OBJECTIVE:To explore the application of targeted mRNA sequencing in fusion gene diagnosis of hematologic diseases. METHODS:Bone marrow or peripheral blood samples of 105 patients with abnormally elevated eosinophil proportions and 291 acute leukemia patients from January 2015 to June 2023 in the First Affiliated Hospital of Soochow University were analyzed and gene structural variants were detected by targeted mRNA sequencing. RESULTS:Among 105 patients with abnormally elevated eosinophil proportions, 6 cases were detected with gene structural variants, among which fusion gene testing results in 5 cases could serve as diagnostic indicators for myeloid neoplasms with eosinophilia. In addition, a IL3∷ETV6 fusion gene was detected in one patient with chronic eosinophilic leukemia, not otherwise specified. Among 119 patients with acute myeloid leukemia (AML), 38 cases were detected structural variants by targeted mRNA sequencing, accounting for 31.9%, which was significantly higher than 20.2% (24/119) detected by multiple quantitative PCR (P < 0.05). We also found one patient with AML had both NUP98∷PRRX2 and KCTD5∷JAK2 fusion genes. A total of 104 patients were detected structural variants by targeted mRNA sequencing in 172 cases with acute B-lymphoblastic leukemia who were tested negative by multiple quantitative PCR, with a detection rate of 60.5% (102/172). CONCLUSION:Targeted mRNA sequencing can effectively detect fusion gene and has potential clinical application value in diagnosis and classificatation in hematologic diseases.

BackgroundAutophagy is a self-renewal mechanism in which cells degrade damaged organelles or abnormal proteins through lysosomes. This process eliminates harmful components within the cell and maintains energy homeostasis. Multiple myeloma (MM) is a hematological malignancy characterized by uncontrolled plasma cell proliferation. Autophagy plays a dual role in tumorigenesis, yet its prognostic implications in MM remain underexplored.MethodsTranscriptomic and clinical data from 1,386 MM patients (training cohort: GSE136337, n = 415; validation cohorts: GSE24080, n = 558; GSE4581, n = 413) were analyzed. A seven-gene signature (ATIC, CDKN1A, DNAJB9, EDEM1, GABARAPL1, RAB1A, VAMP7) was identified using LASSO-Cox regression. Predictive performance of the autophagy-related model was assessed via Kaplan-Meier analysis, ROC curves, and nomograms. Immune infiltration, drug sensitivity, and functional pathways of the autophagy-related model were evaluated using CIBERSORT, ESTIMATE, and GSEA. The gene expression in the autophagy prognostic model was verified by qRT-PCR in the U266 and RPMI8226 cell lines and blood samples of multiple myeloma patients from the First Affiliated Hospital of Wenzhou Medical University.ResultsThe autophagy-related risk score stratified patients into high-risk and low-risk groups with distinct survival outcomes (high-risk HR = 0.391, 95%CI:0.284-0.540, p < 0.001). The model demonstrated robust predictive accuracy (5-year AUC = 0.729) and was independently validated. High-risk patients exhibited elevated immune checkpoint expression (CD48, CD70, BTLA), stromal infiltration, and drug resistance. Functional enrichment linked high-risk profiles to MYC activation and oxidative phosphorylation. Through qRT-PCR, the accuracy of the autophagy-related model has been verified in the U266 and RPMI8226 cell lines, as well as in the blood samples of multiple myeloma patients from the First Affiliated Hospital of Wenzhou Medical University.ConclusionThis autophagy-related gene signature provides a reliable prognostic tool for MM, highlighting immune dysregulation and therapeutic resistance mechanisms. Its integration with clinical parameters enhances risk stratification and treatment planning.

Multiple myeloma (MM) is a heterogeneous disease, the full understanding of whose pathogenesis remains elusive. While B-cell receptors are known to play a pivotal role in myeloma pathogenesis, the characterization of immunoglobulin heavy-chain (IGH) gene repertoire and their clinical significance in Chinese patients has not been fully explored. In this study, we analyzed the profiling of clonal IGH gene rearrangements via NGS assay, and its cytogenetic abnormalities by FISH in a cohort of 301 Chinese patients with newly diagnosed MM. We identified a particular subgroup, which was characterized by a marked overrepresentation of IGHV4-39. Additionally, IGHV4-39 was correlated with a higher somatic hypermutation rate and shorter HCDR3 length. Notably, IGHV4-39 was significantly more prevalent in patients with high-risk cytogenetic abnormalities, particularly the recurrent IGH translocations involving t(4;14). Our findings, represented the largest IGH data in MM series from Asia, and investigated the association between specific IGHV and cytogenetic alterations in Chinese MM patients for the first time.

Acute myeloid leukemia (AML) patients with FLT3-ITD mutations were benefit from hematopoietic cell transplantation (HSCT) in the first complete remission. Previous research suggested that newly diagnosed AML patients with high allelic ratio (AR) of FLT3-ITD have unfavorable survivals, while newly diagnosed AML patients with lower FLT3-ITD AR and concomitant NPM1 mutations have favorable outcomes. In AML patients with FLT3-ITD, co-occurrence with DNMT3A, and NPM1 mutations (triple-mutated AML patients) have the worst prognoses, however, it is little known about how these mutations synergize in these triple-mutated AML patients. Here we showed that hepatic leukemia factor (HLF) gene was more highly expressed in triple-mutated AML patients than in those without the DNMT3A mutations. We found that HLF gene expressions had significant difference in triple-mutated and FLT3-ITD/NPM1 AML patients (double-mutated AML patients). Moreover, in DNMT3A mutated AML patients, correlated with high HLF gene expression, which may be itself associated with poor survival rate and drug resistance. Overall our data establish that HLF gene as a novel biomarker in this genetically defined the triple-mutated AML subgroup.

