neoplastic pDC proliferations associated with AML and BPDCN.Using the same 2% cutoff of pDCs as used by others, the frequency of pDC-AML was about 3% in our study, slightly lower than the previously reported occurrence rate , which iRUNX1 mutations were detected in 64%, a frequency slightly lower than 73% and 78% reported in two earlier reports [ASXL1,  DNMT3A,  SRSF2,  BCOR,  TET2,  NRAS and FLT3. The mutation profile of pDC-AML is distinct from BPDCN cases which showed infrequent to absent mutations of RUNX1,  DNMT3A,  BCOR and FLT3, but mutations of TET2 in over half of cases. The immunophenotype of pDCs in pDC-AML was also distinct from BPDCN, with the most remarkable disparities in the frequency of CD34, CD56 and TCL1 expression. pDCs in pDC-AML are uniformly or partially positive for CD34, and mostly negative for TCL1 and CD56; in contrast, BPDCN was completely negative for CD34 and frequently positive for CD56 and TCL1 . The molecular genetic events contributing to pDC expansion/differentiation in RUNX1 wild type pDC-AML need to be further studied.The mechanisms behind pDC expansion/differentiation in pDC-AML were explored by Xiao et al.  who showCD123 targeted therapy has become a frontline therapy for patients with BPDCN given their high level of CD123 expression . Anti-CDpDC-AML is a rare subtype of AML and shows an immunophenotypic and mutation profile that differs greatly from BPDCN, suggesting different origin. The high expression of CD123 in both pDCs and myeloblasts in pDC-AML can be targeted for therapeutic purpose.


Such changes often disrupt transport of calcium ions (Ca2+) in the endoplasmic reticulum (ER) or mitochondria, impacting apoptosis. Evidence rapidly accumulates that this is similar in blood cancer. Principles of intracellular Ca2+ signaling are outlined in the introduction. We describe different Ca2+-toolkit components and summarize the unique relationship between extracellular Ca2+ in the endosteal niche and hematopoietic stem cells. The foundational data on Ca2+ homeostasis in red blood cells is discussed, with the demonstration of changes in red blood cell disorders. This leads to the role of Ca2+ in neoplastic erythropoiesis. Then we expand onto the neoplastic impact of deregulated plasma membrane Ca2+ channels, ER Ca2+ channels, Ca2+ pumps and exchangers, as well as Ca2+ sensor and effector proteins across all types of hematologic neoplasms. This includes an overview of genetic variants in the Ca2+-toolkit encoding genes in lymphoid and myeloid cancers as recorded in publically available cancer databases. The data we compiled demonstrate that multiple Ca2+ homeostatic mechanisms and Ca2+ responsive pathways are altered in hematologic cancers. Some of these alterations may have genetic basis but this requires further investigation. Most changes in the Ca2+-toolkit do not appear to define/associate with specific disease entities but may influence disease grade, prognosis, treatment response, and certain complications. Further elucidation of the underlying mechanisms may lead to novel treatments, with the aim to tailor drugs to different patterns of deregulation. To our knowledge this is the first review of its type in the published literature. We hope that the evidence we compiled increases awareness of the calcium signaling deregulation in hematologic neoplasms and triggers more clinical studies to help advance this field.Intracellular calcium signaling regulates diverse physiological and pathological processes. In solid tumors, changes to calcium channels and effectors  Spec in HSCs . This coal space . On the g events .2+ signaling. High Ca2+ levels in the endosteal niche have been shown to assist homing of HSCs through their calcium-sensing receptor , taking Ca2+ in from the extracellular space, or extruding it from the cell .Cytoplasmic free Ca systems  and moduthe cell . Various toolkit , 24, is 2+ storage in almost any cell, as this functionality is often deregulated in cancer including blood cancer   receptors (IP3Rs) (2+ from the ER upon binding of IP3 generated by phospholipase C (PLC)   . Many pa (IP3Rs)   . PLC ope C (PLC) , 30. Whey (SOCE) , 32. SOC cytosol , 32 STIM <300 μM . Loss of content . PLC als content , 34  and the membrane potential (ΔΨm ∼ −150 mV) . Ca2+ uptake into mitochondria activates pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase, thereby stimulating the tricyclic acid cycle and energy generation  formed when VDAC1 clusters with adenine nucleotide translocase  and cyclophilin D . The mPTP opening causes depolarization of the inner mitochondrial membrane, which uncouples the respiratory chain leading to increased mitochondrial membrane permeability and the release of cytochrome c  . Small aneration , 40. In poptosis , 42. Prochrome c , 35, 36.2+ storage and release mechanisms (2+ stores (2+ to the cytosol mainly through two-pore channels (TPC1-TPC2) and TRP mucolipin channels (TRPML1-TRPML3) activated by second messengers such as nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca2+-mobilizing second messenger known  as historically, this work provided guidance for research into Ca2+ signaling in selected blood cancers. We then focus on the neoplastic impact of deregulated Ca2+ influx through the plasma membrane and the ER, Ca2+ efflux via Ca2+ pumps and exchangers, and the impact of deregulated Ca2+ sensor and effector proteins in blood cancer. Throughout the review we highlight potential therapeutic strategies being developed to abrogate this deregulation.This review emphasizes importance of abnormal Ca2+ homeostasis in RBCs has a long history and has been regularly reviewed  that pump Ca2+ from the cytosol into vesicles and thus protect the cytosolic and membrane proteins from Ca2+-induced damage   to induce Ca2+ oscillations under specific conditions. Due to the broad variance in channel copy number per cell, there is variation in RBC responses to stimulation, and the numbers of “responding cells” typically range from 10% to 30%    , N-methyl-D-aspartate (NMDA) receptors, and VDACs  channels transduce changes in the plasma membrane potential to intracellular Ca2+ transients that initiate many crucial physiological processes    or in neassemia) , 111. CA+ levels . In norm+ levels . In cont of CALR . Elevate2+, cell dehydration and the presence of organelle remnants in RBCs have the potential to promote thrombosis in PV , nicotinic acetylcholine receptor (nAChR), Piezo1, NMDA receptor, and the Ca2+ selective Orai1 channels  and in AML cell lines  . TRPM2 dTRPV1, TRPV6 and TRPM2 contribute to the growth of cells derived from acute lymphoblastic leukemia (ALL) , 172. TRIn chronic lymphocytic leukemia (CLL) cells, patient-derived and the Jok-1 cell line, TRPC1 plays a role in promoting cell survival. It does so by contributing to the production of anti-inflammatory cytokines and the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways triggered by CD5 activation , 168. TRTRP genes have been reported in 33 cancer types including hematologic malignancies, in particular diffuse large B-cell lymphoma (DLBCL) and AML cells  and bulk AML cells compared to wild-type. The transcription factor cAMP-response element binding protein (CREB), which is involved in calcium signaling, is decreased in P2X7-null LICs and upregulated in AML patients. When CREB is overexpressed in P2X7-null AML cells, the development of leukemia is similar to wild-type AML cells . An increase in extracellular ATP, often due to damage to the plasma membrane or exocytosis of ATP-containing granules, is the principal physiological stimulus for P2X receptor activation . Alteredf P2X7RA . AML devf P2X7RA . Ca2+ inML cells . These rv channels leading to Ca2+ influx  signaling, and the expression of transcripts involved in extracellular matrix remodeling  and are subdivided into three families CaV1, CaV2 and CaV3  and mediate long-lasting (L-type) currents with slow inactivation. In contrast, CaV3 channels are activated by low voltage (around −60 mV with a peak at −20 mV) and mediate transient currents (T-type) with faster kinetics than the L-type currents. CaV2 channels are activated by high voltage and mediate P/Q-type, N-type and R-type Ca2+ currents  in AML and B-ALL samples and of CACNA1A (coding for CaV2.1) in samples from patients with CLL, marginal zone lymphoma and monoclonal gammopathy of unknown significance (CACNA1C transcripts (coding for CaV1.2) was seen in centroblastic lymphoma, CACNA1F (coding for CaV1.4) in anaplastic large cell lymphoma, and CACNA1G (coding for CaV3.1) in mantle cell lymphoma (V channels in a range of lymphoma cell lines and patient-derived samples (V1.2 (CACNA1C) expression was increased in classical Hodgkin lymphoma cell lines when compared to other B-cell lymphoma cell lines. CaV1.3 (CACNA1D) showed higher expression in samples from patients with activated B-cell-like DLBCL (ABC-DLBCL), whereas expression of CaV1.1 (CACNA1S), CaV1.2 (CACNA1C), and CaV1.4 (CACNA1F) were higher in germinal centre B-cell like DLBCL (GCB-DLBCL)  from pediatric patients (V1.2 channel in AML-MSCs using lercanidipine (an anti-hypertensive drug) interfered with leukemia growth ex vivo and in vivo, with no toxic effects on normal MSCs or healthy CD34-positive HSCs  . Therapepatients . Inhibitive HSCs . Further2+. SOCE is triggered when Orai1 and STIM1/STIM2 proteins interact in response to ER Ca2+ store depletion. Increased expression of Orai1 or STIM1/STIM2 has been recorded in cell lines derived from AML  in immature and mature B-cells to modulate cell proliferation and differentiation  is part of the membrane-spanning 4-domain family, subfamily A (MS4A) . The exantiation . CD20 hantiation –201.2+ influx in patient CLL cells and cell lines such as SUDHL-4, BL2, Ramos, BL60, Raji, Daudi, and normal B-cells  are multipotent, proliferative, and self-renewing cells that propagate leukemia . In AML llations , 212. InAML LSCs , 212BCR and TCR recruit kinases such as SYK (spleen tyrosine kinase), BTK (Bruton’s tyrosine kinase), BLNK (B-cell linker kinase), and ZAP70 (zeta chain of T-cell receptor-associated protein kinase 7) to phosphorylate and activate PLC-γ. PLC-γ2 plays a role in CLL, DLBCL, Hodgkin lymphoma, endemic Burkitt lymphoma, MALT-associated gastric lymphoma, and plasma cell myeloma . For exa2+ and increased cell survival  and pro-apoptotic members     , 218. Bcand BAK) , 220. Toand BAK)  , which inhibits mitochondrial Ca2+ uptake, both counteract BIRD-2-induced cell death , expression of which is elevated in patient-derived AML cells and cell lines  and apoptosis  were observed. This suggests a link between lysosomal biogenesis, NMDA receptor function and Meg-01 cell proliferation  (+/Ca2+ exchanger NCX that actively extrudes Ca2+ while taking in Na+ transported passively using the energy of transmembrane Na+ gradient generated by the Na+/K+ ATPase (+(Li+)/Ca2+ exchanger (NCLX) that controls Ca2+ levels in the mitochondria (2+ out of the cytoplasm into the Golgi apparatus through secretory pathway Ca2+ ATPases (SPCA), and to the ER through SERCA  , 242 , calcineurin, CALR, and PKC. Dysregulation of these Ca2+ sensors alters gene regulation associated with cell apoptosis, proliferation, and migration   . Dephosps (NFAT) . The cals (NFAT) . Inappros (NFAT) , 314 (ses (NFAT) .Calcineurin and NFAT have also been implicated in other hematologic malignancies, including Burkitt lymphoma, T-cell lymphoma, T-ALL, DLBCL, CML, CLL, and AML , 316. Th2+-buffering protein that helps maintain intracellular Ca2+ homeostasis and assists the folding of proteins destined for secretion or insertion into the plasma membrane  (2+-toolkit encoding genes were queried as gene sets grouped according to function (see 2+-toolkit encoding genes (5.9%) and patients with low-grade T-cell neoplasms had the highest frequency (48.8%), most carrying multiple variants  , 355. Thvariants Table 3.patients  , TRPC and TRPM channels (11.6% each), CaV and NMDA receptor channels (9.3% each), ryanodine receptors (RYR) (7.0%), and a few others at 4.7% each  , Cad SERCA) Table 3.tabolism . Multipl T-cells , 357–361RYR1-3 were the most common (4.5%), followed by PLC (3.2%), CACNA (2.8%), TRPM (2.6%) and TRPC genes (2.1%), with others present in <2% of patients. Some of these mutations appeared exclusive between each other. In DLBCL, RYR mutations were less common (2.5%) but CACNA variants were more common (3.8%)  and TRP genes  were the most common in AML but no variants exceeded 3% frequency. MDS patients had a lower frequency of variants at 2.3% overall, although data for these patients was limited. In the MPN group, 17% had CALR mutations, consistent with it being a known MPN driver . It was intriguing that these variants were exclusive with CALR mutations and also appeared exclusive with each other, raising the possibility of their independent effects in MPN  (depending on the gene)  may lead to mitochondrial Ca2+ overload triggering apoptosis. Therefore, to escape apoptosis, cancer cells shift to preferentially utilize extracellular Ca2+ transferred into cells by overexpressed or overactive plasma membrane Ca2+ channels. On the other hand, mechanisms that utilize ER-derived Ca2+ may be reduced, and overactive SERCA2 pumps Ca2+ back into the ER. This remodeling of Ca2+ signaling helps cells maintain heightened oncogenic signaling while shielding mitochondria from Ca2+ overload. The examples of mechanisms providing anti-apoptotic effects include Bcl-2-mediated inhibition of IP3R in DLBCL and reduced ER Ca2+ binding by mutated CALR in MPN. In addition, intracellular mediators of Ca2+ signaling may be overexpressed or overactive e.g. PLC, PKC, ORP4L, calcineurin and CaMK. Understanding how Ca2+ signaling remodels in cancer cells creates therapeutic opportunities, with the potential to spare normal cells and tailor therapies according to the underlying mechanism in different cancers/patients , prognosis including responsiveness to chemotherapy , and complications (e.g. thrombosis and bone marow fibrosis in MPN). Deregulation of Ca2+signaling provides an opportunity to design novel therapeutic interventions. Some options are currently investigated mostly at the pre-clinical level in various cancer models . Similar opportunities are being considered in solid tumours, which may facilitate faster clinical translation to blood cancer. Future research to define the role of specific Ca2+ regulatory mechanisms in different blood cancer types will be challenging but such work is likely to advance therapies.In conclusion, multiple CaMLK-Z conceived and designed the work. TI and MLK-Z wrote most of the paper. JL and TNG helped with the literature review, figure preparation, and writing of specific sections; JL wrote the TRP section, TNG wrote about VDACs and helped analyze the cBioPortal data. AB provided advice and wrote about calcium homeostasis in mature red cells. All authors contributed to the article and approved the submitted version.Bone Marrow Cancer Research Trust (Christchurch) (UoA 9102-3720536) and Auckland Medical Research Foundation (Funder reference 1119009) provided salary funding to staff working on this project but had no influence over any aspects of the work or the decision to publish.