P < 0.05) but no significant difference with T1 or T2 rabbits. The expression of cecal pro-inflammatory factors IL-1β and TNF-α was increased in the T4 and T5 than in those of other groups (P < 0.05). Conversely, the tight junction proteins  were decreased to varying degrees in the T4 and T5 groups. The pH value in the cecal digesta of T5 rabbits was lower than that of T1, T2, and T3 (P < 0.05), while the concentration of volatile fatty acids and propionate was higher than those of T1, T2, and T3 rabbits (P < 0.05). In terms of gut microbiota, at the phylum level, the relative burden of Firmicutes and Actinobacteria in T2 rabbits was the highest (P < 0.05), and the relative burden of Proteobacteria in T5 rabbits was higher than that of other groups (P < 0.05). At the genus level, the relative burden of Ruminococcus was higher in T2 and T3 rabbits than that of other groups, and T5 rabbits have the lowest relative burden of Ruminococcus. Combination analysis showed that cecal metabolites were positively associated with fermentation-related phenotypes and the burden of Firmicutes (P < 0.05). In conclusion, different dietary NFC/NDF ratios can affect the intestinal immune response and growth performance of rabbits, and there was a positive effect when dietary NFC/NDF = 1.0–1.3.Carbohydrate is the most common macronutrient consumed across all phases of the diet and acts as a potential regulator in modulating the gut microbiota in animals. However, the influences of dietary non-fibrous carbohydrate (NFC) to neutral detergent fiber (NDF) in different ratios on gut microbiota, metabolites, intestinal immunity, and growth performance have not been fully explored. A total of 135 healthy weaned rabbits (45.1 ± 0.7 d of age) with an average body weight of 1.08 ± 0.07 kg were randomly divided into five groups. Under the same other nutrient levels, rabbits were fed diets with NFC/NDF ratios of 0.7 (T1), 1.0 (T2), 1.3 (T3), 1.6 (T4), and 1.9 (T5). During the 28-day experiment, T3 rabbits showed the highest final body weight and the lowest feed-to-weight ratio than T5 rabbits ( Lactobacillus, Bifidobacterium, Bacteroides, and Faecalibacterium  can be degraded by intestinal microorganisms into VFA to supply energy for the organism. Studies have shown that dietary NDF levels could affect cecal-digesta pH and VFA concentration in rabbits, and the amount of anaerobic fungal increased with the increasing dietary NDF level, VFA produced by anaerobic fungal fermentation can promote the expression of intestinal tight junction proteins to avoid inflammatory reactions in the organism, which is beneficial to intestinal health  in diets are important components of carbohydrates that can provide a large amount of blood glucose to animals with negative energy balance Hall, . As a reRecent studies have shown that an unbalanced NFC/NDF ratio in the diet can affect the disturbances in the rumen environment and a reduction in bacterial diversity of ruminants, which may eventually result in a reduction of growth performance  were randomly divided into five groups, respectively. Each group was further allocated into 27 replicates, with a rabbit per replicate. Under the condition of the same other nutrient levels, rabbits were fed diets with NFC/NDF of 0.7 (T1), 1.0 (T2), 1.3 (T3), 1.6 (T4), and 1.9 (T5). The experiment period is 28 days. During the experiment, all rabbits were allowed to eat and drink freely. The rabbit dormitory was ensured with normal light and ventilation. The diet was formulated according to the nutritional requirements of the National Research Council , using cvia the following formulas. Mortality (%) =  × 100%. Diarrhea (%) =  × 100%. On the 28th day of the trial, six rabbits from each replicate were randomly selected; blood samples were taken (2.5 mL) from the marginal vein of the ear using an anticoagulant-free vacuum test tube (5 mL) and immediately transported for analysis of routine hemogram with an auto hematology analyzer . Afterward, the rabbits were euthanized according to AVMA  and feed consumption were measured; average daily gain (ADG), average daily feed intake (ADFI), and the feed conversion ratio (FCR) were calculated (g feed/g gain) for all phases. The number of dead rabbits with diarrhea was recorded during the experiment, and the mortality and diarrhea rates were calculated −ΔΔCt method. Each sample was analyzed in triplicate and the results are reported as the geometric mean of the three results.The mRNA expression of genes related to gut immunity interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), Claudin-1, Occludin, zonula occludens (ZO-1), and GAPDH in cecum tissues was measured by RT-qPCR. Primers were designed using Premier 5.0 software , and GAP3-N of the cecal content was measured by the phenol-sodium hypochlorite colorimetric method. A measure of 1 mL of sample solution or standard solution was added to 4 mL of HCl (0.2 mol/L) solution and then mixed well. After that, 0.4 mL of the mixture was added to 5 mL of phenol reagent, and 4 mL of sodium hypochlorite was added and mixed well. The sample was water bath-heated at 60°C for 10 min, and after cooling, the absorbance was measured at 545 nm wavelength , according to the integration parameters and calibration curve. The pH value was detected potentiometrically using an automated pH analyzer . The NHA total of six cecal content samples from each group were randomly selected and used for the analysis of the gut microbiota. The workflow included DNA extraction, quality inspection, database sequencing, and 16S rRNA gene sequencing. Detailed methods are available in *Power package (version 3.1.4.), to ensure adequate power for the analysis. Afterward, experimental data were tested for normality by using the Shapiro–Wilk test of normality and for homogeneity of variances by using Levene's test. An overall test for treatment effect was first performed with a one-way analysis of variance (ANOVA). Linear and quadratic trends were conducted using orthogonal polynomial coefficients . The indexes were expressed as means with standard error of the mean (SEM) and significant main effect. P < 0.05 was considered a significant difference. GraphPad-Prism version 7.0  and heatmap illustrator  software were used to draw the graphs and heatmaps, respectively.The results obtained were analyzed for power analysis in the GP < 0.05), and the ADFI of rabbits in T1, T2, and T3 was significantly higher than that in T4 and T5. Polynomial contrast analysis showed that the level of BW , ADG , and ADFI  displayed linear and quadratic patterns of decrease with increasing dietary NFC/NDF inclusion level, with T1, T2, T3, and T4 being higher than T5. However, the F/G ratio in T5 rabbits was higher than that in the other groups , and the F/G ratio of rabbits in T3 was slightly lower than that in T1, T2, and T4 (P > 0.05). No individuals in the T1, T2, and T3 groups were found to have diarrhea or death. However, the diarrhea rates in the T4 and T5 groups were 18.52 and 59.26%, respectively, with the death rates being 7.41 and 14.81%, respectively.As presented in P < 0.05), while there were no significant differences in lymphocyte and monocyte in all groups (P > 0.05).P < 0.05, P < 0.05, P < 0.05, The results are shown in P < 0.05). The pH decreased in a linear (Plin = 0.032) and a quadratic (Pquad = 0.005) fashion with increasing dietary NFC/NDF inclusion levels with T1, T2, and T3 being higher than T4 and T5. The level of VFA in T5 rabbits was higher than that in T1 and T2 (P < 0.05). With the increase in dietary NFC/NDF, the concentration of acetate decreased, which was highest in T1 and T2 rabbits than in other groups, especially compared with T5 (P < 0.05). However, the concentration of propionate in T5 rabbits was higher than that in T1, T2, and T3 (P < 0.05). Polynomial contrast analysis showed that the level of VFA (Pquad = 0.013) and propionate (Pquad < 0.001) displayed a quadratic pattern of increase with increasing dietary NFC/NDF inclusion level. But the expression of acetate displayed quadratic (Pquad = 0.014) and linear (Plin = 0.005) patterns of decrease. Among these five groups, no significant difference was observed in NH3-N and butyrate (P > 0.05).As shown in P < 0.05). The cecal microbiota samples from T1, T2, and T3 were clustered together and clearly separated from the cecal microbiota of T4 and T5 rabbits, as shown in The Alpha diversity of the cecal microbiota of rabbits was influenced by dietary NFC/NDF . CoveragP > 0.05), T3 , T4 , and T5  groups. The relative burden of Bacteroidetes differed (P = 0.003) between the experimental treatments, displaying linear (Plin = 0.011) and quadratic (Pquad = 0.014) patterns of increase with increasing dietary NFC/NDF level, and the relative burden of Bacteroidetes in T2 rabbits was lower than that in T4 and T5 (P < 0.01) groups. The relative burden of Verrucomicrobia in T5 rabbits was higher than that in other groups . Compared with the T2 rabbits, dietary NFC/NDF changed the relative burden of Actinobacteria in the T1 , T3 , T4 , and T5  groups. However, the relative burden of Proteobacteria increased in a linear (Plin < 0.001) and a quadratic (Pquad < 0.001) fashion with increasing dietary NFC/NDF inclusion levels with T4 and T5 being higher than that in T1, T2, and T3 groups. The relative burdens of Patescibacteria, Tenericutes, Cyanobacteria, and Epsilonbacteraeota were all below 1%, and there were significant differences between all groups (P < 0.05).We compared the microbial communities at the phylum level. The predominant phyla were Firmicutes, Bacteroidetes, Verrucomicrobia, and Actinobacteria accounted for 97.14, 96.01, 95.42, 96.77, and 94.84% for T1, T2, T3, T4, and T5 groups, respectively , Table 4Ruminococcus_NK4A214_group, Ruminococcus_UCG-014, and Ruminococcus_UCG-013 in Firmicutes of T2 and T3 rabbits were higher than those of T4 and T5 (P < 0.05). In addition, polynomial contrast analysis showed that the level of the relative burden of Ruminococcus_NK4A214_group (Pquad < 0.001), Ruminococcus_UCG-014 (Pquad < 0.001), Ruminococcus_UCG-013 (Pquad < 0.002), and Ruminococcaceae_unclassified (Pquad < 0.001) displayed a quadratic pattern of decrease with increasing dietary NFC/NDF inclusion level. Compared with the T2 rabbits, dietary NFC/NDF changed the relative burden of Coriobacteriaceae_UCG-002 in the T1 , T3 , T4 , and T5  groups. Increasing dietary NFC/NDF inclusion level resulted in a quadratic pattern of increase for the relative burden of Muribaculaceae_unclassified (Pquad < 0.001) and Akkermansia (Pquad < 0.001) and resulted in a linear pattern of increase for the relative burden of Candidatus_Saccharimonas (Plin = 0.024). Meanwhile, these three microbial at T5 were higher than other groups (P < 0.05). In addition, the relative burden of the Subdoligranulum differed (P < 0.001) between the experimental treatments, displaying linear (Plin = 0.006) and quadratic (Pquad = 0.002) patterns of decrease with increasing dietary NFC/NDF levels. Meanwhile, the linear and quadratic patterns of decrease in the relative burden of Lachnospiraceae_NK4A136_group (Plin = 0.010) with increasing dietary NFC/NDF level were noted. Furthermore, both the relative burden of Subdoligranulum and Lachnospiraceae_NK4A136_group reached the highest at T2 and the lowest at T5.We analyzed the microbial communities with a relative burden of >1% at the genus level , Table 5Clostridiales, Clostridia, Ruminococcaceae, Ruminococcaceae_NK4A214_group, and Ruminococcaceae_ UCG_014 in Firmicutes and Coriobacteriales, Coriobacteria, Coriobacteriaceae_UCG_002, and Eggerthellaceae in Actinobacteria. However, the harmful differential bacteria, Rickettsiales which is symbiotic with the host, are screened in T5 rabbits.The LDA effect size (LEfSe) was mainly used to obtain the final differential species by comparing between the groups. As shown in Ruminococcaceae_NK4A214_group, Ruminococcaceae_UCG−014, and Ruminococcaceae_UCG−013 was positively correlated with acetate and negatively correlated with propionate (P < 0.01) (Lachnospiraceae_NK4A136_group showed a strong positive correlation with butyrate and a negative correlation with VFA (P < 0.05). The genus Subdoligranulum showed a positive correlation with acetate and butyrate (P < 0.05). The abundance of genus Lachnospiraceae_unclassified and Alistipes was positively correlated with pH and negatively correlated with VFA (P < 0.05); moreover, the relative burden of Alistipes was negatively correlated with NH3-N (P < 0.01). Coriobacteriaceae_UCG−002 showed a positive correlation with VFA, acetate, and butyrate and negatively correlated with pH (P < 0.05). The abundance of genus Candidatus_Saccharimonas was positively correlated with VFA and negatively correlated with pH (P < 0.01). In addition, Muribaculaceae_unclassified and Akkermansia were negatively correlated with acetate and butyrate (P < 0.05).Correlation analysis revealed that the burden of genus  < 0.01) . LachnosAn appropriate level of dietary fiber is essential for animal growth and development, and excess or deficiency of fiber would limit the digestion and absorption of nutrients. We found that the ADG and ADFI of rabbits at NFC/NDF ratio = 0.7~1.3 were higher than those at NFC/NDF = 1.6–1.9, but the F/G, diarrhea rate, and mortality were lower than those at NFC/NDF = 1.6–1.9. A higher concentration of dietary fiber results in lower concentrations of other nutrients, and a compensatory increase in appetite as feedback which will lead to a higher F/G ratio. Meanwhile, the fiber can provide substrate for intestinal fermentation, produce VFA, maintain intestinal pH balance, and ensure normal activities of intestinal microorganisms; beneficial intestinal microorganisms and their fermentation products acting on antigen cells can improve the expression of immunoglobulins and macrophages in the body, thus regulating the immune function of the intestine, and nutritional diarrhea will be relieved. Moreover, NFC contains plant cell contents and the readily fermentable part, which will stimulate normal intestinal peristalsis, making the animals feel full. When dietary NFC is higher, the ADFI of rabbits is generally lower  play an important role in immune cell activation and differentiation. However, high levels of IL may also induce an inflammatory response  can be found below: NCBI Sequence Read Archive (SRA) database, The animal study was reviewed and approved by all experimental procedures were approved by the Ethics Committee of Animal Experimentation of Hebei Agricultural University (Protocol 2021083).SL, TL, and BC: conceptualization. SL, TL, and KW: methodology, investigation, software, data curation, and writing—original draft preparation. KW and CL: validation and resources. TL and CL: formal analysis. XC and BC: writing review, editing, and supervision. BC: project administration and funding acquisition. All authors have agreed to the final manuscript.