Erythropoietin (EPO) and erythropoiesis-stimulating agents are primary treatments for anemia in chronic kidney disease. However, their clinical utility is significantly limited by high cost, patient compliance challenges, adverse cardiovascular effects associated with frequent injections, and inherent hyporesponsiveness. These constraints underscore an urgent need for cost-effective, safer, and more potent therapeutic alternatives. Here, we demonstrate that a short-term fasting regimen (3 days) in humans significantly increased red blood cell (RBC) counts with enhanced functionality to youthful levels, particularly in individuals with baseline cytopenia. To identify a more feasible fasting duration capable of augmenting erythropoiesis, we performed a fasting time course in mice. Remarkably, a single 6-hour fasting was sufficient to increase the production of RBCs in both young and aged mice. This effect was sustained throughout 15 days of refeeding, equivalent to approximately 2% of the mouse lifespan. Flow cytometry analysis revealed that short-term fasting selectively increased terminally differentiated erythrocytes within the bone marrow (BM), without altering splenic erythropoiesis. Flow cytometry and single-cell RNA sequencing demonstrated that short-term fasting promoted the self-renewal of BM megakaryocyte-erythroid progenitors (MEPs), accelerated erythroblast maturation (with transcriptional changes preceding surface marker alterations, confirmed by RNA velocity), and increased proportions of MEPs and downstream erythroid cells during refeeding. Hematopoietic stem cell pools and megakaryocyte lineages were not compromised. Surprisingly, short-term fasting-induced erythropoiesis occurred independently of canonical Epo-EpoR signaling or hypoxia. Transcriptomic profiling of mouse BM MEPs post-fasting revealed significant alterations in cell cycle pathways. BrdU/Ki67 assays confirmed accelerated MEP cycling without disrupting quiescence, aligning with downregulation of cell cycle inhibitory regulators. Bulk transcriptomic analysis revealed that short-term fasting profoundly downregulated the G1-S checkpoint gene Ms4a3 in MEPs. Functional studies confirmed that nutrient starvation in an in vitro mouse erythroid cell model (mouse erythroleukemia cells, MEL), or MS4A3 silencing in MEL cells, human cord blood-derived HSC erythroid progenitors (HUDEP-2), and primary mouse MEPs significantly enhanced erythroid differentiation and maturation, establishing MS4A3 downregulation as a key facilitator. Further in vivo studies showed that short-term fasting activates autophagy in mouse MEPs. The erythropoietic response to short-term fasting phenocopied rapamycin-induced effects and was abolished by pharmacological (3-methyladenine, 3-MA) or genetic (Atg5 or Atg7 knockout) inhibition of autophagy in mice. In autophagy-deficient MEPs in mice, fasting failed to modulate Ms4a3 expression or accelerate the G1-S transition. Thus, short-term fasting downregulates MS4A3 via autophagy to enhance erythropoiesis, independent of EPO elevation. Proteomic analysis in starved MEL cells ± 3-MA identified the transcription factor JunD as a potential autophagy-sensitive regulator of Ms4a3. Short-term fasting reduced JunD protein levels in wild-type mouse BM MEPs, but not in autophagy-deficient MEPs, and autophagy mediated JunD degradation. Combined Cleavage Under Targets & Tagmentation (CUT&Tag) and qPCR analyses confirmed JunD as a direct transcriptional activator of Ms4a3 in mouse MEPs. JunD silencing reduced Ms4a3 expression and promoted erythroid lineage progression in both MEL cells and primary MEPs. In summary, we identify a novel mechanism whereby short-term fasting potently augments erythropoiesis. Short-term fasting triggers autophagy-mediated degradation of the transcription factor JunD, leading to suppression of its target, MS4A3, a critical negative regulator of erythroid maturation. Rather than activating the energy-intensive canonical EPO signaling “gas pedal”, short-term fasting disables the JunD-MS4A3 “brake” operating under steady-state conditions, enabling energy-efficient erythropoiesis. These findings reveal a non-invasive, physiological intervention to significantly improve RBC output and propose novel therapeutic strategies targeting the autophagy-JunD-MS4A3 axis for erythroid disorders, particularly EPO-insensitive anemias.

Acute graft-versus-host disease (aGvHD) is considered a result of "cytokine storm." Targeted therapeutic interventions on cytokines via ubiquitination regulatory pathways may provide a potential approach for aGvHD treatment. Ubiquitin-specific peptidase 11 (USP11) has been reported to play key roles in a variety of physiopathological processes by regulating the stability and function of several vital protein molecules. However, its role in aGvHD remains unclear. In this study, we identified USP11 was associated with aGvHD in patients. In the aGvHD mouse model, the colon and liver were more seriously affected in recipient mice who received USP11 wt bone marrow (BM) cells and eased after the donor was treated with a USP11 inhibitor or received USP11 ko BM cells. In mouse models, IL-6 was identified as a major effecter in accelerating aGvHD induced by USP11. In the cell model, IL-6 mRNA transcript was affected by USP11. In addition, USP11 also inhibited IL-6 degradation by affecting IL-6 ubiquitination. Furthermore, the positive correlation between USP11 and IL-6 was confirmed in the GvHD patients' samples. Collectively, all results indicated that USP11 played a critical role in the onset and progression of aGvHD. USP11 might be a potential target for aGvHD treatment.