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Efficient neutron transport is a key ingredient to the performance of ultracold neutron (UCN) sources, important to meeting the challenges placed by high precision fundamental physics experiments. At the Paul Scherrer Institute’s UCN source we have been continuously improving our understanding of the UCN source parameters by performing a series of studies to characterize neutron production and moderation, and UCN production, extraction, and transport efficiency to the beamport. The present study on the absolute UCN transport efficiency completes our previous publications. We report on complementary measurements, namely one on the height-dependent UCN density and a second on the transmission of a calibrated quantity of UCN over a  Ultracold neutrons (UCNs) are neutrons with kinetic energies below about 300 neV, equivalent to temperatures below 3 mK. They are key probes in particle physics experiments at the low-energy frontier. UCNs are reflected at any angle of incidence from certain materials like steel, beryllium, nickel, diamond-like carbon (DLC) or alloys like nickel-molybdenum (NiMo), since they cannot penetrate a sufficiently thick nuclear potential barrier of the material which is higher than their kinetic energy. Hence, UCNs can be stored in bottles made from or coated with these materials and can be observed for hundreds of seconds . This maThe high intensity UCN source at the Paul Scherrer Institute (PSI) , 14–17 hThe UCN intensity at the PSI source was considerably increased over the last years. At the same time, we have been improving our understanding of the UCN source parameters. In a series of studies, we covered neutron production and moderation, UCN production and extraction out of the sDN optics , 19–21. UCN transport characteristics of the individual neutron guides were measured prior to mounting, and the results reported in Ref. . A previous work , this wamentclass2pt{minimous work  that, alThe obtained results provide fundamental input to better analyze the UCN source performance, help to identify sections for further improvements, and allow for more precise estimations of the statistical sensitivity of experiments. In Sect. In the following we describe the two main experiments: (a) UCN transmission from beamport West-1 to beamport South, allowing to constrain the loss and diffuse reflection parameters of the guide coatings, and (b) UCN storage at different heights above beamport level, aiming to find the vertical position maximizing UCN density, and helping to constrain the shape of the energy spectrum of the UCN exiting the sDThe measurements presented in this section test the neutron optics above the central storage vessel flaps. The idea of the measurement, with the involved parts illustrated in Fig. In order to quantify how many UCNs started towards the beamport South after their release from the external storage vessel at beamport West-1, a reference measurement was necessary. In this reference measurement the UCNs were released by opening shutter 2, on the other side of the external chamber, towards detector West-1. By using identical filling and storage times as in the ping-pong procedure, and by performing the measurements within a short time to assure UCN source stability, we made sure that the correct initial number of UCNs was obtained.The transmission efficiency of the UCNs from one beamport to the other beamport then depends on all cumulative losses in the guides, windows, storage vessel, and possible slits between the components. Although all UCN guides were tested prior to installation and commissioning of the UCN source , this inA 25 L external storage vessel made of a 1 m long piece of UCN guide (NiMo coated glass tube I.D. 180 mm) and two shutters  coated with DLC were mounted at beamport West-1. UCNs could be emptied in both directions, either towards the UCN storage vessel of the source via shutter 1 or towards a small .t = 10.7 s, end of the proton pulse.tduring and after the proton beam pulse the external storage vessel is filled for 8.6 s with shutter 1 open and shutter 2 closed (this filling time was optimized beforehand for a maximum number of stored UCN).t = 19.3 s, shutter 1 closes.t = 20.7 s, i.e. 1.4 s later, the main flaps of the UCN storage vessel open and for 110 s the UCNs can fall down towards the solid deuterium vessel where they are absorbed. Hence, the storage vessel and connected guides are quickly emptied. The UCN count rate in detector South drops rapidly after opening the flaps and reaches the background level of t = 130.7 s, the storage vessel flaps close again.t = 149.3 s, after a storage time of 130 s, one shutter of the external storage vessel opens, releasing the stored UCNs. When shutter 2 opens, a reference measurement of the stored UCNs is done. When shutter 1 opens, a transport measurement to beamport South is done. The detector behind the closed shutter 2 measures the background simultaneously. After traversing the source UCN guides and the source storage vessel, a fraction of the UCNs reaches the detector at beamport South. The observed number of UCN counts with respect to the time after the beam pulse is shown in Figs. t = 330 s, all shutters are reset into the initial position, shutter 1 open, shutter 2 closed, storage vessel flaps open.t = 360 s, next proton pulse.The time sequence of the measurement was the following :Figure The big and small Cascade detectors have different detection efficiencies, due to their difference in size, different connection to the UCN guide, and different aluminum foils serving as entrance window, which can have different transmissions . This reWith detectors directly attached to the external storage bottle at beamport West-1, we measured their relative detection efficiencies, R of UCNs asThe different detection efficiencies were taken into account when we calculate the corrected fraction, The corrected fraction of UCNs reaching the detector at beamport South is mentclass2pt{minimThe UCN transmission of the safety window in the center of the magnet decreases with higher field-values as expected, as UCNs with the “right” spin state are accelerated by the magnetic field (‘high field seekers’) and pass, while UCNs with the “wrong” spin state (‘low field seekers’) are decelerated, and can only pass if their kinetic energy is higher than the repelling potential of 60.3 neV/T. The polarizing magnet can fully polarize the UCN beam, because the maximum magnetic field-strength of 5 T results in a potential barrier of 300 neV for the “wrong” spin state that is higher than the Fermi potential of all guide walls (about 220 neV). However, the effective transmission rate of the magnet, taking into account the vacuum safety window made of a 100 mm thick AlMg3 foil, mounted at the point of highest magnetic field in the center of the magnet, does not decrease to 0.5. This is because high-field seeking UCNs are accelerated and hence have a higher transmission through the foil.The measured and corrected fractions of UCNs with respect to the magnetic field strength in the polarizing magnet are shown in Fig. Fitting the time distribution of the UCN counts from the external storage bottle of the reference measurement, see Fig. As additional input for the simulation we also measured the UCN transmission through the AlMg3 foils from the same batch as the guide safety windows , which was built to compare UCN densities at various UCN sources  and hencThe A sketch of the setup is shown in Fig. The measurements were performed over a period of several days and thus the UCN source performance changed during this time, e.g., due to