P< 0.001; median follow-up: 83 days) and was well tolerated. Here, we report final safety from PROVENT at ∼15 months of follow-up.In the PROVENT phase 3 pre-exposure prophylaxis study, AZD7442 (tixagevimab/cilgavimab) reduced symptomatic COVID-19 by 76.7% vs placebo at primary analysis (In PROVENT (NCT04625725), adults without prior SARS-CoV-2 infection or COVID-19 vaccination, with increased risk of inadequate response to vaccination and/or SARS-CoV-2 exposure, were randomized 2:1 to either a 300-mg intramuscular dose of AZD7442 or placebo. Results are reported from the February 22, 2023 final data cut-off. The primary safety endpoint was assessment of adverse events (AEs), serious adverse events (SAEs), medically attended AEs (MAAEs), and AEs of special interest (AESIs). A cardiovascular events adjudication committee independently reviewed and adjudicated 5 event types . Efficacy data have previously been reported.Table). Most AEs were mild to moderate in severity; 256 (7.4%) and 125 (7.2%) of participants in the AZD7442 and placebo groups, respectively, reported an AE of grade 3 (severe) or higher. SAEs occurred in 6.2% and 5.6% of AZD7442 and placebo participants, MAAEs in 28.6% and 25.3%, respectively, and deaths in 0.6% and 0.6%, respectively. AESIs occurred in 3.0% and 2.5% of AZD7442 and placebo participants, respectively, including 0.5% and 0.4% with cardiovascular events categorized as AESIs. Of 88 (2.5%) and 39 (2.2%) participants in AZD7442 and placebo groups with cardiovascular events evaluated by an adjudication committee, 40 (1.2%) and 12 (0.7%) had positively adjudicated events.Across both the AZD7442 and placebo groups, 3669 (69.8%) participants completed the study. Median follow-up was 456 days in the AZD7442 group and 455 days in the placebo group. AEs occurred in 58.2% and 58.0% of participants administered AZD7442 and placebo, respectively (This analysis provides further evidence to support the long-term safety of AZD7442 as prevention for COVID-19.Myron J. Levin, MD, Dynavax: Advisor/Consultant|GSK: Advisor/Consultant|GSK: Grant/Research Support|GSK: Data safety monitoring/Advisory board|Johnson & Johnson: Grant/Research Support|Merck & Co.: Advisor/Consultant|Moderna: Grant/Research Support|Novavax: Grant/Research Support|Pfizer: Advisor/Consultant|Seqirus: Advisor/Consultant Andrew Ustianowski, MD, PhD, Gilead: Honoraria|Gilead: Advisory Board|GSK: Honoraria|Janssen: Honoraria|Merck: Honoraria|Merck: Advisory Board|Sanofi: Honoraria|ViiV Healthcare/GSK: Advisory Board Stéphane De Wit, MD, AstraZeneca: Financial support for the conduct of this study Rohini Beavon, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Jesse Thissen, MSc, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Seth Seegobin, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Katie Streicher, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Alexandre Kiazand, MD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Mark T. Esser, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds


Background: There are multiple genes that are co-amplified along with human epidermal growth factor receptor 2 (HER2) in chromosome 17. GRB7 and PGAP3 are two such genes. We hypothesize that the protein products of these genes may serve as immunohistochemistry (IHC) markers for detecting HER2 amplification in breast cancer.Methods: Tissue sections from one hundred and thirty-five primary breast carcinoma cases were subjected to immunohistochemical staining for antibodies against HER2, GRB7, and PGAP3 and graded on a scale of 1 to 3. Both membranous staining and cytoplasmic staining were assessed for GRB7 and PGAP3. For equivocal HER2 IHC positivity, fluorescent in situ hybridization was performed to get the final HER2 status.Results: IHC staining for GRB7 and PGAP 3 was a moderate to strong predictor for HER2 status (area under the curve (AUC) of 0.768, 0.868,0.754, and 0.790 for GRB7 membranous staining, GRB7 cytoplasmic staining, PGAP3 membranous staining, and PGAP3 cytoplasmic staining respectively). A combination of GRB7 cytoplasmic and PGAP3 membranous staining resulted in an AUC of 0.905 (95% CI 0.855-0.954), while a combination of GRB7 and PGAP3 cytoplasmic staining resulted in an AUC of 0.902 (95% CI 0.851-0.953).Conclusion: The point estimates for the AUC of GRB7 and combined GRB7 and PGAP3 in predicting the AUC suggest a strong predictive ability of these markers to predict HER2. With further refinement in technique, cytoplasmic staining and membranous IHC staining for GRB7 and PGAP3 have potential to serve as surrogate markers for HER2 status. The strategy of using protein products of co-amplified genes of HER2 is likely to be successful in technical validation. Human epidermal growth factor receptor 2 (HER2) amplification testing is an essential part of breast cancer workup . HER2-amThe HER2 amplification region on chromosome 17 contains other genes that are co-amplified -5. TheseOur study was approved by the Institutional Ethics Committee of All India Institute of Medical Sciences, Rishikesh. One hundred and thirty-five breast cancer cases were selected, and their core-biopsies and blocks were retrieved. The IHC status of these cases was confirmed with respect to their IHC and/or ISH status. Of these cases, two were excluded  tumor not having an ISH report, and another one did not have sufficient material for further testing). Sections were cut from these blocks and then immuno-stained with GRB7 antibodies  and PGAP3 antibodies . GRB7 staining failed in one case, which was removed for statistical analysis involving GRB7 staining as one of the variables. PGAP3 staining failed in a separate case, which was similarly removed for statistical analysis involving PGAP3 staining. The HER2 antibodies  were evaluated according to commonly followed guidelines .For GRB7 and PGAP3, both cytoplasmic positivity and membranous positivity were noted separately. Membranous positivity was interpreted like IHC for HER2. Cytoplasmic evaluation was done subjectively by categorizing cases as 0 (negative), 1 (mild), 2 (moderate), or 3 (marked). A tumor was graded as cytoplasmic grade 0 if there was no staining or weak, non-granular, staining in a minority of the cells. Grade 1 staining was defined as a weak, non-granular staining affecting most of the cells. Grade 2 staining was defined as the presence of a mixture of weak, non-granular cytoplasmic staining as well as strong granular staining in most of the cells. Grade 3 cytoplasmic staining was defined as the presence of strong, granular staining in most of the cells. Representative pictures of cytoplasmic staining grading are given in Figure The association between HER2 status and GRB7/PGAP3 was assessed by the Fisher exact test, logistic regression, and receiver-operating-characteristic (ROC) curve analysis. The area under the ROC curve (AUC) was calculated with the 95% confidence interval (CI) by the DeLong method. By multiple logistic regression and stepwise selection based on the Akaike Information Criterion (AIC), the best independent predictors of HER2 status were selected. Finally, as a simple model, the scores of the independent predictors were summed, and the diagnostic performance of the summed variable was evaluated. The statistical calculations were done by R statistical environment version 4.1.1 along with the package “pROC” and the R commander plugin EZR ,13.The clinical characteristics of the cancer samples studied are given in Table All four predictors  were significantly associated with HER2  also showed a high AUC suggesting strong predictive ability . The summed score of GRB7 cytoplasmic and PGAP3 cytoplasmic stain also suggested strong predictive ability with an AUC of 0.902 and 95% CI of 0.851-0.953. The diagnostic accuracy of the summed scores is given in Table There were 20 HER2+ tumors on IHC. All twenty of them turned out to be negative on FISH. Hence, sensitivity could not be calculated in these cases due to the absence of true positive cases. However, the specificity could be calculated for GRB7, PGAP3, and different combinations of these biomarkers which is given in Table Both GRB7 and PGAP3 have been found to be significant predictors of HER2 status. Taken individually, the predictive strength is modest, with only cytoplasmic staining of GRB7 attaining an AUC of greater than 0.85. However, when the combined score of GRB7 and PGAP3 is taken, the prognostic strength increases markedly, attaining an AUC value of greater than 0.9. This itself is proof of the predictive strength of GRB7 and PGAP3 and suggests that with improvement in retrieval techniques, better prediction will be attained. This itself is not surprising since amplification of the HER2 locus leads to amplification of these genes as well, being present in the same amplicon [The main advantage of using IHC for different protein products of genes co-amplified with HER2 is that IHC is of an order of magnitude cheaper than ISH. In addition, ISH requires a specialized, more sophisticated laboratory and more expensive technical staff. IHC is therefore potentially cost-saving for those laboratories that have IHC but do not have FISH. Also, staining for GRB7 and PGAP3 may serve as quality control indicators of IHC staining in cases where there may be scarcity or non-availability of proper control tissue.One additional takeaway of this study is that the cytoplasmic staining of GRB7 and PGAP3 may be more informative than membranous staining alone. Examination of the protein atlas suggests that GRB7 and PGAP3 are localized both in the cytoplasm as well as on the plasma membrane ,10. The It needs to be emphasized that this is a large proof-of-concept study. Due to the considerable number of cases, this can also serve as an initial validation study of GRB7 and PGAP3 in predicting HER2 status. These initial results are promising with a concordance approaching 85% but still have failed to demonstrate a concordance of greater than 95% with the reference standard when taking the optimal cut-off Table . This isThe primary limitation of this study was that the method of assessment of cytoplasmic staining was subjective. During future validation, clearly defined criteria for cytoplasmic staining will be laid down during validation. Fine granular staining of the cytoplasm, coarse granular staining, and the extent of masking of nuclei are being investigated. Furthermore, detailed assessment of antibody clones and techniques is also required. Another limitation is that we could not assess the diagnostic accuracy in the subgroup of HER2 2+ (on IHC) tumors due to all such tumors being negative for HER2 amplification on FISH. The specificity of the markers in HER2 2+ tumors was generally high, but further validation of GRB7 and PGAP3 antibodies in a subset containing an adequate number of such HER2 amplified HER2 2+ tumors is needed after further standardization of technique. This study has demonstrated strong predictive ability of GRB7 and PGAP3 in combination for detecting HER2 amplified breast cancers. The predictive ability should get only stronger with further standardization and refinement in technique. Both GRB7 and PGAP3 are possible and promising alternative biomarkers for detecting amplification of the HER2 related site on chromosome 17.