The data are available from the corresponding author upon reasonable request. This study was approved by the ethics committee of the First Affiliated Hospital of Soochow University according to Helsinki declaration, and informed consent was obtained from all subjects. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND:Fasting is known to influence the immune functions of leukocytes primarily by regulating their mobilization and redistribution between the bone marrow and the peripheral tissues or circulation, in particular via relocalization of leukocytes back in the bone marrow. However, how the immune system responds to the increased risk of invasion by infectious pathogens with fewer leukocytes in the peripheral blood during fasting intervention remains an open question.RESULTS:We used proteomic, biochemical and flow cytometric tools to evaluate the impact of short-term intensive fasting (STIF), known as beego, on red blood cells by profiling the cells from the STIF subjects before and after 6 days of fasting and 6 days of gradual refeeding. We found that STIF, by triggering the activation of the complement system via the complement receptor on the membrane of red blood cells, boosts fairly sustainable function of red blood cells in immune responses in close relation to various pathogens, including viruses, bacteria and parasites, particularly with the pronounced capacity to defend against SARS-CoV-2, without compromising their oxygen delivery capacity and viability.CONCLUSION:STIF fosters the immune function of red blood cells and therefore, it may be considered as a nonmedical intervention option for the stronger capacity of red blood cells to combat infectious diseases.

OBJECTIVE:To detect the relative expression of IGLL1 (immunoglobulin lambda-like polypeptide 1) mRNA in bone marrow of children with T-cell acute lymphoblastic leukemia (T-ALL), and analyze its correlation with the clinical characteristics and prognosis of the patients, so as to clarify the clinical significance of IGLL1 in pediatric T-ALL patients.METHODS:A total of 56 pediatric T-ALL patients hospitalized in Children's Hospital of Soochow University from June 2012 to December 2017 and treated with CCLG-ALL 2008 regimen were selected. Transcriptome sequencing technology was used to detect the transcription level of IGLL1 gene in children with T-ALL. According to 25% of the IGLL1 transcription level (cutoff value:448), the enrolled children were divided into IGLL1 low expression group (17 cases) and IGLL1 high expression group (39 cases). Combined with clinical data, the correlation between the expression level of IGLL1 and prognosis of the patients was analyzed.RESULTS:The comparative analysis showed that the transcription level of IGLL1 was not correlated with the clinical characteristics of the patients, such as sex, age, bone marrow blast, white blood cell (WBC) count at initial diagnosis. The 5-year OS rate of patients with high IGLL1 expression was significantly higher than that of patients with low IGLL1 expression (76.9%±6.7% vs 47.1%±12.1%, P =0.018). Further comparison of relapse-free survival (RFS) rate between the two groups showed that the 5-year RFS rate of patients with high IGLL1 expression was higher than that of patients with low IGLL1 expression, but the difference between the two groups was not statistically significant (P =0.095). Multivariate COX analysis was conducted on common clinical prognostic factors (age, sex, WBC count at diagnosis, prednisone response on the 7th day, bone marrow response on the 15th day after treatment) and IGLL1 expression level, and the results showed that IGLL1 expression (P =0.012) and prednisone response (P =0.017) were independent risk factors for overall survival in pediatric T-ALL patients.CONCLUSION:In pediatric T-ALL, the OS rate of children with high expression of IGLL1 gene was significantly higher than that of children with low expression of IGLL1 gene, and the expression level of IGLL1 gene was an independent factor affecting the survival of children with T-ALL, which suggests that IGLL1 is a marker of good clinical prognosis of children with T-ALL.

SUMMARYFasting is known to improve health, but the precisely beneficial effects of specific types of fasting and their underlying mechanisms are not fully understood. We herein report that in humans, occasional short-term intensive fasting (STIF), a traditional fasting format favored by Asians, promotes erythropoiesis and boosts the function of red blood cells (RBCs) in oxygen transportation, ATP generation, antioxidant capacity, and innate immune response. The rejuvenation of erythropoiesis is more pronounced in humans with low RBC counts. Using mouse models and a human erythroid progenitor cell model, we found that occasional STIF rejuvenates erythropoiesis by enhancing megakaryocyte-erythroid progenitor selfrenewal and erythroid-biased differentiation without compromising normal hematopoiesis. Molecularly, STIF relies on an autophagy-dependent but erythropoietin (EPO) upregulation-independent MS4A3-CDK2 module to augment the production of RBCs. Our findings thus suggest that STIF can occasionally be practiced as an efficient noninvasive intervention for better erythropoiesis, particularly for adults with low RBC counts.Graphical abstractIn briefXu et al showed that short-term intensive fasting (STIF), when occasionally practiced, boosts red blood cell function and promotes erythropoiesis by regulating MS4A3-CDK2 module to enhance megakaryocyte-erythroid progenitor selfrenewal and erythroid-biased differentiation.HighlightsOccasional STIF boosts the function of red blood cellsOccasional STIF promotes erythropoiesis, which is more significant in adults with low red blood cell countsOccasional STIF selectively enhances MEP selfrenewal and erythroid-biased differentiation via MS4A3-CDK2 moduleThe erythropoietic MS4A3-CDK2 response to STIF is autophagy-dependent but EPO upregulation-independent.