changing proton current or sD2 surface degradation . In orde in Ref.  it was f in Ref. . This beThe height dependence of the UCN density measured after storage times of 2 s, 20 s, 50 s is plotted in Fig. The elevated height was measured from the bottom edge of the UCN guide at the exit of shutter 2 to the bottom of the UCN guide at the entrance of the standard storage bottle at shutter 3. UCN densities between  in Ref. , measureThe storage-time constant in any storage bottle depends on the kinetic energy spectrum of the UCNs. This spectrum in turn depends on the elevation of the volume above the beamport, since the UCNs are slowed down by gravity. This implies lower bounce-rate and thus smaller losses. The storage-time constants obtained from discrete storage times were confirmed using the leakage rate of UCNs through shutter 4 during the storage measurements of times up to 100 s and fitting with a single-exponential function.Figure The UCN transmission of the crank-shaped UCN guide was measured in storage mode by comparing the horizontal storage measurement to a storage measurement without the crank-shaped guide. At the height of the beamport, a transmission of The MCUCN code used in this work was developed at PSI. The code and its physical parameters are described in detail in . The simExperiments (i)–(iv) were used to determine the wall loss coefficient, mentclass2pt{minimWe need two independent model sections for the simulation of the two measurement setups described in the previous chapters. Both are added after the beamport in separate simulations, attaching them to the first part of the MCUCN model that includes the entire system of UCN source and its UCN guides. On the one hand, the coating parameters of the guides up to the beamports − constrained from the ping-pong experiment − influence the energy spectrum of the UCNs which can be stored in the second experiment, with the standard storage bottle placed at various heights. On the other hand, this energy spectrum will determine the transmission of UCNs which is detected in the ping-pong experiment. Therefore, in the simulations we performed several iterations in scanning the parameter space aiming to minimize the deviation between measured and simulated UCN counts.The MC simulation of the ping-pong measurement followed the same steps as taken in the experiment: 8.6 s filling of the external storage volume, 130 s of UCN storage, cleaning the source volume via the main flapper valves, and counting of UCNs released after storage towards the detector at beamport South. In the simulation model the reference counts were simulated by opening shutter 2 towards the big Cascade detector at beamport West-1 and using this to fit the model parameters to best match the corrected fractions from the measurement listed in Table The time-dependent fraction of the opened area of the two UCN shutters of the external storage vessel , 25 was The parameters of the source storage vessel were set to the values obtained in . UCN depWe performed MC scans in the 2D parameter space, see Fig. We identify in the 2D plot a linearly-shaped valley displaying a linear anti-correlation between the loss parameter and the fraction of diffuse reflections.A second constraint was obtained from a very similar parameter scan in which we fitted the emptying time-profile in detector South in a supplementary measurement to ping-pong, see Figs.  in Ref. . This caThe combined results from the previous constraints are shown in Fig.  in Ref. .The violet area in Fig. In a next set of simulations, the count fractions in detector South as a function of the magnetic field in the polarizer were compared to the measurements. The results were compiled in Table As a cross-check of the ping-pong simulation results, independent of the MC fitting procedure, we compared the measured and simulated time-profiles in the ping-pong experiment. For this we used the final fit parameters. The simulated peak in the detector at beamport South, shown in Fig. The transmission of UCNs through AlMg3 foils for the safety windows was measured as reported in . We used in Ref. . The difIn the MCUCN simulation model of the storage bottle measurement at various heights we reproduced the full storage measurement procedure, including setting the height above the beamport by rotation of the crank-shaped UCN guide, according to details described in the experimental section. We used separate parameters for this setup starting at the beamport with the stainless steel guide bend, and the source part upstream of the beamport with parameters determined in the ping-pong measurement.Since we were interested in simulations of relative counts, the vertical detector arm connected by a 90 deg bend to the standard storage bottle was simplified in the model to an ideal UCN counter  just after the diameter-reduction piece. A detailed model of the L-shaped tube would have needed additional uncalibrated parameters e.g. for diffuse reflections and gaps. We assumed a negligible change in the detection efficiency of the measurement at different heights thanks to the about 1 m vertical acceleration of the UCNs before the detector foil.The simulated height dependence of the UCN density after 2 s, 20 s, and 50 s of storage is shown in Fig. a loss parameter for steel, an optical potential of e 9.2 in , but witin order to reproduce the measured transmission of the crank-shaped guide, an exponent From Figs. The fit parameters and their uncertainties for the stainless steel walls and the spectral exponent, 3.0\pm 0.\times 10By comparing the MCUCN simulation model to a series of experimental data, we were able to reproduce various measurements within about 2Based on the benchmarks presented in the previous sections, see calibrated parameters in Table  cument}2 , 21. TheThe spectrum is cleaned from neutrons above the UCN energy range about 12 s after the start of the proton pulse, as shown in Fig. N is the ratio of the counts integrated over all arrival times and, It can also be useful for the analysis of the UCN source output to calculate the transmission of UCNs between the sDmentclass2pt{minimOur UCN transport and storage measurements, and their reproductions in simulations presented here constitute an important part of a series of studies aiming at the characterization of the UCN source components at PSI in view of further improvement of their performance.Measurements of the UCN transmission from beamport-to-beamport through the guide system and central storage vessel of the PSI UCN source were performed with high statistics. The timing was chosen such that signal and background regions were well separated. The transmitted fraction of UCNs agrees well with detailed Monte Carlo simulations, which were benchmarked along with additional measurements.A second type of experiment, UCN storage at different heights above the beamport in a standard storage bottle, allowed maximizing the UCN density in this storage bottle by optimizing its vertical position. The UCN density shows a flat maximum at around 800 mm of height. This optimization procedure enabled estimating an approximate function for the energy spectrum of the UCNs exiting the moderator. This obtained function indicates a loss of neutrons in the lowest energy range, as expected, considering the poly-crystalline structure of the sDoderator , 33.By comparing the MCUCN simulation model to experimental data, we were able to reproduce various measurements and extract UCN loss and diffuse reflection parameters for the South and West-1 source guides. This demonstrates that the UCN optics of the PSI UCN source is well understood. The present study provides a guideline for possible improvements of source components.We could also derive the energy spectrum of UCNs arriving at the detector at the beamport. It is clearly shown that UCNs with energies well above the neutron optical potential of the UCN guides arrive at the beamport despite of the UCN guide length of about 8 m. We have shown that these neutrons disappear within about 12 s and a storable UCN energy spectrum remains.As main application, the benchmarked MCUCN model presented here was employed to predict the statistical sensitivity of the n2EDM apparatus connected to the UCN source model and to find the optimal height of the double-chamber, assuming typical parameters for the UCN optics of the experiment . Our sim