Grey matter (GM) reduction is a consistent observation in established late stages of schizophrenia, but patients in the untreated early stages of illness display an increase as well as a decrease in GM distribution relative to healthy controls (HC). The relative excess of GM may indicate putative compensatory responses, though to date its relevance is unclear.343 first-episode treatment-naïve patients with schizophrenia (FES) and 342 HC were recruited. Multivariate source-based morphometry was performed to identify covarying ‘networks' of grey matter concentration (GMC). Neurocognitive scores using the Cambridge Neuropsychological Test Automated Battery (CANTAB) and symptom burden using the Positive and Negative Symptoms Scale (PANSS) were obtained. Bivariate linear relationships between GMC and cognition/symptoms were studied.Compared to healthy subjects, FES had prominently lower GMC in two components; the first consists of the anterior insula, inferior frontal gyrus, anterior cingulate and the second component with the superior temporal gyrus, precuneus, inferior/superior parietal lobule, cuneus, and lingual gyrus. Higher GMC was seen in adjacent areas of the middle and superior temporal gyrus, middle frontal gyrus, inferior parietal cortex and putamen. Greater GMC of this component was associated with lower duration of untreated psychosis, less severe positive symptoms and better performance on cognitive tests.In untreated stages of schizophrenia, both a distributed lower and higher GMC is observable. While the higher GMC is relatively modest, it occurs across frontoparietal, temporal and subcortical regions in association with reduced illness burden suggesting a compensatory role for higher GMC in the early stages of schizophrenia. Progressive loss of grey matter (GM) tissue is one of the presumed pathophysiological processes targeted by the paradigm of ‘early intervention’ in psychosis  and matched healthy subjects (n = 342). As patients with a more severe illness often receive a higher antipsychotic dose, which is independently associated with GM reduction  (age from 16 to 45 years) from the Mental Health Center in West China Hospital and 342 HC (age from 16 to 45 years) were enrolled in the present study. Patients were interviewed and assessed using the Structured Clinical Interview for the DSM-IV  (SCID-I/P)  . 210 of 343 NN-FES and 253 of 342 HC had CANTAB data available. A total of 214 NN-FES and 292 HC were assessed using the short version of the seven-subtest  Chinese Revised Version of the Wechsler Adult Intelligence Scale (WAIS-RC)   Gong, . CANTAB Patients were scanned after written informed consent was obtained, but before treatment initiation; in all cases, this was on the same day of initial assessment, along with other laboratory assessments required for routine clinical care. Patients were not included in the study if any exposure to antipsychotics took place before the scanning. 206 patients and 170 HC were scanned on a Signa 3.0-T MR scanner  using an eight-channel phased-array head coil in the Department of Radiology at West China Hospital. The scan parameters are listed in the Supplementary Methods. 137 patients and 172 HC were scanned on a 3.0 T MR scanner  using an eight-channel phased-array head coil in the department of Radiology at West China Hospital Tibet People's Government in Chengdu Office Branch. The data from the 2 scanners (datasets 1 and 2 respectively) were combined for aggregated analysis; see online supplementary materials for scanner-related variations.To improve the registration of the MRI images, T1 Images were processed using Diffeomorphic Anatomical Registration Through Exponentiated Lie algebra (DARTEL) toolbox in Statistical Parametric Mapping (SPM) 8, and followed the standard approach of Ashburner . The dethttp://mialab.mrn.org/software/gift/) was used to perform the independent component analysis decompositions on the aggregated dataset consisting of 343 NN-FES and 342 HC from 2 scanners  . These source maps were scaled to unit standard deviation (SBM Z map) and thresholded at a value of |The GM intensity in each voxel in each subject  is the sum of 30 product values obtained from multiplying the voxel loading for each component with its coefficient for that subject. The sign of the loading coefficients of a component in a subject does not directly provide the direction of change in absolute GM intensity in a region .t tests (continuous variables) or χ2 tests  were used to evaluate differences in demographic and clinical characteristics between NN-FES and HC. All statistical tests were carried out using SPSS, Version 18.0. .Independent two-sample p < 0.05 after correcting for multiple testing using the false discovery rate (FDR) method was used to infer statistical significance for group comparisons. SBM analysis was repeated on the two scanner-specific datasets separately to confirm the results from the combined analysis (See online Supplement section 4).A multivariate analysis of covariance (MANCOVA) model was used with SBM loading coefficients as dependent variables, diagnosis as a factor, and scanner, age, gender, years of education and intracranial volume as covariates in SPSS 18.0. A threshold of Principal component analysis (PCA) was used for dimensionality reduction across the 16 scores from 6 CANTAB subtests. The latent variables from the PCA were assumed to reflect underlying cognitive dimensions. For this PCA, a parallel analysis (PA) helped determine the most appropriate number of components to be retained. As the components were expected to be correlated, we employed a varimax rotation (Further details in the Supplement).To exclude the effects of scanner, age, sex, and years of education on loading parameters of components, multiple regression analyses were performed on each SBM component as dependent variables, and scanner, age, gender, educational years and intracranial volume as independent variables, in the HC and NN-FES separately. Standard residual scores of each component from these regressions were then substituted for the raw values in the correlation analyses with clinical and neurocognitive variables.As the duration of the untreated psychosis (DUP) showed a skewed distribution, Spearman's test was used to assess the relationship between DUP and the SBM components showing group difference. Pearson's test was used to evaluate the relationship between the SBM components and positive, negative, and disorganisation factor scores from PANSS  and component 26  had areas with higher GMC in NN-FES than HC  based on the transformed locations of the largest clusters in the component maps. All four components had similar diagnostic differences when scanner-specific subsets of data were analysed, but a component with spatial similarity to component 13 was not recovered independently from the Philips scanner .The tissue maps of GMC were decomposed into 30 SBM components. Eight components were suggestive of obvious artefacts, i.e. having spatial patterns primarily around the edges of the brain, in white matter regions or CSF and these were discarded . From the remaining components, we observed four spatial patterns showing a significant effect of diagnosis. These four components and their spatial patterns are depicted in  than HC . The bran ms delay and Pattern Recognition Memory per cent correct in immediate test (factor loadings 0.48–0.82) . . NN-FES showed impaired neurocognition in all four components when compared to HC  representing sustained attention, memory and planning ability (Higher score is better for the neurocognitive ability of PC1) (factor loadings 0.53–0.90); PC2 included the CANTAB subtest variable IED reflecting cognitive set-shifting  (factor loadings 0.95–0.97); PC3 was comprised of RVP Probability of false alarm (RVP-PFA) and RVP B″ (measure the tendency to respond regardless of whether the target sequence is present) (factor loadings 0.93) (higher is better); PC4 was comprised of DMS per cent correct, DMS per cent correct in ed to HC .Table 3r = 0.152, FDR-corrected for clinical features p = 0.04), positive symptoms  and to neurocognitive factor 2 (adjusted for IQ)   and component 4  among patients. The observed direction of this relationship indicated that younger patients had higher GMC for both components, but the R2 of these models were small (4.7–6.04%) . No other significant correlation was found between GM spatial components, clinical or neurocognitive variables.Component 26  significantly related to the duration of psychosis (= 0.056) . This inr = −0.441, FDR-corrected p < 0.0001) and component 13  and component 26 . The loading on component 24 was significantly related to component 13 . As component 4 was a predominantly positively loading spatial component while components 24, 13 and 26 were predominantly negative components, these correlations indicated that patients with lower GMC in one component (4 or 24) are likely to also have higher GMC in either component 13 (with lower GMC in component 4 or 24) or component 26 (with lower GMC in either component 4). No significant correlation was found between component 24 and component 26 as well as between component 13 and component 26 (online Supplementary Table S7), indicating that patients did not share the distribution of relative higher in GMC; while some patients had predominant higher GMC of component 13, others had a predominant higher GMC in component 26.In the patient group, the loading on component 4 was significantly related to component 24 . However, when we used a more liberal threshold, higher GMC in the NN-FES group were found in caudate, cerebellum and a temporal pole at p < 0.001 at voxel level with FWE-corrected p < 0.05 at the cluster level, overlapping, and thus validating the interpreted direction of greater GMC in patients from the SBM analysis .Compared with HC, patients with NN-FES had lower GMC in the bilateral cingulate gyrus, insular, right superior temporal gyrus, medial orbital frontal, rectus, caudate, precentral gyrus and lingual gyrus at FWE-corrected Using multivariate SBM in a sample of treatment-naïve first-episode patients with schizophrenia, we report three major findings. Firstly, we found both a prominently lower grey matter concentration in frontoinsular, temporal and parietal regions, and a higher GMC in a distributed cortical/subcortical component , and cerebellum (tonsil and inferior semi-lunar lobule). Second, the higher GM in the cortico/subcortical component was seen in patients with shorter DUP and was associated with less severe positive symptoms and better performance on set-shifting ability, suggesting an adaptive advantage for patients with relative GM increase in these regions. Third, there was a significant relationship between the spatial components showing higher and lower GMC, supporting a coordinated reorganisation process. Taken together, these findings delineate a distributed pattern of GM changes in schizophrenia before any antipsychotic exposure.p < 0.05 for voxel level, but this became obvious at a lower threshold at p < 0.001 uncorrected at voxel level . This supports our notion that in order to detect GM increases in schizophrenia, mass univariate approaches that depend on spatially contiguous clusters are less sensitive than approaches like SBM that leverage more distributed changes. Though the diagnostic effect of the cerebellar component did not independently replicate in the smaller of the 2 datasets, our observation of higher GM in NN-FES involving the cerebellum, middle and superior temporal gyrus, middle frontal gyrus, inferior parietal lobule and putamen is consistent with several studies with large sample size (>100 subjects) . This result was consistent with our previous finding of extensive and excessive age-related cortical thickness loss in frontal, temporal, parietal, and cingulate gyri in patients  sample whereby the variance due to referral pathways and catchment demographics were minimal. Our continuous sampling of all referrals to the hospital eliminated the bias of selecting only the most unwell subjects that are accepted to treatment programmes. Furthermore, with a higher upper bound for age of inclusion in our first-episode programme, we achieved better sex balance in the sample  when compared to neurodegenerative or early developmental disorders, as well as to the heterogeneity of its clinical course.


FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML), with FLT3 internal tandem duplication (ITD) mutations being associated with a more aggressive clinical course. While two large, randomized clinical trials have shown a survival benefit with the frontline use of an oral FLT3 inhibitor (midostaurin or quizartinib) in patients with FLT3-mutated AML, the role of FLT3 inhibitors in older adults with newly diagnosed FLT3-mutated AML remains unclear. A definitive improvement in survival has not been observed in intensively treated patients over 60 years of age receiving frontline FLT3 inhibitors. Furthermore, many patients with FLT3-mutated AML are unsuitable for intensive chemotherapy due to age and/or comorbidities, and this population represents a particular unmet need. For these older patients who are unfit for intensive approaches, azacitidine + venetoclax is a new standard of care and is used by many clinicians irrespective of FLT3 mutation status. However, FLT3-ITD mutations confer resistance to venetoclax and are a well-established mechanism of relapse to lower-intensity venetoclax-based regimens, leading to short durations of remission and poor survival. Preclinical and clinical data suggest synergy between FLT3 inhibitors and venetoclax, providing rationale for their combination. Novel strategies to safely incorporate FLT3 inhibitors into the standard hypomethylating agent + venetoclax backbone are now being explored in this older, less fit population with newly diagnosed FLT3-mutated AML, with encouraging early results. Herein, we discuss the frontline use of FLT3 inhibitors in older adults with FLT3-mutated AML, including the potential role of FLT3 inhibitors in combination with intensive chemotherapy and as part of novel, lower-intensity doublet and triplet regimens in this older population. FLT3) mutations—particularly FLT3-internal tandem duplication (ITD)—are a major mechanism of resistance to venetoclax-based therapies [FLT3-mediated resistance are needed. While intensive chemotherapy plus a FLT3 inhibitor is now standard of care for younger, fit patients with newly diagnosed FLT3-mutated AML [FLT3 mutations in older adults with AML and the historical outcomes of FLT3-mutated AML in this population. We review the role of upfront FLT3 inhibitors and specifically highlight the ongoing efforts to develop more effective frontline therapies for older adults with FLT3-mutated AML who are unfit for intensive chemotherapy, including novel triplet regimens that integrate FLT3 inhibitors into lower-intensity backbones.Since 2017, there has been a remarkable expansion of new drug approvals for the treatment of acute myeloid leukemia (AML), both in the frontline and relapsed or refractory settings –5. Fortuherapies , 13, andated AML , 15, theFLT3 mutations are identified in approximately one-third of patients with newly diagnosed AML, are enriched in AML with a normal karyotype, and are frequently co-mutated with NPM1 and/or DNMT3A mutations [FLT3-ITD mutations are identified in 20–25% of patients with newly diagnosed AML, whereas point mutations in the tyrosine kinase domain (TKD) are identified in 5–10%, approximately half of which occur at D835 in the activation loop [FLT3 mutations varies across age groups, and they are relatively less common in older patients in AML [FLT3-ITD mutations ranged from 23% in patients <45 years of age to 15% in those >70 years of age. Similarly, the incidence of FLT3-TKD mutations ranged from 9% to 4% across these age groups, respectively [FLT3 mutations are less common in older adults in relative terms, due to the higher incidence of AML in older patients, the population-based absolute incidence of FLT3-mutated AML actually increases with age, with patients >60 years of age representing approximately 60% of all patients with FLT3-ITD mutated AML  [FLT3-mutated AML in this older population and the unmet clinical need that it represents.utations –20. FLT3ion loop , 22. Hows in AML . In one ectively . However . These fFLT3-ITD mutations have been consistently shown across most studies to be associated with a more aggressive phenotype and worse survival in AML, while most studies have shown no—or only minimal—prognostic impact of FLT3-TKD mutations [FLT3-ITD-mutated AML, a higher allelic ratio is associated with inferior outcomes, particularly when frontline FLT3 inhibitors are not used [FLT3 mutations in older patients. In this older population, the prognostic impact of FLT3 mutations has been less consistent. In one analysis of 243 patients >60 years of age with newly diagnosed AML with normal karyotype treated with intensive chemotherapy, the presence of a FLT3-ITD mutation was associated with shorter disease-free survival (DFS)  and OS  in patients 60–69 years of age, but not in those patients who were >70 years of age [FLT3-ITD mutations were independently associated with worse OS in a multivariate analysis, with high FLT3-ITD allelic ratios associated with a higher risk of death than were lower allelic ratios  [FLT3-ITD mutations in intensively treated older adults with newly diagnosed AML [utations , 27–30. not used . Most ofs of age . In a lactively) . Howeverosed AML , 34, 35.FLT3 mutations in older patients treated with lower-intensity therapies are even more limited. A subgroup analysis of the AZA-AML-001 study—which randomized patients >65 years of age with newly diagnosed AML to azacitidine versus conventional care regimens (CCR)—found no difference in outcomes of FLT3-mutated versus FLT3 wild type patients, although only 18 patients were included in this analysis, limiting its power [FLT3-mutated AML (including both ITD and TKD) versus FLT3 wild type AML when treated with the venetoclax-based combination [FLT3 mutation, those with FLT3-ITD-mutated AML had particularly poor outcomes (median OS 9.9 months), which was shorter than the FLT3 wild type population (median OS 14.7 months) and the FLT3-TKD-mutated population (median OS 19.2 months). Patients who were FLT3-ITD-mutated but NPM1 wild type AML had the shortest duration of remission among the subgroups evaluated (median 10.1 months versus 18.4 months for FLT3 wild type AML). Together, these data suggest that FLT3-ITD mutations may be a poor prognostic factor even when azacitidine + venetoclax is given.Studies of ts power . In a pobination . HoweverFLT3-mutated AML, based on positive data from the RATIFY study that evaluated midostaurin—and, more recently, from the QuANTUM-First study evaluating quizartinib [FLT3-mutated AML (either ITD or TKD) to standard chemotherapy (i.e. induction with “7 + 3” chemotherapy and high-dose cytarabine consolidation) plus either midostaurin  or placebo. Patients also received 1-year of maintenance with midostaurin or placebo. Midostaurin did not significantly improve the complete remission (CR) rate  but was associated with a significant improvement in median OS . The survival benefit of midostaurin was seen in both FLT3-ITD and FLT3-TKD mutations, including in patients with either high or low FLT3-ITD allelic ratios. The greatest benefit to midostaurin therapy was observed in patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT) in first remission [FLT3-mutated AML and led to the Food and Drug Administration (FDA) approval of midostaurin in combination with standard induction and consolidation chemotherapy in adults with newly diagnosed FLT3-mutated AML, although the FDA did not approve its use as maintenance therapy. Despite the impressive survival benefit of midostaurin in the RATIFY study, it is important to note that the study did not enroll patients 60 years of age and older, and so the generalizability of these data to this older population is uncertain.The combination of a FLT3 inhibitor with frontline intensive chemotherapy is the standard of care for fit adults with newly diagnosed zartinib , 37. TheFLT3-ITD-mutated AML who were deemed suitable for intensive chemotherapy [P = 0.032), which was driven largely by lower relapse rates . Interestingly, in a post hoc analysis, the OS benefit of quizartinib appeared to be limited to patients <60 years of age  and was not observed in patients ≥60 years of age . In July 2023, quizartinib was approved by the FDA for use in combination with standard induction and consolidation chemotherapy in patients with newly diagnosed FLT3-ITD-mutated AML; importantly, it was also approved as monotherapy maintenance for up to 3 years in patients who do not undergo subsequent HSCT. Together, the RATIFY and QuANTUM-First studies provide important clinical data showing that upfront integration of FLT3 inhibitors in patients with newly diagnosed FLT3-mutated AML improves outcomes. Specifically, both studies show that the combination of a FLT3 inhibitor with intensive chemotherapy results in improved OS in patients up to 60 years of age, although the data supporting this approach in older adults are still limited.The randomized phase III QuANTUM-First study later evaluated the same backbone chemotherapy regimen in combination with quizartinib  or placebo in adults up to 75 years of age with otherapy . Five-huHistorically, intensive chemotherapy was the only treatment option for newly diagnosed AML with a reasonable chance of durable remission, and therefore patients up to approximately 75 years of age were routinely recommended to receive intensive AML-directed therapy. However, appropriate selection of patients for intensive chemotherapy is imperative, particularly for patients ≥60 years of age, a group that can have unacceptably high rates of morbidity and mortality with this approach .FLT3-ITD-mutated AML received intensive chemotherapy plus midostaurin, followed by allogeneic HSCT and 1 year of midostaurin maintenance [P < 0.001) and the median OS was 22.7 months , suggesting benefit to adding midostaurin in this group.Patients >60 years of age were excluded from the RATIFY study , and thentenance . The finFLT3-mutated AML. Data from these intensive chemotherapy-based prospective studies in older adults, as well as data from prospective studies of lower-intensity regimens are summarized in Table As previously described, while older patients who were deemed suitable for intensive chemotherapy were included in the QuANTUM-First study, a survival benefit was not observed in the subgroup of patients. While the precise reasons for the lack of benefit in the older population is not entirely clear, the quizartinib arm had a higher rate of death in the first 3 months of therapy in this group, which may have at least in part offset a potential survival benefit. Until further data are available, these findings provide some skepticism for the use of upfront intensive chemotherapy plus a FLT3 inhibitor in older adults with FLT3-mutated AML [FLT3-mutated AML [As many older adults are not suitable candidates for intensive chemotherapy, several clinical trials have investigated lower-intensity therapies incorporating a FLT3 inhibitor in this population. Based on encouraging data from a phase I/II study of azacitidine + sorafenib in relapsed/refractory ated AML , this coated AML . Twenty-FLT3-ITD-mutated AML, including patients with relapsed/refractory disease or newly diagnosed patients who were >60 years of age and/or considered to be unfit for intensive chemotherapy [FLT3-mutated AML, although survival remains modest with these approaches.Another study evaluated azacitidine or low-dose cytarabine (LDAC) in combination with quizartinib in patients with otherapy . Thirty-FLT3 mutation status) to LDAC with or without quizartinib [FLT3-ITD mutation and 6 patients (3%) with a FLT3-TKD mutation. In the global population there was no difference in response rates or survival outcomes. However, in a subgroup analysis of the 27 