Clinical and Translational MedicineVolume 12, Issue 5 e892 LETTER TO EDITOROpen Access Dynamic change of variant allele frequency reveals disease status, clonal evolution and survival in pediatric relapsed B-cell acute lymphoblastic leukaemia Shuiyan Wu, Shuiyan Wu Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, China Pediatric Intensive Care Unit, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorLixia Liu, Lixia Liu Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Tianjin, ChinaSearch for more papers by this authorXinran Chu, Xinran Chu Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorJiajia Zheng, Jiajia Zheng Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorZixing Chen, Zixing Chen Department of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorLi Gao, Li Gao Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorPeifang Xiao, Peifang Xiao Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorJun Lu, Jun Lu Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorQi Ji, Qi Ji Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorJing Ling, Jing Ling Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorShanbo Cao, Shanbo Cao Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Tianjin, ChinaSearch for more papers by this authorJian Pan, Jian Pan Institute of Pediatric Research, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorJiayue Qin, Corresponding Author Jiayue Qin jyqin@live.cn orcid.org/0000-0003-4721-0016 Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Tianjin, China Correspondence Shaoyan Hu, Department of Hematology and Oncology, Children's Hospital of Soochow University, No. 92, Zhongnan Street, Suzhou 215002, China. Email: hushaoyan@suda.edu.cn Jiayue Qin, Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Building D4, International Enterprise Community, Changyuan Road, Wuqing District, Tianjin 301799, China. Email: jyqin@live.cnSearch for more papers by this authorShaoyan Hu, Corresponding Author Shaoyan Hu hushaoyan@suda.edu.cn orcid.org/0000-0002-3386-6957 Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, China Correspondence Shaoyan Hu, Department of Hematology and Oncology, Children's Hospital of Soochow University, No. 92, Zhongnan Street, Suzhou 215002, China. Email: hushaoyan@suda.edu.cn Jiayue Qin, Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Building D4, International Enterprise Community, Changyuan Road, Wuqing District, Tianjin 301799, China. Email: jyqin@live.cnSearch for more papers by this author Shuiyan Wu, Shuiyan Wu Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, China Pediatric Intensive Care Unit, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorLixia Liu, Lixia Liu Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Tianjin, ChinaSearch for more papers by this authorXinran Chu, Xinran Chu Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorJiajia Zheng, Jiajia Zheng Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorZixing Chen, Zixing Chen Department of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorLi Gao, Li Gao Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorPeifang Xiao, Peifang Xiao Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorJun Lu, Jun Lu Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorQi Ji, Qi Ji Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorJing Ling, Jing Ling Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorShanbo Cao, Shanbo Cao Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Tianjin, ChinaSearch for more papers by this authorJian Pan, Jian Pan Institute of Pediatric Research, Children's Hospital of Soochow University, Suzhou, ChinaSearch for more papers by this authorJiayue Qin, Corresponding Author Jiayue Qin jyqin@live.cn orcid.org/0000-0003-4721-0016 Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Tianjin, China Correspondence Shaoyan Hu, Department of Hematology and Oncology, Children's Hospital of Soochow University, No. 92, Zhongnan Street, Suzhou 215002, China. Email: hushaoyan@suda.edu.cn Jiayue Qin, Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Building D4, International Enterprise Community, Changyuan Road, Wuqing District, Tianjin 301799, China. Email: jyqin@live.cnSearch for more papers by this authorShaoyan Hu, Corresponding Author Shaoyan Hu hushaoyan@suda.edu.cn orcid.org/0000-0002-3386-6957 Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, China Correspondence Shaoyan Hu, Department of Hematology and Oncology, Children's Hospital of Soochow University, No. 92, Zhongnan Street, Suzhou 215002, China. Email: hushaoyan@suda.edu.cn Jiayue Qin, Department of Medical Affairs, Acornmed Biotechnology Co., Ltd., Building D4, International Enterprise Community, Changyuan Road, Wuqing District, Tianjin 301799, China. Email: jyqin@live.cnSearch for more papers by this author First published: 23 May 2022 https://doi.org/10.1002/ctm2.892 Shuiyan Wu, Lixia Liu, Xinran Chu and Jiajia Zheng contributed equally. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Dear Editor, Changes in variant allele frequency (VAF) in different mutated genes played an important role in predicting patient disease status and assessing patient prognosis in adult myelodysplastic syndrome and acute myeloid leukaemia,1-3 but were not well established in pediatric relapsed B-cell acute lymphoblastic leukaemia (B-ALL) patients.4, 5 Thus, we evaluated the mutation profile and dynamic change of VAF in 68 serial bone marrow (BM) samples based on deep targeted next-generation sequencing (NGS) in 24 Chinese pediatric relapsed B-ALL patients at diagnosis, remission and relapse, providing insight into predicting disease status, clonal evolution and survival. The details of clinical data and methods are described in Tables S1 and S2. A total of 71 mutated genes and 204 genetic mutations were detected at diagnosis and/or relapse in the 24 patients (Figure S1 and Table S3). Note that, 90.5% (19/21) of the patients at relapse were often accompanied by new mutations, which is consistent with the results of Malinowska-Ozdowy et al.6 For 21 patients with diagnosis-relapse pairs, 81.0% (17/21) of the patients had mutations at diagnosis and mutations were detected in all patients at relapse (Figure 1A). Compared with the mutations at diagnosis, the mutations detected at relapse were significantly involved in transcription factor pathway (61.9% vs. 23.8%, p = 0.013) (Figure 1B), which plays an essential role in the pathogenesis of pediatric ALL.7, 8 Moreover, the number of mutations in pediatric B-ALL patients at relapse tended to be higher than that at diagnosis (median 3 vs. 2, p = 0.080), indicating that the mutation profile of B-ALL patients at relapse is more complex. We further examined the mutation types and no significant difference was shown in the distribution of conversion and transversion mutations between the two time points (p = 0.843) (Figure S2). FIGURE 1Open in figure viewerPowerPoint Comparative analysis of mutation distribution in 21 patients with diagnosis-relapse paired samples. (A) Histogram showing the comparison of mutated genes at diagnosis and relapse. Mutation at diagnosis and relapse is colored blue and red, respectively. (B) Volcano plot showing the comparison of functional pathways in which the mutated genes are involved at diagnosis and relapse. The horizontal axis represents the magnitude of association (log2 odds ratio), and the vertical axis indicates the -log2 p-value. Each circle shows a functional pathway and the size of each circle indicates the frequency of the mutated gene involved in functional pathways. (C) Distribution of three mutation types, including diagnosis-specific type (only present at diagnosis), relapse-specific type (only present at relapse) and overlap type (present at both diagnosis and relapse) according to different pathways Based on the 153 mutation sites at diagnosis and/or relapse, we categorized mutation sites into three types, including diagnosis-specific type (only present at diagnosis), relapse-specific type (only present at relapse) and overlap type (present both at diagnosis and relapse). According to the enrichment analysis of mutation sites involved in different gene