Furthermore, our model could capture delayed afterdepolarizations (DADs), early afterdepolarizations (EADs), and contraction abnormalities in terms of aftercontractions triggered by either drug action or special pacing modes. Finally, we further validated the mechanical results of the model against previous experimental and in silico studies, e.g., the contractility dependence on pacing rate. Adding a new level of applicability to the normative models of human cardiomyocytes, BPSLand represents a robust, fully-human in silico model with promising capabilities for translational cardiology.Contractility has become one of the main readouts in computational and experimental studies on cardiomyocytes. Following this trend, we propose a novel mathematical model of human ventricular cardiomyocytes electromechanics, BPSLand, by coupling a recent human contractile element to the BPS2020 model of electrophysiology. BPSLand is the result of a hybrid optimization process and it reproduces all the electrophysiology experimental indices captured by its predecessor BPS2020, simultaneously enabling the simulation of realistic human active tension and its potential abnormalities. The transmural heterogeneity in both electrophysiology and contractility departments was simulated consistent with previous computational and  The future of diagnosis and treatment in cardiology progressively depends on advanced methods in imaging, gene profiling, and pharmaceutical technologies. Despite the recent advances in health technologies, the current empirical clinical investigations face serious challenges as the complexity of therapeutic interventions, prognosis, and the possibility of classifying the treatments grow. Specifically, identifying the optimal treatment strategy with a degree of statistical significance poses serious challenges to current empirical routes in cardiology . Furtherin silico model of human ventricular cell electrophysiology  and includes specific mechanisms not simulated by ToR-ORd . Given these improvements in simulating electrophysiology phenomena, it is worth investigating how we can expand the spectrum of BPS2020 simulation, by using it as the basis for a new electromechanical human CM in silico model.Our recently published BPS2020 model of the human adult ventricular CM electrophysiology , holds sin silico model meeting the computational expectations from both departments, the electrophysiology and contractility, with the potential for facilitating the translation of biophysical and pharmacological functions into pre-clinical readouts.We have integrated one of the most recent human contractile machinery (LandCE)  into BPS50, TaRT95) and the time-to-peak (TaTTP). AP biomarkers are the duration at 30%, 40%, 50%, 70% and 90% , the maximum upstroke velocity (dV/dtmax), the peak voltage (VPeak) and the resting membrane potential (RMP). In silico biomarkers were computed as in +]o = 4 mM. Conversely, as no information was reported on the in vitro Ta biomarker ranges, we run our simulations at the standard concentration [K+]o = 5.4 mM.To calibrate the BPSLand model, we used a dataset of isometric active tension (Ta) biomarkers recorded from human isolated ventricular CMs  and a da50 and CaT relaxation time at 50% (CaRT50) rate adaptation data  APD rate dependence, restitution and accommodation data in control condition and with current blockers from endocardial CMs  see Sup; 2) TaPeion data ; 3) TaPeion data .+ currents , transient outward K+ current (Ito), L-type Ca2+ current (ICaL), also with its Na+ and K+ components , the rapid, slow and inward rectifying K+ currents , the Na+/Ca2+ exchanger divided in its cytosolic and subspace components , the Na+/K+ pump (INaK), Na+, K+ and Ca2+ background currents  and the sarcolemmal Ca2+ pump (IpCa). Ca2+ fluxes from/to SR are the RyR-sensitive Ca2+ release (Jrel), the SERCA pump (Jup) and a leakage flux (Jleak).The original BPS2020 model  was base2+ concentration [Ca2+]i computed by BPS2020, to update a new state variable CaTRPN, representing the fraction of troponin C units which bound to Ca2+.We integrated LandCE into BPS2020 following the approach presented in . Shortly2+ buffered by troponin C, [Ca2+]TRPN, to update the intracellular Ca2+ concentration.2+ concentration that can bind to troponin C.BPS2020 receives as feedback the amount of Cabi,sim is the ith simulated biomarker, LBi the ith experimental lower bound for bi,sim, UBi the ith experimental upper bound for bi,sim, wi the weight for each biomarker’s cost , followed by the simplex optimization [Matlab function fminsearchbnd (2+ sensitivity ([Ca2+]T50), for which we chose  instead of . As the original range  increased substantially the CaT peak, i.e. the systolic Ca2+ (+22%), we decided not to affect the BPS2020 electrophysiology and chose  as it preserved the original BPS2020 CaT peak.After integrating LandCE into BPS2020, we first optimized the LandCE parameters using a hybrid approach combining first a genetic optimization  and the minimum of the diastolic active tension, systolic and diastolic intracellular free Ca2+ (CaSys and CaDiast). The acceptable ranges for these biomarkers were taken from the original Land publication , time-to-peak (TaTTP), relaxation time to 50% and 95% of the diastolic level (TaRTlication  for TaTT2+ fluxes of the SERCA pump (Jup) and the RyR-sensitive release (Jrel), using all the constraints in max), AP duration at 40%, 50% and 90% , and the triangulation metric (Tri9040), we set the lower and upper bounds as ±5% of their values in the original BPS2020 model, which were fit against the experimental data  and 3.13 (mM/ms), respectively. At the end of this second step, the electromechanical model correctly simulated TaRT95 and TaPeak while the remaining contractility biomarkers were close to their respective lower bounds. However, we missed one of the key features of BPS2020, i.e., the inverse relationship [Ca2+]o – APD90, which was otherwise simulated at the end of the first step.In order to capture the remaining contractility biomarkers, we then run a second simplex optimization on the Catal data  at [K+]o2+]o – APD90 relationship, we added one final step to our pipeline, where we did a minor manual re-tuning of Jrel,max (0.0240 →0.0220 1/ms) and Jup,max (3.1333 → 3 mM/ms), still considering their lower and upper bounds as in rel,max and Jup,max on the [Ca2+]o – APD90 relationship.In order to restore the [Ca+]o = 5.4 mM, [Ca2+]o = 1.8 mM and [Na+]o = 144 mM as extracellular ion concentration. We then compared simulated TaPeak, TaRT50 and CaRT50 with the in vitro data by To test the active tension dependence on the applied pacing rate, we paced BPSLand at 0.5, 1, 1.5, 2, 2.5 and 3 Hz for 1,000 beats to reach the steady state, using o = 5.4 mM, [Ca2+]o = 1.8 mM, [Na+]o = 144 mM) and cycle length (CL) of 4,000 ms. The second EAD protocol simulated