patients with a FLT3-ITD mutation, the quizartinib-containing arm was associated with higher rates of CR/CRi  and superior OS . While the number of patients in this subgroup analysis are relatively small, the findings are suggestive of a benefit to the addition of a FLT3 inhibitor to a lower-intensity backbone in older adults with newly diagnosed FLT3-mutated AML who are unfit for intensive chemotherapy.Building upon the encouraging data with a quizartinib-based doublet, a subsequent study randomized older adults with newly diagnosed AML . The azacitidine + gilteritinib doublet was associated with a significantly higher rate of CRc , with an improvement in CRc rates that was particularly notable in patients with a FLT3-ITD allelic ratio >0.5 . However, despite the increase in CRc rates with the doublet, there was no difference in OS (the primary endpoint of the study) between the 2 arms. The median OS was 9.8 and 8.9 months, respectively , and the study was terminated early due to futility. The lack of survival benefit may be explained—at least in part—by different rates of subsequent AML-directed treatment after discontinuing protocol therapy. Fifteen patients (20.3%) in the azacitidine + gilteritinib arm received subsequent therapy, 3 of whom received a subsequent FLT3 inhibitor, compared with 22 patients (44.9%) in the azacitidine arm, 14 of whom received a FLT3 inhibitor. However, it is notable that EFS was also not significantly different between the 2 arms, and therefore the lack of survival benefit cannot be explained entirely by differences in post-protocol therapy. Ultimately, the LACEWING study failed to show a significant benefit to the addition of gilteritinib to azacitidine in older patients with newly diagnosed FLT3-mutated AML and suggests that use of these doublets may not be optimal in this population.Gilteritinib is a potent FLT3 inhibitor that is FDA-approved for the treatment of relapsed/refractory AL study . To evalAL study . One hunP < 0.001) and survival , as compared with azacitidine alone [FLT3-mutated AML—as well as some other high-risk subgroups—remain suboptimal. For example, in a study of 81 patients who received either azacitidine or LDAC in combination with venetoclax, expansion of pre-existing FLT3 clones or development of new FLT3 mutations were commonly seen at the time of relapse [FLT3-ITD mutations lead to overexpression of BCL-xL and MCL-1, both anti-apoptotic proteins shown to contribute to venetoclax resistance [FLT3-mutated and FLT3 wild type AML, the clonal expansion of FLT3-mutated cells in response to these therapies suggest that these mutations are an important mechanism of relapse.The outcomes of older adults with AML have greatly improved with the clinical development of venetoclax-based lower-intensity therapies. In the randomized phase III VIALE-A study, patients ≥75 years of age or unsuitable for intensive chemotherapy with newly diagnosed AML who received azacitidine + venetoclax had improved response rates . Across both groups, 41 patients had a FLT3-ITD mutation alone, 21 had a FLT3-TKD mutation alone, and 2 patients had both. This subgroup analysis confirmed that azacitidine + venetoclax improves response rates in patients with FLT3-mutated AML . The combination regimen was also associated with numerically superior OS . Among patients treated with azacitidine + venetoclax, there was no difference in OS between those FLT3-mutated or FLT3 wild type AML. However, there was evidence of differential outcomes between the different types of FLT3 mutations, which may have confounded this analysis. The CRc rates for patients with a FLT3-ITD or FLT3-TKD mutation were 63% and 77%, respectively. The median OS in patients with FLT3-TKD-mutated AML was 19.2 months, compared with only 9.9 months in those with FLT3-ITD-mutated AML. This relatively poor survival highlights that effective treatment of FLT3-ITD-mutated AML represents a particular unmet need even in the era of venetoclax-based lower-intensity therapies.Given the survival benefit observed with azacitidine + venetoclax, many clinicians offer this regimen to older, unfit adults with newly diagnosed AML, regardless of regimens . In thisFLT3-ITD mutations have been shown preclinically to reduce BCL-2 dependence of AML cells and to lead to upregulation of BCL-xL and MCL-1, antiapoptotic proteins that are established to impart resistance to venetoclax [FLT3-mutated AML cells to venetoclax [FLT3-mediated resistance to venetoclax and synergy between venetoclax and FLT3 inhibitors that may lead to more durable responses in FLT3-mutated AML are highlighted in Fig. FLT3 wild type cell lines [FLT3-mutated AML.netoclax –53. Sevenetoclax , 51–53. ll lines . GilteriFLT3-mutated AML [FLT3-mutated AML, 47 patients (84%) had a FLT3-ITD mutation . The modified CRc rate  was 75%, and the median OS was 10.0 months. While it is challenging to compare across clinical trials, the response rates compare favorably to the ADMIRAL trial, where the CRc rate was 54.3%. The higher response rates with venetoclax + gilteritinib were observed despite this study enrolling more heavily pretreated patients than the ADMIRAL study, including 36 patients (64%) with prior FLT3 inhibitor exposure (versus only 13% in ADMIRAL) and 38% of patients having received ≥3 lines of therapy . This study therefore provides clinical rationale for the further development of venetoclax and FLT3 inhibitor combinations in FLT3-mutated AML.These preclinical observations of synergistic activity of venetoclax and FLT3 inhibition were subsequently evaluated in a phase 1b clinical trial of venetoclax + gilteritinib in patients with relapsed/refractory ated AML . Among 5FLT3-mutated AML. In a single-center retrospective analysis, 87 patients with newly diagnosed FLT3-mutated AML were treated with either triplet therapy (n = 27) or doublet therapy with a lower-intensity therapy plus a FLT3 inhibitor, without venetoclax (n = 60) [P < 0.01) and CR/CRi (93% versus 70%), which translated to superior OS with the triplets , a survival benefit that was confirmed on multivariate analysis .Building upon the efficacy of the HMA + venetoclax regimen in patients with newly diagnosed AML, several studies are now evaluating lower-intensity therapy + venetoclax + FLT3 inhibitor triplets in patients with newly diagnosed (n = 60) . PatientFLT3 mutations were enrolled, for whom the treating physician could add a FLT3 inhibitor [FLT3-mutated AML were treated . The FLT3 inhibitor added was sorafenib in 5 patients, gilteritinib in 5 patients, and midostaurin in 2 patients. The CRc rate (CR + CRp + CRi) was 92%, and the median OS was not reached after a median follow-up of 14.5 months. Early data from an ongoing study of decitabine, venetoclax, and quizartinib have also been reported [FLT3-ITD-mutated AML have been treated, all of whom responded (2 CR and 3 CRi). In both studies, no early mortality was observed, suggesting that triplet regimens can be delivered safely in this older population.Subsequent prospective studies have suggested benefits of these triplet regimens, as compared with historical expectations with lower-intensity therapy plus either venetoclax or a FLT3 inhibitor. In a phase II study of a 10-day regimen of decitabine + venetoclax in older/unfit adults with newly diagnosed AML, a subgroup of patients with nhibitor . Twelve reported . Five paFLT3-mutated AML [FLT3-ITD mutation, and 8 patients (27%) had a FLT3-TKD mutation. A gilteritinib dose of 80 mg daily was used in the combination based on safety established from a preceding phase I study in relapsed/refractory FLT3-mutated AML. Increased myelosuppression was observed with the addition of gilteritinib to the azacitidine + venetoclax backbone, and therefore a bone marrow was performed on day 14 of cycle 1, and the gilteritinib and venetoclax were held to allow for count recovery if it showed marrow remission or aplasia. In consolidation, azacitidine was given for 5 days and venetoclax for 7 days to minimize prolonged myelosuppression. With these mitigation strategies in place, the modified CRc rate was 100%, with 27 patients (90%) achieving CR. With a median follow-up of 17 months, the 1-year OS and 2-year OS rates were 84% and 71%, respectively. These results appear to compare favorably to historical expectations with doublet regimens in a similar older population, including with azacitidine + venetoclax, where the CRc rate was 67% and the 2-year OS in patients with FLT3-mutated AML was 20–40%. Thus, using the appropriate mitigation strategies to prevent excessive myelosuppression, the triplet regimen of azacitidine, venetoclax, and gilteritinib can be delivered safely and represents a highly effective treatment option for older or unfit adults with newly diagnosed FLT3-mutated AML.The triplet combination of azacitidine, venetoclax, and gilteritinib has also been evaluated in older adults and those unfit for intensive chemotherapy with newly diagnosed ated AML . This isFLT3-mutated AML in older adults who are unfit for intensive chemotherapy represents a particular clinical challenge, given the lack of an approved FLT3 inhibitor for use in this setting. FLT3 mutations (particularly FLT3-ITD mutations) are associated with a more aggressive clinical course and resistance to commonly used regimens in this population, including an HMA + venetoclax. Triplet regimens of an HMA, venetoclax, and a FLT3 inhibitor have yielded promising outcomes; however, many questions still remain. Despite the encouraging response and early survival data with these regimens, the negative results from the randomized LACEWING study temper enthusiasm somewhat [FLT3-containing subclones, it is possible that reducing doses of the other agents could predispose to FLT3-negative relapses, which have been reported to account for 40-50% of relapses with frontline regimens incorporating FLT3 inhibitors [FLT3-mutated AML will attempt to identify the optimal doses of these agents when given in combination (NCT05520567). The best FLT3 inhibitor use in the frontline setting is also not clear, and randomized studies comparing intensive chemotherapy plus midostaurin to either gilteritinib (NCT04027309) or crenolanib (NCT03258931) in younger, fit patients with newly diagnosed FLT3-mutated AML are ongoing. The findings from these studies could also inform future approaches in older and less fit patients.The treatment of somewhat . Thus, rhibitors . A folloFLT3 mutations and/or those with FLT3-TKD-mutated AML is not well-established in older patients. The eligibility cutoffs in the RATIFY and QuANTUM-First studies were a FLT3 allelic ratio ≥0.03 and a FLT3 variant allelic frequency ≥0.05, respectively, and thus randomized data supporting the use of FLT3 inhibitors in the context of very low-burden FLT3-mutated AML are lacking. In one retrospective analysis of patients  with newly diagnosed FLT3-mutated AML , the best outcomes were observed in those who received a frontline FLT3 inhibitor and underwent subsequent HSCT, suggesting that this is an effective approach for fit patients with low-burden FLT3 mutations [FLT3 mutations who are not candidates for HSCT remains uncertain. For FLT3-TKD-mutated AML in younger, fit adults, the RATIFY study showed that this group benefitted from the addition of midostaurin; however, it is unclear whether this applies in the context of lower-intensity venetoclax-based regimens in older adults. While FLT3-ITD mutations are clearly important mechanisms of resistance to an HMA + venetoclax, these regimens result in relatively favorable outcomes in patients with FLT3-TKD-mutated AML. Thus, it remains unclear whether the potential added toxicity of a FLT3 inhibitor and the need to reduce doses of the HMA and venetoclax in order to deliver the triplet combination will ultimately improve outcomes for these patients, as compared with use of azacitidine + venetoclax alone.Finally, the added benefit of FLT3 inhibition in patients with very low-burden utations . How the