pathways, diagnosis-specific type, relapse-specific type and overlap type were the most common in telomere maintenance pathway, cell metabolism/apoptosis-related genes/cohesion complex pathway and spliceosome/drug metabolism/cell cycle regulation pathway, respectively, suggesting that these functional pathways play an important role in relapsed B-ALL (Figure 1C). We next investigated VAF changes with serial NGS assessments and discovered delta VAF from diagnosis was significantly associated with response to treatment. In 15 patients with diagnosis-remission pairs, patients achieving complete remission (CR) had significant VAF reduction (p < 0.001) (Figure 2A), revealing that several potential markers could be used for detecting minimal residual disease (MRD), including TP53, NRAS, PTPN11 and NF1, as these mutations disappeared in CR. Moreover, in 18 patients with remission-relapse pairs, VAF was significantly increased in relapsed patients, indicating delta VAF can be used to evaluate patients for recurrence (p < 0.001) (Figure 2B). FIGURE 2Open in figure viewerPowerPoint Dynamic change of variant allele frequency (VAF) with serial next-generation sequencing (NGS) assessments. (A) Comparison of VAFs at diagnosis and remission in 15 patients with diagnosis-remission paired samples. (B) Comparison of VAFs at remission and relapse in 18 patients with remission-relapse paired samples. (C) Comparison of VAFs at three different relapse stages in 24 relapsed patients, including very early, early and late relapse stage. Each point in different color represents an independent VAF mutation site. VAF, variant allele frequency; NGS, next-generation sequencing Depending on the time of relapse, we divided patients into three groups, including very early (<18 months), early (18–36 months) and late (>36 months) relapse stages.9 We found that the VAF in the late relapse stage was significantly higher than that in both very early and early relapse stages (p = 0.001; p < 0.001), while no statistical difference was discovered in VAF between the very early and early stages (p = 0.721, Figure 2C), suggesting that continuous gene mutation monitoring during the CR period would facilitate the early detection of clones, and early clinical intervention might help to prolong the CR time. Clonal evolution diagrams were produced based on the mutations. We found three relapse-related clonal evolution patterns, including relapse evolving from a genetically distinct clone (Pattern A), a major clone at diagnosis (Pattern B), and a subclone at diagnosis (Pattern C), respectively (Figure 3). For patient 19 (P19), belonging to Pattern A, flow cytometry showed MRD negative with 3% of BM blasts, and the genetic results showed lower VAF mutations in the TP53, PTPN11 and PAX5 genes at a four-month time point prior to relapse (Figure 3B,C). Compared with the genetic results at relapse, we found that the novel enlarged clonal mutation led to the eventual relapse, suggesting serial gene mutation detection in CR period is helpful for early detection of molecular recurrence. However, this phenomenon was not observed in P16 and P18. For P16, belonging to Pattern B, TP53 p.R248Q mutation was detected at initial diagnosis, disappeared at remission, 2.5 months after remission, 1.5 months before relapse, and 1 month before relapse, and reappeared at relapse (Figure 3B,C), suggesting that TP53 is an effective therapeutic monitoring indicator. For P18, belonging to Pattern C, had mutations in KRAS p.A146V, PTPN11 p.E76K and MAPK1 p.7_8del that were cleared after treatment, but the mutation in PTPN11 p.D61H expanded at relapse (2.9% vs. 21.6%), indicating that the proportion of RAS signaling pathway subclones might be reduced during treatment, expanded in the later stage and caused relapse (Figure 3B,C), which is consistent with the results of Ma et al.10 FIGURE 3Open in figure viewerPowerPoint Relapse-related clonal evolution patterns based on variant allele frequency (VAF). (A) Schematic diagram of three relapse-related clonal evolution patterns, including relapse evolution from a genetically distinct clone, a major clone at diagnosis, and a subclone at diagnosis, named clonal evolution patterns A, B and C, respectively. Longitudinal analysis of VAF (B) and relapse-related clonal evolution pattern (C) in three representative pediatric B-ALL patients. The horizontal axis represents follow-up time, and the vertical axis indicates VAF. Each point in different color represents an independent VAF mutation site. VAF, variant allele frequency; BM, bone marrow In order to explore the prognostic value of VAF, we analyzed the association between VAF at relapse and survival. Based on the maximally selected log-rank statistic, mean VAF was found to be the cutoff point for differentiating patient prognosis. Compared with patients carrying a lower mutation load (mean VAF < 20%), patients with a higher mutation load (mean VAF ≥ 20%) had poorer OS (median survival 3.7 vs. 14.1 months, p = 0.032) (Figure 4A) and OS censored at transplantation (median survival 2.0 vs. 18.9 months, p = 0.022) (Figure 4B), which suggests that mean VAF at relapse can stratify the prognosis of relapsed patients. FIGURE 4Open in figure viewerPowerPoint Prognostic impact of variant allele frequency (VAF) on survival. Overall survival (OS) (A) and OS censored at the time of transplantation (B) of 24 relapsed pediatric B-ALL patients with mean VAF ≥20% versus patients with mean VAF < 20% at relapse. VAF, variant allele frequency; OS, overall survival; SCT, transplantation In conclusion, our study highlights that dynamic change of VAF reveals the disease status, clonal evolution, and survival in pediatric relapsed B-ALL patients, and serial molecular detections during the remission period contribute to early detection of relapse, which plays an important role in early intervention and precision therapy. ACKNOWLEDGEMENTS This study was supported by the Natural Science Foundation of China (81970163, 82170218, and 81800185), Jiangsu Province Projects (BE2021654 and BK20210097), Suzhou projects (SS201809 and GSWS2020039) and Jiangsu Provincial Government Scholarship and Jiangsu Provincial Health International (Regional) Exchange Support Program. CONFLICT OF INTEREST The authors declare no conflict of interest. Supporting Information Filename Description ctm2892-sup-0001-SuppMat.docx1.4 MB Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. REFERENCES 1Yun S, Geyer SM, Komrokji RS, et al. Prognostic significance of serial molecular annotation in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Leukemia. 2021; 35(4): 1145- 1155. 2Uy GL, Duncavage EJ, Chang GS, et al. Dynamic changes in the clonal structure of MDS and AML in response to epigenetic therapy. Leukemia. 2017; 31(4): 872- 881. 3Zhao P, Qin J, Liu W, et al. Using circulating tumor DNA to monitor myelodysplastic syndromes status. Hematol Oncol. 2019; 37(4): 531- 533. 4Li B, Brady SW, Ma X, et al. Therapy-induced mutations drive the genomic landscape of relapsed acute lymphoblastic leukemia. Blood. 2020; 135(1): 41- 55. 5Antic Z, Yu J, Bornhauser BC, et al. Clonal dynamics in pediatric B-cell precursor acute lymphoblastic leukemia with very early relapse. Pediatr Blood Cancer. 2022; 69(1):e29361. 6Malinowska-Ozdowy K, Frech C, Schonegger A, et al. KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia. Leukemia. 2015; 29(8): 1656- 1667. 7Oshima K, Khiabanian H, da Silva-Almeida AC, et al. Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia. Proc Natl Acad Sci USA. 2016; 113(40): 11306- 11311. 8Mar BG, Bullinger LB, McLean KM, et al. Mutations in epigenetic regulators including SETD2 are gained during relapse in paediatric acute lymphoblastic leukaemia. Nat Commun. 2014; 5: 3469. 9Parker C, Waters R, Leighton C, et al. Effect of mitoxantrone on outcome of children with first relapse of acute lymphoblastic leukaemia (ALL R3): an open-label randomised trial. Lancet. 2010; 376(9757): 2009- 2017. 10Ma X, Edmonson M, Yergeau D, et al. Rise and fall of subclones from diagnosis to relapse in pediatric B-acute lymphoblastic leukaemia. Nat Commun. 2015; 6: 6604. Volume12, Issue5May 2022e892 FiguresReferencesRelatedInformation