dofetilide, similarly to what we did in +]o = 5 mM, [Ca2+]o = 2 mM, [Na+]o = 137 mM), using the IKr drug binding values reported by We assessed the occurrence of early-afterdepolarization (EADs) and aftercontractions in the BPSLand following three different protocols. First, we simulated the administration of quinidine considering the drug effects on ITo assess the occurrence of delayed afterdepolarizations (DADs) we used the same protocol as in 2+]i, [Ca2+]ss and Ta traces simulated at [K+]o = 5.4 mM in 2+]o to highlight the inverse APD90-[Ca2+]o dependence, which was described first by in vitro and in vivo  and amplitude (CaAmp). All the AP biomarkers are within the experimental ranges, as well as TaPeak and TaRT95. Conversely, TaRT50 and TaTTP are very close to their respective experimental lower bounds, although out of the in vitro ranges. The comparison of AP, Ca2+ and Ta traces simulated with [K+]o = 4 mM and [K+]o = 5.4 mM is presented in We report the AP, . Convera (CPVT) . In facta (CPVT) . These va (CPVT) , impairea (CPVT) , arrhytha (CPVT) . Furthera (CPVT) . Finallya (CPVT)  that cana (CPVT) : with noin silico model of human adult CMs, combining both electrophysiology and contractility. As the electrophysiology description by BPS2020 carried a few novelties, especially the APD-[Ca2+]o relationship and an extended and more reliable description of Ca2+ handling, including the generation of DADs, it was important to us to create a model able to translate such novelties also to contractility. Of note, we did not aim to simulate here specific pathological conditions affecting contractility, as this will be topic for future works.Therefore, the goal of our work was to provide the new and validated BPSLand 2+ concentration computed by BPS2020, to compute the fraction of troponin C units bound to Ca2+, and this new flux of Ca2+ towards the sarcomere is then included in the equation regulating the BPS2020 cytosolic Ca2+ concentration, to close the loop. In terms of mathematical formulation, the process was straightforward, as BPS2020 and ToR-ORd are both based on the original ORd model. For the optimization of the model, we built our cost function with the same biomarkers  and experimental ranges as in Land et al. and Margara et al., and we tuned the same parameters  within the same ranges, except for the Ca2+ sensitivity [Ca2+]T50. In both Land et al. and Margara et al., [Ca2+]T50 was optimized within . However, values in that range would have altered too much the CaT amplitude of BPS 2020. Land et al. already reported that such parameter “needs to be fit depending on the calcium transient used to drive the model,” as it is not consistent inter-species and also variable in their experiments on skinned human CMs. Therefore, we optimized [Ca2+]T50 in the range , which allowed us to keep the same CaT morphology and magnitude of the original BPS 2020. As we reported in 2+ fluxes again to keep the BPSLand Ca2+ handling the most similar to BPS2020. Already at this stage, the resulting model would have satisfactorily simulated the considered AP and Ta biomarkers. However, it lost the ability to simulate the inverse APD-[Ca2+]o dependence for high [Ca2+]o values. Such dependence was one of the key-novelties of BPS2020 . The final BPSLand model satisfactorily simulates AP and Ta biomarkers, together with the APD-[Ca2+]o inverse dependence . However, to our current knowledge, there is no such kind of data collection.We first validated BPSLand against the same AP data (APD rate dependence and restitution in control condition and with current blocker) used to validate BPS2020 and the original ORd model. The rationale is we want BPSLand to work as well as BPS2020 in simulating electrophysiology data. As we already presented in detail those simulations in in vitro experiments performed on human CMs and cardiac preparations  and TaPeak (M > EPI > ENDO) sequences, although the TaPeak values are greater in ToR-ORd+Land than in our model . In fact, although we used the same TaPeak range as in in vitro values , larger GCaL (thus being prone to more significant reactivation of ICaL during phase 3 of the AP) and Jrel,max . From the in vitro point of view, the higher sensitivity of M cells to drugs affecting repolarizing ion currents compared to endocardial and epicardial was reported by CaL reactivation-driven and RyR spontaneous opening-driven EADs, as we also report here in CaL-driven EADs. Similarly to RyR spontaneous opening-driven EADs, also DADs are source of aftercontractions (in vitro following the administration of diverse compounds or in presence of mutations in several cardiac preparations, e.g., cardiac tissues and trabeculae following dofetilide administration (Abnormalities in the ionic regulations of cardiomyocytes e.g., EADs and DADs can trigger the occurrence of a contractile irregularity in form of aftercontractions  the inciractions . BPSLandstration , in myocstration , or in Cstration .2+ regulation and mechanics. Despite this, the experimental in vitro human data, taking into account both electrical and mechanical aspects, are still few, and urgently required to ensure better insight in electromechanical coupling and design more accurate models. The BPSLand model itself has some limitations. Preload and afterload conditions contribute to contractility response and should be considered in future model developments by including a mathematical description of dynamic changes in sarcomere length, since now only the isometric condition can be simulated. Other previously published mechanical models, e.g., 50in vitro data, it slightly underestimates the TaRT50, as BPSLand simulates very similar CaRT50 and TaRT50 for each tested rate. One reason could be a slightly too fast relaxation dynamic in the contractile element. However, we replicated the same test with a second in silico model (The proposed computational model can be used to better understand the electromechanical interactions and the strong relationship between Caco model  and an ein vitro experiments. In addition to replicate correctly the results produced by its predecessor BPS2020, BPSLand adds an accurate simulation of active tension and contractility abnormalities, which can be triggered by drugs or specific pacing protocols. Therefore, BPSLand expands the domain of applicability of in silico model, which traditionally focus mainly on the simulation of the cardiac cell electrophysiology.In this paper, we presented our new electromechanical model of human adult ventricular cardiomyocyte, built and validated using several sets of human