Here, we investigated the beneficial effects and the underlying mechanisms of Rb1 on hypercontraction induced by high glucose (HG) and endothelial dysfunction (ED). The isometric tension of aortic rings was measured by myography. The rings were treated with NG-nitro-L-arginine methyl ester (L-NAME) to induce chemical destruction of the endothelium, and Rb1 was added after HG induction. The agonist-induced vasoconstriction was significantly higher in the aortic rings treated with L-NAME + HG50 than in those treated with HG50 or L-NAME (p = 0.011) alone. Rb1 significantly reduced the hypercontraction in the aortic rings treated with L-NAME + HG50 (p = 0.004). The ATP-sensitive K+ channel (KATP) blocker glibenclamide tended to increase the Rb1-associated reduction in the agonist-induced vasoconstriction in the rings treated with L-NAME + HG50. The effect of Rb1 in the aortic rings treated with L-NAME + HG50 resulted from a decrease in extracellular Ca2+ influx through the receptor-operated Ca2+ channel  and the voltage-gated Ca2+ channel , whereas Rb1 did not interfere with Ca2+ release from the sarcoplasmic reticulum. In conclusion, we found that Rb1 reduced hyper-vasoconstriction induced by HG and ED by inhibiting the ROCC and the VGCC, and possibly by activating the KATP in rat aorta. This study provides further evidence that Rb1 could be developed as a therapeutic target for ED in diabetes.Acute hyperglycemia induces oxidative damage and inflammation, leading to vascular dysfunction. Ginsenoside Rb Pathological conditions can result in acute hyperglycemia. For example, acute pancreatitis can not only impair glucose metabolism  but can Araliaceae [1, Rb2, Rc, and Rd, and protopanaxatriol-type compounds, including Rg1, Rg2, and Rh1, with both of these types possessing a dammarane triterpenoid structure [1) and Rg1  used in the present study are representative components of protopanaxadiol-type and protopanaxatriol-type, respectively [1 and Rg1 differ in aspects such as the positions of sugar moieties [1 and Rg1 are known to have antidiabetic effects [1 accounts for the largest proportion of ginsenoside saponins contained in red ginseng [1 was shown to stimulate the production of nitric oxide in vascular endothelial cells [1 was also found to down-regulate the Wnt/β-catenin pathway, which is associated with vascular calcification in rat vascular smooth muscle cells [1 was reported to inhibit store-operated Ca2+ entry (SOCE) by suppressing stromal interaction molecule activation rather than by altering Ca2+ release from the sarcoplasmic reticulum (SR), and thereby to decrease endothelin-1-induced vasoconstriction [1 for three weeks was reported to decrease SOCE and vasoconstriction in the pulmonary arteries of rats with pulmonary hypertension [1 on vasoconstriction under hyperglycemia and ED conditions remained unclear. Therefore, in the present study, we investigated the effects of Rb1 on hyper-vasoconstriction induced by HG and ED in rat aorta, and its underlying mechanisms.Red ginseng (Ginseng Radix Rubra), a plant belonging to the family aliaceae , has beealiaceae . It has aliaceae . Ginsenotructure . The ginectively . The chemoieties . Both Rb effects ,15. Espe ginseng ,16. Rb1 al cells . Rb1 wasle cells . In pulmtriction . Furtherrtension . HoweverG-nitro-L-arginine methyl ester  and L-NAME + HG25 (1.18 ± 0.06 g). In contrast, the agonist-induced vasoconstriction was significantly higher in the rings treated with L-NAME + HG50 (1.32 ± 0.06 g) than in the control (p = 0.007) and L-NAME-treated (p = 0.011) rings. The agonist-induced vasoconstriction of aortic rings was not significantly different between the L-NAME + Mannitol (1.22 ± 0.03 g) group and the control group. These results suggest that vasoconstriction was significantly increased only after simultaneous treatment with HG50 and L-NAME for denudation of the endothelium, and thus was attributable to the HG condition rather than a hyperosmotic effect  or 50 mM (HG50) did not significantly affect the agonist-induced vasoconstriction of rings with intact endothelium . Also, there was no significant difference in the agonist-induced vasoconstriction between the rings treated with Nc effect A.−6 or 10−5 M Rb1  or Rg1 , or 10−5 M N-acetylcysteine (NAC 10) on hyper-vasoconstriction induced by HG and ED were evaluated. The agonist-induced vasoconstriction in the aortic rings treated with L-NAME + Rb1 1 (1.09 ± 0.04 g), L-NAME + Rb1 10 (1.11 ± 0.05 g), and L-NAME + Rg1 10 (1.14 ± 0.05 g) did not differ significantly from the agonist-induced vasoconstriction in the rings treated with L-NAME alone (1.10 ± 0.06 g). Similarly, the agonist-induced vasoconstriction in the aortic rings treated with L-NAME + HG25 + Rb1 1 (1.18 ± 0.04 g), L-NAME + HG25 + Rb1 10 (1.11 ± 0.05 g), and L-NAME + HG25 + Rg1 10 (1.19 ± 0.04 g) did not differ significantly from the agonist-induced vasoconstriction in the aortic rings treated with L-NAME + HG25 (1.18 ± 0.06 g). Compared to the aortic rings treated with L-NAME + HG50 (1.32 ± 0.06 g), the agonist-induced vasoconstriction in the rings treated with L-NAME + HG50 + Rb1 10  was significantly reduced to a level similar to that seen in the L-NAME + HG50 + NAC 10 (1.14 ± 0.06 g) group; however, such a reduction was not seen in the rings treated with L-NAME + HG50 + Rg1 10 (1.28 ± 0.05 g) (The effects of 10 0.05 g) B.1 10 on agonist-induced vasoconstriction, aortic rings were treated with several K+ channel blockers. Tetraethylamine , barium chloride , and 4-aminopyridine  did not significantly alter the agonist-induced vasoconstriction in the aortic rings treated with L-NAME + HG50 + Rb1 10 (1.11 ± 0.05 g), whereas the agonist-induced vasoconstriction tended to be higher in the rings treated with L-NAME + HG50 + Rb1 10 + Glibenclamide  than with L-NAME + HG50 + Rb1 10 . These results suggested that activation of the ATP-sensitive K+ channel (KATP) may be involved in the inhibitory effect of Rb1 on hyper-vasoconstriction induced by HG and ED  and L-NAME + HG50 + Rb1 10 (0.32 ± 0.02 g)   (To further explore the mechanism of the action of Rb 0.02 g) B. In con= 0.001) C and the< 0.001) D.2+ levels through SOCE [2+ concentrations may be increased in the vascular smooth muscle cells of patients with acute pancreatitis, resulting in vascular hypercontraction.The present study found that the agonist-induced vasoconstriction of aortic rings with intact endothelium was not affected by the HG concentration. In the presence of ED, however, the agonist-induced vasoconstriction was significantly increased by the addition of 50 mM glucose. This concentration is relevant to that seen in the hyperosmolar hyperglycemic state  leading ugh SOCE . Becauseugh SOCE , intrace1 and Rg1 to reduce hyper-vasoconstriction induced by HG and ED was also evaluated. Although neither Rb1 nor Rg1 altered vasoconstriction in the aortic rings treated with L-NAME and L-NAME+HG25, Rb1 significantly reduced hyper-vasoconstriction induced by 50 mM glucose and ED, whereas Rg1 was ineffective. Rb1 was more effective than Rg1 in mitigating vascular smooth muscle dysfunction in the presence of angiotensin 2-induced abdominal aortic aneurysm [1 and Rg1 may reflect differences in their chemical configurations, such as the positions of sugar moieties and aglycone structures [The ability of Rbaneurysm  and inhianeurysm . These dructures .1 on hyper-vasoconstriction induced by HG and ED were not altered by the K+ channel inhibitors, TEA, BaCl2, and 4-AP. In contrast, the KATP blocker Gliben tended to suppress the Rb1 inhibition of hyper-vasoconstriction induced by HG and ED. HG has been found to inhibit KATP activity in vascular smooth muscle, an inhibition mediated by increased superoxide production in the human omental artery [ATP is inhibited when intracellular ATP is decreased, followed by the opening of the VGCC, resulting in increased cytosolic Ca2+ levels [1 may inhibit extracellular Ca2+ influx through the activation of the KATP, thereby reducing hyper-vasoconstriction induced by HG and ED.The effects of Rbl artery . Further+ levels ,31. Thus1 has been found to reduce intracellular Ca2+ concentrations in the myocardial H9C2 cell hypoxia model, through a mechanism involving the downregulation of the calcium/calmodulin-dependent protein kinase II and the ryanodine receptor 2 [1 selectively suppressed the L-type VGCC in cultured rat hippocampus neurons [1 on hyper-vasoconstriction induced by HG and ED were associated with Ca2+ flow, the effects of Rb1 on vasoconstriction induced by sarcoplasmic SR Ca2+ release or extracellular Ca2+ influx were measured. The ability of Rb1 to ameliorate the increased vasoconstriction induced by treatment with L-NAME and 50 mM glucose was found to be independent of SR Ca2+ release. In contrast, Rb1 was found to reduce hyper-vasoconstriction induced by HG and ED through inhibiting extracellular Ca2+ influx via the ROCC and the VGCC, regarded as the main Ca2+ channels for controlling vascular tension in vascular smooth muscle [Rbceptor 2 . Further neurons . To deteh muscle ,35.1 was found to inhibit Ca2+ increase through SOCE in pulmonary arterial smooth muscle cells [1 significantly inhibited Ca2+ influx through SOCE only in vascular smooth muscle cells, but not in vascular endothelial cells, exposed to 30 mM glucose for 48 h [1 did not affect the agonist-induced vasoconstriction in aortas with HG and intact endothelium. This lack of effect may be due to endothelium-derived contracting factors, including angiotensin II [Pretreatment with Rble cells . Moreovefor 48 h . In agreensin II , thromboensin II , and endensin II , release1 is expected to have therapeutic potential in diseases accompanied by hyperglycemia and ED. The present study indicates that Rb1, which possesses anti-diabetic properties [2+ influx and/or possibly through the KATP channel activation.Rboperties , reduced1 is effective as a preventive and therapeutic intervention for hyper-vasoconstriction induced by HG and ED in rat aorta provides a potential rationale for clinical trials aimed at evaluating the efficacy of Rb1 in patients with both hyperglycemia and ED.These findings indicate the importance of maintaining healthy endothelium to prevent hypercontraction of vascular smooth muscles, especially in patients with hyperglycemia. If endothelial vessels are damaged, however, it is crucial to manage hypercontraction of vascular smooth muscle in hyperglycemic conditions. The finding that RbN = 4–11). The experimental protocol in this study was approved by the Institutional Committee for Animal Research Ethics of Korea University (KUIACUC-2021-0088).Three-week-old male Sprague Dawley rats (weighing 65 ± 15 g) were purchased from Young Bio  and housed at a temperature of 21–23 °C and under a 12 h photoperiod. The rats were given free access to tap water and a standard chow diet comprised of 60% carbohydrate, 27% protein, and 13% fat . After acclimatization, the rats were assigned to distinct groups  and the connective tissue was removed carefully. Each aorta was cut into rings 2–3 mm in length and placed in 37 °C chambers, which were continually aerated with a mixture of 5% CO2 and 95% O2. The aortic rings were connected to a DMT 620M  with tungsten wires, and then stabilized at 1.0 g tension for 80 min. The aortic rings were pretreated with 10−4 M L-NAME to denudate the endothelium chemically and thus to assess only the vasoconstrictive responses of vascular smooth muscle. For the HG condition, the total