Beego is a traditional Chinese complete water-only fasting practice initially developed for spiritual purposes, later extending to physical fitness purposes. Beego notably includes a psychological induction component that includes meditation and abdominal breathing, light body exercise and ends with a specific gradual refeeding program before returning to a normal diet. Beego has regained its popularity in recent decades in China as a strategy for helping people in subhealthy conditions or with metabolic syndrome, but we are unaware of any studies examining the biological effects of this practice. To address this, we here performed a longitudinal study of beego comprising fasting (7 and 14 day cohorts) and a 7-day programmed refeeding phase. In addition to detecting improvements in cardiovascular physiology and selective reduction of blood pressure in hypertensive subjects, we observed that beego decreased blood triacylglycerol (TG) selectively in TG-high subjects and increased cholesterol in all subjects during fasting; however, the cholesterol levels were normalised after completion of the refeeding program. Strikingly, beego reduced platelet formation, activation, aggregation and degranulation, resulting in an alleviated thrombosis risk, yet maintained haemostasis by sustaining levels of coagulation factors and other haemostatic proteins. Mechanistically, we speculate that downregulation of G6B and MYL9 may influence the observed beego-mediated reduction in platelets. Fundamentally, our study supports that supervised beego reduces thrombosis risk without compromising haemostasis capacity. Moreover, our results support that beego under medical supervision can be implemented as non-invasive intervention for reducing thrombosis risk, and suggest several lines of intriguing inquiry for future studies about this fasting practice (http://www.chictr.org.cn/index.aspx, number, ChiCTR1900027451).