Despite many recent advances in treatment options, acute myeloid leukemia (AML) still has a high mortality rate. One important issue in optimizing outcomes for AML patients lies in the limited ability to predict response to specific therapies, duration of response, and likelihood of relapse. With evolving genetic characterization and improving molecular definitions, the ability to predict outcomes and long-term prognosis is slowly improving. The majority of the currently used prognostic assessments relate to molecular and chromosomal abnormalities, as well as response to initial therapy. These risk categories, however, do not account for a large amount of the variability in AML. Laboratory techniques now utilized in the clinic extend beyond bone marrow morphology and single gene sequencing, to next-generation sequencing of large gene panels and multiparameter flow cytometry, among others. Other technologic advances, such as gene expression analysis, have yet to demonstrate enough predictive and prognostic power to be employed in clinical medicine outside of clinical trials, but may be incorporated into the clinic in the future. In this review, we discuss the utility of current biomarkers, and present novel biomarker techniques and strategies that are in development for AML patients. Measurable residual disease (MRD) is a powerful prognostic tool that is increasingly being incorporated into clinical practice, and there are some exciting emerging biomarker technologies that have the potential to improve prognostic power in AML. As AML continues to be a difficult-to-treat disease with poor outcomes in many subtypes, advances in biomarkers that lead to better treatment decisions are greatly needed. FLT3 was the first commonly-mutated gene identified in AML for which a targeted therapy became available [In a heterogeneous disease such as AML, both predictive and prognostic biomarkers are essential for treatment planning and patient education. Early biomarkers in AML dating back to the 1970s include the French-American-British (FAB) classification system which provided morphology-based differentiation between subtypes of AML that had therapeutic and prognostic implications ,3. The Evailable . Testingvailable . Here, wFLT3 exist in roughly one third of AML diagnoses [FLT3 has a crucial regulatory role in hematopoiesis, and mutations in this gene therefore are important in the pathophysiology of AML [FLT3 mutations have been discovered, including the internal tandem duplication of the JM domain-encoding region (FLT3-ITD), representing roughly 25% of AML cases, and mutations around the D835 residue of the TK domain (FLT3-TKD), representing approximately 10% of AML cases [FLT3-ITD was dependent on allelic ratio and NPM1 mutational status [FLT3-ITD disease is considered intermediate risk [Activating mutations of iagnoses ,8. FLT3 y of AML . Variousl status , but as ate risk .FLT3 mutations, substantial research has focused on the creation of FLT3 inhibitors for use in frontline, relapsed/refractory, and maintenance settings. The phase III RATIFY study (CALGB 10603), for example, looked at the addition of the tyrosine kinase inhibitor midostaurin to intensive chemotherapy in newly-diagnosed AML with FLT3 mutations (either ITD or TKD) and showed an overall survival (OS) benefit with midostaurin compared to placebo  [FLT3 inhibitor, gilteritinib, to salvage chemotherapy in relapsed/refractory AML [Because of the high frequency and generally poor prognostic significance of ctively) . The phatory AML . The giltory AML .FLT3-ITD AML who achieved a complete response (CR) after allogeneic stem cell transplantation (SCT) and found that sorafenib maintenance reduces disease relapse and death versus placebo by more than half [p = 0.028), but there was no significant difference between MRD-positive patients. In contrast, following SCT, MRD-positive patients had an improved RFS with sorafenib maintenance compared to placebo (p = 0.015) while MRD-negative patients did not (p = 0.191) [The phase II German study SORMAIN tested the benefit of adding the tyrosine kinase inhibitor sorafenib as maintenance therapy in adult patients with han half . This tr= 0.191) .IDH1 and IDH2) genes are found in roughly 8% and 12% of AML cases, respectively, making up one of the most common genetic mutations following FLT3 [IDH1 and IDH2 lead to higher levels of histone hypermethylation, consequently blocking proper cellular differentiation and maturation, and promoting development of AML [Mutations of isocitrate hydrogenase 1 and 2  and fit individuals [NPM1-mutated leukemic cells also tend to express the CD33 cell surface antigen, gemtuzumab ozogamicin can be added to standard intensive regimens to improve survival in favorable- and intermediate-risk AML [NPM1 mutation, however, wanes with increasing age [NPM1, various agents are under investigation, including menin inhibitors, retinoic acid, arsenic trioxide, and dactinomycin [Intensive chemotherapy is the currently recommended therapeutic approach for ividuals . Becausesing age ,35. Venesing age . While tinomycin .CD33 is a cell surface antigen that is normally found on immature myeloid precursors and downregulated as part of normal cell maturation . In AML,Gemtuzumab ozogamicin (GO) is a CD33-specific antibody-drug conjugate that was introduced as the first targeted therapy for AML in 2000 . The ageThe decision to add GO to standard intensive chemotherapy is based on risk stratification as per the ELN genetic risk classification system, where GO has been shown to provide a significant survival benefit in cases of favorable-risk AML, and modest benefit for intermediate-risk . IncorpoTP53 codes for a tumor suppressor protein, which is essential for normal cell cycle regulation and response to DNA damage [TP53 is present in roughly 5–10% of de novo AML [TP53-mutated AML falls under the adverse risk category and confers a remarkably poor prognosis, with a 2-year OS of only 12.8% [TP53-mutated AML resistant to standard intensive chemotherapy, but also it has shown resistance to HMAs, such as azacitidine and decitabine, and the BCL-2 inhibitor venetoclax [Located on chromosome 17p13, novo AML  but muchnovo AML . Within ly 12.8% ,56. Not netoclax . Rates onetoclax . Thus, tTP53-mutated AML are currently under investigation. Recently, a Phase Ib study investigating the use of magrolimab and azacitidine in frontline mutant TP53 AML in patients unsuitable for standard intensive chemotherapy has shown promise in achieving durable responses and improving OS [TP53. Combination therapy with azacitidine and APR-246 has demonstrated a synergistic cytotoxic effect and some encouraging preliminary data [Targeted therapies for oving OS . Furtheroving OS , restoriary data ,61,62, bASXL1 gene have been identified across different myeloid malignancies, including AML [ASXL1 is located on chromosome 20q11 and is involved in the control of gene transcription [ASXL1 have been described in approximately 10% of cases of AML [ASXL1 mutations as an adverse risk factor associated with aggressive disease and resistance to initial chemotherapy leading to worse clinical outcomes [ASXL1 have shown sensitivity to BET inhibitors in mouse models [Initially reported in myelodysplastic syndrome (MDS), mutations in the ding AML ,64. ASXLcription . Mutatiocription . Mutatios of AML ,68. Priooutcomes ,70. Recee models . Furthere models .RUNX1 has a key function in regulating normal hematopoiesis [RUNX1 mutations can be divided into germline mutations, responsible for familial platelet disorders and predisposition to AML, and somatic mutations, which occur in both lymphoid and myeloid cancers, including AML [RUNX1 are found in roughly 10% of AML cases and occur in tandem with a complex array of gene mutations, including epigenetic regulators such as IDH2 and ASXL1 [RUNX1. One promising area of research involves combining protein translation inhibitors, such as omacetaxine, with venetoclax; AML cells with mutated RUNX1 are more sensitive to this combination than their wild-type counterparts [RUNX1-mutated leukemic blast cells, leading to improved survival in xenograft models of AML with mutant RUNX1 [opoiesis . RUNX1 mding AML . Mutationd ASXL1 , which cnd ASXL1 ,76,77. Rterparts . There int RUNX1 .Cytogenetic analysis continues to be very important for the prognosis of patients with AML. The 2017 ELN guidelines outlined specific cytogenetic abnormalities according to their overall risk category . In the NPM1-mutated AML in conjunction with poor-risk cytogenetic changes confers adverse risk [The recently published 2022 ELN guidelines include the following additions and modifications : first, rse risk . Second,rse risk ,81. The rse risk . While trse risk ,52,83.6 to one in 104 cells, as compared with one in 100 seen by eye on morphologic assessment. The technologies for measuring MRD, such as quantitative real-time PCR and flow cytometry, have existed for many years, but standardization of the testing methods is ongoing. There is also debate about when and how frequently MRD status should be determined.Measurable residual disease (MRD) refers to the presence of leukemia cells down to levels of one in 10NPM1 who had MRD assessed by multiparameter flow cytometry, and found that outcomes were similar between the patients who had a partial remission and patients who had a CR but remained MRD-positive  [Not surprisingly, the presence of residual disease is a negative prognostic indicator in AML, even if the amount of residual disease present is below the threshold for morphologic detection . For exactively) . A meta-ctively) . The negctively) .While MRD has clear prognostic importance, it is not necessarily clear how to incorporate it into treatment decisions. The ELN AML MRD expert panel issued an updated series of recommendations for the clinical use of MRD in December of 2021 . These rMECOM, ERG, WT1, GATA2, BAALC, MEIS1 and SPI1 in the bone marrow of 560 newly diagnosed AML patients and found that lower expression of MECOM and MEIS1 correlated with better CR rates, OS, and disease-free survival (DFS) [One promising avenue for AML biomarkers involves the identification of gene signatures by gene expression analysis. For example, one group analyzed 268 AML patients with cytogenetically normal AML obtaining a CR after induction chemotherapy, in hopes of identifying which genes could predict relapse by RNA sequencing (RNA-seq) . They idal (DFS) .The technique of single-cell RNA-seq (scRNA-seq) allows for an up-close view of the heterogeneity within the tumor microenvironment or a population of leukemia cells. Van Galen et al. presented a nanowell-based method of analyzing both DNA mutations and transcriptional information from sixteen AML patients and five healthy donors . They weCD44, HLAs, and PTMA [Stetson et al. used scRNA-seq to analyze differences between matched samples  in five different AML patients, and found characteristic RNA changes with AML progression, such as high expression of and PTMA .One limitation of gene expression analyses and scRNA-seq is that transcription profiles are dynamic and may change dramatically depending on the tissue microenvironment, phase of the cell cycle, and in response to different treatments. The ability to monitor these changes in real time would vastly increase the power of this technique and the importance of its incorporation into clinical practice.Immunotherapy has proved effective in many different tumor types but is still lagging in AML. Potential biomarkers for immunotherapy include receptor expression on tumor or immune cell surfaces, numbers or types of T cells present, and cytokine expression within the tumor microenvironment . The onlTP53-mutated disease trended towards better response to flotetuzumab than wild-type TP53  though this difference did not reach statistical significance [Other than the case of GO, immunotherapy has yet to show significant efficacy in AML, though multiple agents are being studied. One of these is flotetuzumab (MGD006), a CD123 × CD3 dual affinity retargeting protein (DART). Based on a phase I/II study of flotetuzumab in patients with relapsed/refractory AML, Uy et al. defined a 10-gene expression signature that predicted response to flotetuzumab . Anotherificance .If immunotherapy becomes more commonplace in AML treatment, it will be essential to develop predictive biomarkers to optimally select patients for this type of therapy.TET2) and DNA methyl transferase proteins  are common in AML. Hypomethylating agents are now frequently used in AML treatment, without first quantifying global or specific areas of methylation in the genome prior to use.Epigenetics have long been known to have an important function in AML as well as other cancers. Indeed, dysregulated DNA methylation is characteristic of AML , and mutC1R) as a prognostic biomarker for OS in AML patients, related to chromatin organization rather than gene expression levels of C1R [C1R at >27% was associated with a median OS of 53 versus 11 months. Another group found that the methylation status of ATP11A, ITGAM, and ZNRF2 served as accurate prognostic markers in AML patients [CEBPA, PBX3, LZTS2, and NR6A1) as predictive for longer survival, as well as two other genes (DLX4 and GPX3) [Experimentally, DNA methylation is readily evaluated by methods such as bisulfite sequencing. One group used The Cancer Genome Atlas to identify methylation of a CpG site at complement component 1 subcomponent R (s of C1R . Methylapatients . Sestakond GPX3) . Despiteclinicaltrials.gov, including one where treatment is chosen based on ex vivo sensitivity to venetoclax (NCT04267081). However, there are many limitations in ex vivo drug sensitivity testing at this time, such a suboptimal representation of the bone marrow niche, lack of drug metabolism in the tissue culture system, and the difficulty of modeling clonal selection over time in short-term in vitro models.There are some studies showing the ability of ex vivo drug testing to predict outcomes in patients with AML, as well as multiple commercial products available for ex vivo testing. Thus far, the literature consists mostly of small studies using patient bone marrow samples or peripheral blood to test sensitivity to chemotherapy drugs and targeted agents in tissue culture systems. For example, Lin et al. utilized bone marrow samples from 38 patients with AML to test responses to different AML drugs, by incubating bone marrow cells with various concentrations of drugs, and determining cell viability 72 hours later; they were able to successfully predict clinical treatment outcome in 32/38 cases (84.21%) . Kita etAnother proposed category of biomarkers is proteomic biomarkers. Dowling et al. collected bone marrow samples from AML patients with favorable, intermediate, and unfavorable risk AML, and compared protein expression by mass spectrometry . They foThere are countless clinical trials underway in AML that incorporate novel biomarkers, evidence of the need for improved biomarkers as well as their importance. A few examples of such biomarkers include the intracellular nucleotide pool as a predictive biomarker for response to induction chemotherapy (NCT03234985), and gene expression in peripheral blood samples on day four after salvage chemotherapy treatment as a predictive marker for treatment response (NCT02527447).Emerging and future biomarkers are summarized in FLT3 and IDH1/2 mutation status, have now become predictive biomarkers with the integration of targeted therapies into common clinical use. However, the complexities in AML extend beyond single gene aberrations, and there is a clear need for additional biomarkers. MRD testing is becoming increasingly sensitive and commonly used, though standardization of these techniques will be important moving forward. As laboratory techniques continue to evolve, there will be an increasing capacity to identify characteristics of disease, and ultimately, it will be up to clinicians to determine the best way to synthesize and utilize this complex information in the management and treatment of AML.Our knowledge of the mutational landscape in AML and implications of these mutations in AML prognosis has expanded significantly in the past few decades. A few of these prognostic biomarkers, such as