glucose concentration in each chamber was adjusted to 25 mM [−6 or 10−5 M Rb1 or Rg1, or with 10−5 M NAC, (an antioxidant) [−5 M PE. When the maximum vasoconstriction plateau was reached, 10−5 M acetylcholine was added to confirm endothelial denudation.Rats were anesthetized with isoflurane, and their thoracic aortas were dissected. The aortic tissue was immersed in Krebs solution  , and con1 on hyper-vasoconstriction under HG and ED conditions was associated with the activation of the K+ channel, the aortic rings were pretreated with K+ channel blockers. Briefly, the aortic rings with chemically denuded epithelium were treated with 10−3 M TEA to block the Ca2+-activated K+ channel (KCa2+), 10−6 M Gliben to block the KATP, 10−3 M BaCl2 to block the inward rectifier K+ channel (KIR), or 10−3 M 4-AP to block the voltage-dependent K+ channel (KV) [−5 M Rb1 prior to agonist-induced vasoconstriction.To investigate whether the inhibitory effect of Rbnel (KV) . The rin1 was involved in PE-induced SR Ca2+ release, which causes the exposure of myosin-binding sites on actin and vascular smooth muscle contraction [2+-free Krebs solution supplemented with 10−4 M L-NAME, followed by the addition of 10−5 M PE to induce vasoconstriction due to SR Ca2+ release. The aortas were rinsed with Krebs solution three times to restore lost intracellular Ca2+, followed by rinsing with Ca2+-free Krebs solution two times. The rings were subsequently pretreated with 10−5 M Rb1 for 10 min, followed by the addition of 10−5 M PE. PE-induced vasoconstriction was calculated to estimate the amount of Ca2+ release from the SR [To determine whether Rbtraction , the aorm the SR .2+ channels, ROCC and VGCC, were selected to determine whether the effects of Rb1 included extracellular Ca2+ influx, thereby affecting vasoconstriction under HG and ED conditions. The aortic rings were immersed in Ca2+-free Krebs solution containing L-NAME, followed by the addition of 10−5 M PE (for Ca2+ influx via the ROCC) or 60 mM KCl (for Ca2+ influx via the VGCC) to induce vasoconstriction. The rings were subsequently treated with 10−6–10−2 M CaCl2 to obtain concentration–response curves. After rinsing with Ca2+-free Krebs solution, to evaluate the inhibitory effect of Rb1 on hyper-vasoconstriction induced by HG and ED via the ROCCs or VGCCs, the aortic rings were treated with 10−5 M Rb1 for 10 min prior to the addition of 10−5 M PE or 60 mM KCl and subsequent 10−6–10−2 M CaCl2. The percent PE- or KCl-induced vasoconstriction was calculated based on the maximal contractile response to 10−2 M CaCl2 [Two types of Ca M CaCl2 .2, 4-AP, Gliben, glucose, L-NAME, mannitol, NAC, PE, Rb1 and Rg1, TEA, and dimethylsulfoxide (DMSO), were purchased from Sigma-Aldrich . Rb1, Rg1, and Gliben were dissolved in DMSO, whereas all other reagents were dissolved in distilled water.All chemical reagents, including ACh, BaCl2+ influx- and SR Ca2+ release-induced vasoconstriction were compared by paired t-tests. The other data were analyzed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test for post hoc comparison. p < 0.05 was deemed statistically significant.All data were expressed as mean ± SEM and analyzed using SPSS statistics version 26 software . The data for extracellular Ca1 effectively reduced hyper-vasoconstriction induced by HG and ED (p = 0.004) via the inhibition of Ca2+ influx through the ROCC  and the VGCC  and partially through the activation of the KATP in rat aorta for the first time (1 could be recommended as a therapeutic agent for ED in diabetes and provides a guideline for clinical studies evaluating the importance of managing vascular function in patients with hyperglycemia and ED, including in patients with acute pancreatitis.In this study, we have demonstrated that Rbrst time . Our res


Complex karyotype (CK), defined as ≥3 unrelated chromosomal abnormalities, is associated with a poor prognosis in both acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Despite their high genetic complexity, complex chromosomal abnormalities in AML and MDS may share dysregulated gene expression signatures, leading to poorer outcomes compared to those of standard chemotherapy. We aimed to investigate a subset of genes and their signatures to predict CK in AML and MDS with excess blast (MDS-EB) using gene expression data. The CK signature (CKS) was established and validated, and its prognostic impact on overall survival (OS) was evaluated in comparison with previously reported risk stratification models using gene expression. A 10-gene CKS demonstrated high predictive accuracy for CK and was associated with shorter OS with comparable performance to previously established risk stratification models.SOD1) gene, which is potentially amenable to SOD1 inhibitors.Complex karyotype (CK) is associated with a poor prognosis in both acute myeloid leukemia (AML) and myelodysplastic syndrome with excess blasts (MDS-EB). Transcriptomic analyses have improved our understanding of the disease and risk stratification of myeloid neoplasms; however, CK-specific gene expression signatures have been rarely investigated. In this study, we developed and validated a CK-specific gene expression signature. Differential gene expression analysis between the CK and non-CK groups using data from 348 patients with AML and MDS-EB from four cohorts revealed enrichment of the downregulated genes localized on chromosome 5q or 7q, suggesting that haploinsufficiency due to the deletion of these chromosomes possibly underlies CK pathogenesis. We built a robust transcriptional model for CK prediction using LASSO regression for gene subset selection and validated it using the leave-one-out cross-validation method for fitting the logistic regression model. We established a 10-gene CK signature (CKS) predictive of CK with high predictive accuracy . CKS was significantly associated with shorter overall survival in three independent cohorts, and was comparable to that of previously established risk stratification models for AML. Furthermore, we explored of therapeutic targets among the genes comprising CKS and identified the dysregulated expression of superoxide dismutase 1 ( Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are clonal disorders of myeloid hematopoiesis. The genetic signature of MDS, specifically MDS with excess blasts (MDS-EB), overlaps with that of secondary AML ,2,3,4,5.TP53 mutations is considered the hallmark of CK [CK, defined as ≥3 unrelated chromosomal abnormalities in both AML and MDS, comprises 10–12% of de novo AML and primary MDS cases ,9. CK isrk of CK ,14, otheExtensive transcriptomic analyses of myeloid neoplasms for the development of prognostic gene expression signatures have been beneficial in prognosis prediction and the detection of potential actionable targets ,19,20,21Despite their high genetic complexity, complex chromosomal abnormalities in AML and MDS-EB may share dysregulated gene expression signatures, leading to poorer outcomes compared to those of standard chemotherapy. In this study, we aimed to investigate a subset of genes and their signatures to predict CK in AML and MDS-EB using gene expression data. Consequently, we integrated gene expression data from our cohort with the gene expression profiles of AML and MDS-EB with known karyotype results. The CK signature (CKS) was established and validated, and the prognostic impact of CKS on overall survival (OS) was evaluated in comparison with previously reported risk stratification models using gene expression. SOD1) gene, which is potentially amenable to SOD1 inhibitors.Additionally, to seek expanded utilization of CKS for future treatment selection, we conducted an exploration of therapeutic targets among the genes comprising CKS and identified the dysregulated expression of superoxide dismutase 1 (KMT2A [Our cohort (KUMC cohort) included 47 adults diagnosed with AML or MDS-EB. As the definition of CK in previous studies excludes WHO-designated balanced abnormalities, including t8;21), inv(16)p13.1q22) or t, tq22;q11–12), any translocations involving ;21 involved high-intensity cytarabine and idarubicin (7 + 3), or daunorubicin (including one case combined with midostaurin), followed by consolidation with high-dose cytarabine or low-intensity decitabine . The salvage treatment regimen for the 37.84% of AML patients who did not respond to the initial induction consisted of Fludarabine, cytarabine, G-CSF, and idarubicin regimen for the majority of the cases (78.57%) and a venetoclax + decitabine regimen for the rest. In 8.11% of AML patients, salvage therapy was administered multiple times, involving alterations in the sequence of the previously mentioned regimen or the use of a regimen that included mitoxantrone, intermediate-dose cytarabine, and etoposide. Allogeneic peripheral blood stem cell transplantation  was performed in 21.62% of AML patients. The therapy for MDS-EB patients primarily consisted of 5-day decitabine or 7-day azacitidine, occasionally supplemented with low-dose cytarabine or alloPBSCT in 20% of patients, respectively.To establish CKS, datasets from The Cancer Genome Atlas (TCGA) AML , Beat AMThe diagnostic BM samples were subjected to RNA-seq analysis. RNA was extracted using the QIAamp RNA Blood Mini Kit  and reverse-transcribed to complementary DNA (cDNA) using the SuperScript VILO cDNA synthesis kit . RNA-seq libraries were prepared using the TruSeq Standard Total RNA LT Sample Prep Kit  and sequenced on the NovaSeq Sequencing system . Sequenced reads were aligned to the reference genome GrCh37 (hg19) using HISAT2 (version 2.1.0) , and alip value < 0.05 and absolute log2 fold change > 0.5. The GSE15601 dataset included microarray data from the GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array). DEGs between the CK and non-CK groups were identified using the limma R package [p value < 0.1 and absolute log2 fold change > 1.5 was applied. The DEGs that were common in at least two cohorts were selected. Overrepresentation analysis was performed using ConsensusPathDB (release 35) [Samples in the TCGA AML and Beat AML datasets were measured using the RNA-seq platform. RNA-seq data from the KUMC, TCGA AML, and Beat AML cohorts were subjected to differential gene expression analysis between patients with and without CK within each cohort using the DESeq2 package (version 1.32.0) . The raw package , and theease 35) . Pathwayn = 175; 35 patients with CK and 140 patients with non-CK) and validation  cohorts stratified based on the CK status to generate a CK prediction model and then subsequently validate the model. For the KUMC, TCGA AML, and Beat AML cohorts, normalized values based on reads per kilobase per million mapped reads (RPKM) were used for downstream analysis. Quantile normalization was performed using the preprocessCore package (version 1.58.0). As for GSE15061, data processing was performed using the trimmed mean of differences between perfectly matched and mismatched intensities with the quantile normalization (DQN) algorithm [An overview of the study design and implementation is presented in lgorithm . For siglgorithm . To selelgorithm  and valiThe values for the leukemic stem cell 17 signature score (LSC17) and AML prognostic score (APS) were calculated using model coefficients described by Ng et al. and Docking et al., respectively . Briefly2. HL-60 cells were seeded in 6-well plates and treated with LCS-1  at various time points. After incubation, the cells were collected and centrifuged at 300× g for 10 min. The cell pellet was washed with cold Dulbecco’s phosphate-buffered saline (DPBS) and homogenized using an Ultra-Turrax T8 homogeniser . Thereafter, the samples were centrifuged at 300× g for 10 min, and the supernatant was used to measure SOD