Relapse is the major cause of mortality in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Effective preventive intervention in high-risk AML may be crucial. In this study, we investigated the clinical efficacy and safety of low dose decitabine (DAC) as part of a modified Busulfan-Cyclophosphamide (Bu-Cy) regimen for high-risk AML patients undergoing allo-HSCT to reduce relapse rate. Fifty-nine patients received DAC (20 mg/m(2)/d, i.v.) for 5 days, followed by modified Bu-Cy (DAC group). A matched-pair control (CON) group of 177 patients (matched 1:3) received modified Bu-Cy only. The differences were more substantial among patients with active disease: 2-year OS, 80.7% (DAC) versus 43.5% (CON), P = 0.011 and 2-year LFS, 64.9% (DAC) versus 39.2% (CON), P = 0.024. Median time to relapse was 8 months (DAC) versus 5 months (CON) for the entire groups and 6.5 months (DAC) versus 3.5 months (CON) for patients with active disease. In summary, our data indicated that the conditioning regimen containing low dose DAC may confer a survival advantage in high-risk AML patients with active disease undergoing allo-HSCT, and a prospective randomized trial is warranted to confirm these observations.

OBJECTIVE:To analyze the effect of clinical features, routine laboratory examination and related gene mutation on the OS of patients with myelodysplastic syndrome (MDS) after hematopoietic stem cell transplantation (HSCT).METHODS:121 patients diagnosed as MDS and underwent hematopoietic stem cell transplantation in the First Affiliated Hospital of Soochow University from October 2013 to August 2018 were selected. Basic information of the patients was collected, and blood cells, bone marrow blasts at initial diagnosis, chromosomal karyotypes and gene mutations of the patients were detected.The effect of different factors on overall survival (OS) was analyzed by statistical method.RESULTS:Kaplan-Meier univariate analysis shows that OS was significanly different among different age groups. The 3-year OS rate of patients aged 0-29 years was (83.3±7.7) %, the 3-year OS rate in patients aged 30-49 years was (58.1±7.7 %), and the 3-year OS rate of patients aged 50-69 years was (31.0±22.6) %, which was statistically different (P＜0.05) between different groups. There were also significant differences in OS among patients with different transplantation types. 3-year OS rate: HLA-matched sibling HSCT＞unrelated HLA-matched HSCT＞haploidentical HSCT＞micro HSCT. The OS rate of patients with bone marrow blasts≥10% seems lower than blasts＜10%, but there was no statistical difference.The 3-year OS rate of patients with chromosomal karyotype complex abnormality was (47.7±11.5) %, and that of patients without complex abnormality was (80±4.2) % which was statistical difference (P＜0.05). Patients with DNMT3A, NRAS, TP53 and GATA2 mutations had shorter OS time compared with patients without mutation of these genes, which shows statistically significant (P＜0.05). COX multivariate analysis showed that age, chromosome karyotype, DNMT3A, TET2, GATA2 and NRAS were the independent factors influencing OS of patients after HSCT, with statistically significant difference.CONCLUSION:age of patients, donor selection of HSCT, chromosome karyotype, DNMT3A, NRAS, TP53, GATA2 and TET2 gene mutations are all independent factors affecting the OS of patients after HSCT. Therefore, the assessment of the OS of MDS patients with transplantation requires comprehensive consideration.

Beego is a traditional Chinese complete water-only fasting practice initially developed for spiritual purposes, later extending to physical fitness purposes. Beego notably includes a psychological induction component that includes meditation and abdominal breathing, light body exercise, and ends with a specific gradual refeeding program before returning to a normal diet. Beego has regained its popularity in recent decades in China as a strategy for helping people in subhealthy conditions or with metabolic syndrome, but we are unaware of any studies examining the biological effects of this practice. To address this, we here performed a longitudinal study of beego comprising fasting (7 and 14 day cohorts) and a 7-day programmed refeeding phase. In addition to detecting improvements in cardiovascular physiology and selective reduction of blood pressure in hypertensive subjects, we observed that beego decreased blood triacylglycerol (TG) selectively in TG-high subjects and increased cholesterol in all subjects during fasting; however, the cholesterol levels were normalized after completion of the refeeding program. Strikingly, beego reduced platelet formation, activation, aggregation, and degranulation, resulting in an alleviated thrombosis risk, yet maintained hemostasis by sustaining levels of coagulation factors and other hemostatic proteins. Mechanistically, we speculate that downregulation of G6B and MYL9 may influence the observed beego-mediated reduction in platelets. Fundamentally, our study supports that supervised beego reduces thrombosis risk without compromising hemostasis capacity. Moreover, our results support that beego under medical supervision can be implemented as noninvasive intervention for reducing thrombosis risk, and suggest several lines of intriguing inquiry for future studies about this fasting practice (<http://www.chictr.org.cn/index.aspx>, number,ChiCTR1900027451). ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial <http://www.chictr.org.cn/index.aspx>, number, ChiCTR1900027451 ### Funding Statement This study was supported by the National Natural Science Foundation of China (to JW) by grants 91649113 and 31771640, and by Soochow University (to JW) by grant H190443, and by the Natural Science Foundation of Jiangsu Province, China (to YF) by grant SBK2020043689, and by The Postdoctor Science Foundation of Jiangsu Province (to YF) by grant 2020Z064, and by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Province, China. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Approval No. ECSU-2019000153 by Ethics Review Board at Soochow University, China All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The data that support the findings of this study are available from the corresponding authors upon request. * AIx : augmentation index APTT : activated partial thromboplastin time AT III : antithrombin III testing BDNF : brain-derived neurotrophic factor CNS : central nervous system COGs : cluster of orthologous groups DBP : diastolic blood pressure DDA : data-dependent acquisition DIA : data-independent acquisition DTT : dithiothreitol ELISA : Enzyme-linked immunosorbent assay FIB : Fibrinogen FITC : fluoresceine isothiocyanate GO : Gene ontology HDL-C : high-density lipoprotein cholesterol INR : international normalized ratio LC-MS/MS : liquid phase chromatography LDL-C : low-density lipoprotein cholesterol MPV : Mean platelet volume PCT : platelet hematocrit PDW : platelet distribution width PE : phycoerythrin PLT : platelet PPI : protein-protein interaction PRP : platelet-rich plasma PT : prothrombin time PWA : pulse wave PWV : pulse wave velocity rFD : refeeding days RM : repeated measures ROS : reactive oxygen species RT : room temperature SBP : systolic blood pressure SEM : standard error of the mean SOP : standard operating procedures SPE : solid phase extraction TC : total cholesterol TG : triacylglycerol TPO : thrombopoietin TT : thrombin time wFD : water-only fasting day.