enzymatic activity using a SOD Colorimetric Activity Kit (Invitrogen). The assay was conducted according to the manufacturer’s protocol, and the unit of SOD activity was calculated relative to the standard reagent provided in the kit.Human AML HL-60 cells karyotyped as CK were purchased from the Korean Cell Line Bank  and maintained in RPMI 1640  supplemented with 10% fetal bovine serum  and 25 mM HEPES at 37 °C in 5% COFor cell viability assay, HL-60 cells were seeded in 96-well plates and treated with various concentrations  of LCS-1 diluted in serum-free RPMI 1640 for 24 h. After incubation, a thiazolyl blue tetrazolium bromide (MTT) solution  was added to each well of the plate and incubated at 37 °C for 4 h. Subsequently, dimethyl sulfoxide (Sigma-Aldrich) was added to each well and incubated for 0.5 h. The absorbance at 450 nm was measured using Varioskan LUX multimode microplate reader (Thermo Fisher Scientific). The control was set to 100%, and the absorbance of the other samples were calculated relative to the control. In addition, HL-60 cells were stained with 5 μg/mL Propidium iodide (PI) (Sigma-Aldrich) for 20 min and washed with DPBS. The stained cells were analyzed using the EVOS™ FL Auto 2 imaging system (Thermo Fisher Scientific). PI fluorescence was quantified using ImageJ software (version 1.52a), and each treated sample was calculated relative to the control samples.Intracellular reactive oxygen species (ROS) was measured using 2′,7′-dichlorofluorescin diacetate , which is a cell-permeable, non-fluorescent probe that exhibits green fluorescence upon oxidation. Mitochondrial ROS was measured using MitoSOX™ , a fluorescent dye specific for detecting mitochondrial superoxide production in cells. Briefly, the cells were treated with 1 μM DCFDA or 5 μM MitoSOX™ in serum-free medium and incubated for 20 min at 37 °C. The cells were then washed with DPBS. The cells stained with fluorescent dyes were detected using an EVOS™ FL Auto 2 Imaging system (Thermo Fisher Scientific). The fluorescence values were quantified using ImageJ software, and each condition sample was calculated relative to the control samples.All statistical analyses and visualizations were performed using the R software (version 3.4.3). For model validation using LOOCV, the receiver operating characteristic (ROC) curves and the area under the curve (AUC) were analyzed. For analysis of patient characteristics, comparisons between the two groups were performed using the Kruskal–Wallis rank sum test for continuous variables and the Fisher’s exact test for categorical variables. Survival was estimated using the Kaplan–Meier method, log-rank test, and Cox proportional hazard regression with the Survminer (version 3.4.3) package.p = 0.3855). In our cohort (KUMC cohort), a total of 47 patients with AML or MDS-EB (AML/MDS-EB) were included. The main clinical and hematological characteristics of the patients are presented in The diagnosis distribution within the KUMC cohort following the introduction of the International Consensus Classification (ICC) 2022 is presented in The gene expression data of KUMC, TCGA AML, Beat AML, and GSE15061 were subjected to differential gene expression analysis between patients with and without CK within each cohort, including 69 patients with CK and 279 patients with non-CK. DEGs common in at least two of the four cohorts were selected. A total of 404 DEGs were identified, including 222 upregulated and 182 downregulated genes .n = 38, 9.41% of DEGs), chromosome 7 , or chromosome 17 . More than half of the DEGs located on the 17q were distributed in the 17q21 band (n = 9), while on chromosomes 5 and 7, DEGs were distributed throughout the long arm. For upregulated genes, 20 genes each (4.9% of DEGs) were located on the long arm of chromosome 11 and short arm of chromosome 1, respectively, and were most frequently observed in the 11q13 (n = 7) and 1p34 (n = 8) bands. Notably, the downregulated genes were mostly localized on chromosome 5 or chromosome 7 and the upregulated genes were localized on chromosome 1 or chromosome 11 a. When eBUB1, ORC1, YWHAH, ESPL1, WEE1, CDC20, TFDP1, CHEK1), heme biosynthesis , and cellular responses to stress   . DysreguCK group ,36. UprePRX × 1.7021) + (MMD × 1.5629) + (KIAA1549 × −1.2916) + (GIPC1 × 0.9794) + (RAB33A × 2.1416) + (SOD1 × 2.1254) + (MED7 × −1.8714) + (COPG2 × −2.0169) + (RPS14 × −1.204) + (PSMB10 × −1.6775). The CKS score in the training cohort showed an LOOCV-estimated AUC of 0.952 (n = 167/175) for CK prediction. As the CKS scores differed significantly between the CK and non-CK groups (p < 0.0001), we further evaluated the CKS model using the validation cohort. In the validation cohort, the AUC was 0.977 (n = 161/173), and the CKS score was sufficient to differentiate between the CK and non-CK groups (p < 0.0001).Based on the differential gene expression profiles of the CK group, we investigated whether the expression data from 404 DEGs could be used to establish a more robust CKS for CK classification. To establish the CKS gene set for predicting CK, the final set of ten DEGs were extracted using LASSO regression to fit a multivariate logistic regression model. The model development process using the training cohort defined the CKS calculated for each patient using gene expression weighted by regression coefficients as follows: CKS score = −6.157 + , demonstrating that the CKS was capable of predicting CK in patients with AML and MDS-EB.There were ten non-CK cases with a CK gene expression signature, resulting in a specificity of 96.42%. No differences were observed in terms of clinical outcome from a comparison between these commonly referred to as false positive cases and true negative cases. The most common unbalanced chromosomal abnormalities in CK involve the loss of 5q, 7q and 17p ,14. Notap = 0.032; BeatAML: 20.9 vs. 5.0, p = 0.010). The discrepancy that compromised sensitivity could reflect cases with more favorable clinical outcomes. In the KUMC cohort, there was a single instance of a false negative which precluded meaningful statistical analysis. Among the six samples with available karyotype information, a subclone of the CK clone was observed in only two to three metaphases out of the entire metaphase population in two thirds of these samples  with available survival data were included in the Beat AML cohort. We evaluated the prognostic impact of the CKS determined using the CKS score in predicting clinical outcomes. Three cohorts with survival data, namely, the KUMC, TCGA AML, and Beat AML cohorts, were analyzed. In the KUMC and TCGA cohorts, all patients included in the CKS model development were also included in the survival analysis, whereas 107 patients  (p = 0.028)   (Patients with CK in the KUMC cohort were characterized by lower white blood cell (WBC) counts (× 109/L) . CK was = 0.028) c. Surviv. 55.7%) d. We per 0.0402) .p = 0.033; 1-year OS, 56.1% vs. 39.1%), and a trend toward shorter OS in the Beat AML cohort   , and a stronger association was observed with CKS    c, wherea. 19.6%) d. In bot 0.0024) . Among c 0.0050) .p = 0.0068; APS: HR = 1.4957, p = 0.0486) and Beat AML cohorts   . However 0.0065) . SOD1, one of the components of CKS, is overexpressed in the CK group and is associated with the development of various types of cancers [To investigate the feasibility of utilizing CKS for therapeutic selection, we have explored the potential of genes constituting CKS as prospective therapeutic targets. Through a literature review, we have observed that  cancers ,40. As aTo determine if specific SOD1 inhibition would affect the cellular proliferation of the AML cells HL-60, we treated these cells with the specific SOD1 inhibitor LCS-1. As shown in CK is associated with a poor prognosis and resistance to standard chemotherapy in both AML and MDS-EB ,11,12. IIn accordance with prior studies of the most frequently reported chromosome arms of genomic imbalances (losses and gains) in CK-AML patients, the losses were reported with following regions and frequencies: 5q, 17p, 7q, 18q, 16q, and 17q and conversely, gains, while less prevalent than deletions, were reported with following frequencies: 8q, 11q, 21q, 22q, 1p, and 9p . In thisFBXL17, PCBD2, TIGD6; 7q: TPK1, ZCWPW1) and/or gene expression  [RPS14 has been implicated in the pathogenic mechanisms of cytopenias, including MDS with 5q deletion and congenital macrocytic anemias [The disease mechanism of CK is currently not fully elucidated; however, in MDS or AML with 5q and/or 7q deletions, haploinsufficiency of genes in the deleted region is considered one of the prevailing mechanisms resulting in disease development and malignant progression. DEGs localized at 5q or 7q were previously suggested to be putative haploinsufficient genes based on the location of genes in the commonly deleted region (5q:  ZNF227) ,43,44,45 anemias . In addi anemias ,47,48. HTP53 alterations and inhibitors targeting this pathway have shown promise in vitro and in vivo [SOD1, which is overexpressed in the CK group, is also part of the dysregulated pathway identified through overrepresentation analysis—specifically, the cellular stress response pathway. The treatment strategy for high-risk AML patients with CK primarily involves intensive chemotherapy, followed by allogeneic hematopoietic stem cell transplantation. Nevertheless, over 50% of CK-AML patients experience disease relapse, necessitating the exploration of novel treatment . One of SOD1 codes for Cu/Zn superoxide dismutase associated with intracellular ROS regulation and is regulated by NF-E2 related factor (Nrf2). The interference with the Nrf2 pathway has been identified as one of the mechanisms of action of the Bcl-2 inhibitor venetoclax, which is currently used for the treatment of AML patients who are not eligible for intensive induction chemotherapy in combination with hypomethylating agents. Overexpression of Nrf2 has been linked to venetoclax resistance [sistance ,52,53. TSOD1 overexpression has been associated with an adverse prognosis [PLK1, with SOD1 inhibitors could serve as a promising avenue for future research [SOD1 has been reported in various human cancer types ,55 and trognosis , and indrognosis . We haverognosis , in becoresearch .Owing to the relatively small proportion of patients with MDS-EB included in our study , it is possible that CKS may not fully encompass the molecular genetic characteristics of MDS-EB, even though hierarchical clustering of the 404 DEGs could not differentiate between MDS-EB and AML based on the gene expression patterns and the proportion of inaccurate predictions showed no difference between MDS-EB and AML group . Further application of this formula to a larger MDS-EB group in the future may help validate its efficacy. Another limitation is that the cohort of patients used in this study was composed of diverse patient groups who received various treatments. Therefore, further studies are required to ensure the use of the CKS score for predicting the clinical outcome in the patient group using specific treatment regimens. Lastly, our cell culture was conducted targeting a single gene in a single cell line. While we have presented the potential for SOD1 inhibition following existing research , furtherConclusively, we performed gene expression analysis of patients with AML and MDS-EB with CK and developed a model to predict CK. The significance of this study lies in its ability to diagnose CK using gene expression profiling. The results of this study imply that previously established haploinsufficiency due to the deletion of 5q or 7q possibly underlies the pathogenesis of CK. Moreover, we observed that the CKS could be used as a model to unravel potential therapeutic targets such as SOD1.