Summary Beego is a traditional Chinese complete water-only fasting practice initially developed for spiritual purposes, later extending to physical fitness purposes. Beego notably includes a psychological induction component that includes meditation and abdominal breathing, light body exercise, and ends with a specific gradual refeeding program before returning to a normal diet. Beego has regained its popularity in recent decades in China as a strategy for helping people in subhealthy conditions or with metabolic syndrome, but we are unaware of any studies examining the biological effects of this practice. To address this, we here performed a longitudinal study of beego comprising fasting (7 and 14 day cohorts) and a 7-day programmed refeeding phase. In addition to detecting improvements in cardiovascular physiology and selective reduction of blood pressure in hypertensive subjects, we observed that beego decreased blood triacylglycerol (TG) selectively in TG-high subjects and increased cholesterol in all subjects during fasting; however, the cholesterol levels were normalized after completion of the refeeding program. Strikingly, beego reduced platelet formation, activation, aggregation, and degranulation, resulting in an alleviated thrombosis risk, yet maintained hemostasis by sustaining levels of coagulation factors and other hemostatic proteins. Mechanistically, we speculate that downregulation of G6B and MYL9 may influence the observed beego-mediated reduction in platelets. Fundamentally, our study supports that supervised beego reduces thrombosis risk without compromising hemostasis capacity. Moreover, our results support that beego under medical supervision can be implemented as noninvasive intervention for reducing thrombosis risk, and suggest several lines of intriguing inquiry for future studies about this fasting practice ( http://www.chictr.org.cn/index.aspx , number, ChiCTR1900027451).

Leukemia stem cells (LSC) are responsible for the occurrence, therapeutic resistance, and relapse of leukemia. LSC are typically found in a dormant state and thus are less likely to respond to standard chemotherapeutic agents, which preferentially eradicate actively cycling cells and can have significant toxicity to normal hematopoietic cells. Identifying agents that target both LSC and leukemia cells is thus an important step in the development of novel therapies for leukemia. Despite the uncertainty over the phenotype, biological properties, and hierarchical organization of B-cell acute lymphoblastic leukemia (B-ALL) LSC, CD34 and CD19 together are robust markers for B-ALL LSC. Notably, these LSC have been shown to occur more frequently in high-risk B-ALL than in other hematopoietic malignancies, making it more difficult to target them. An in vitro study indicated that Tenovin-6 induces apoptosis in B-ALL cells and eliminates CD133 B-ALL LSC. LSC of patient-derived ALL are sensitive to TNF-related apoptosis inducing ligand (TRAIL), and the sensitivity of B-ALL LSC to recombinant human CD19L-sTRAIL is greater. Nevertheless, attempts at targeting B-ALL LSC or precursor cells have been limited compared to efforts combating acute myeloid leukemia stem cells. Recent immunotherapies

血小板是血液中行使凝血和止血功能的重要成分,近年来研究发现血小板还有日益增多的各种功能.血小板的数量和功能缺陷本身可导致某些疾病,而某些疾病中出现的血小板数量和功能缺陷,也严重影响疾病的预后.自1906年首次确认血小板来源于巨核细胞后,血小板的各种病理改变就必然与巨核细胞的发育成熟过程密切相关.本文拟对造血系统中巨核细胞系列的发育成熟和血小板生成释放的过程及其调控机理,以及在多种疾病中针对巨核细胞、血小板的靶向干预策略做一简明扼要的综述,使读者获得此领域比较全面综合的理解.

Abstract　　The physiological hematopoiesis depends on the programmed expression of a series of gene regulated by mechanisms at various levels. Currently, the epigenetic regulation has been considered as the most important mechanism during hematopoietic differentiation, resulting in a specific epigenomic landscape in the hematopoietic stem/progenitor cells. We try to concisely review the epigenetic mechanisms, including the genomic methylation, the histone modifications and the expression profiles of noncoding RNA, illustrating briefly the differentiation from the hematopoietic stem/progenitor cells to to the erythroid, myeloid and lymphoid cells.

Long non-coding RNAs (lncRNAs) are important in cancer biology. In this study, we analyzed differentially expressed genes in CD34 + hematopoietic cells and identified a novel lncRNA, H22954, which was down-regulated in acute myeloid leukemia (AML) patients. In cultured AML cells and mouse xenograft models, H22954 expression inhibited cell proliferation and tumor growth, respectively. Bioinformatic analysis and RNA antisense purification assay indicated that H22954 targeted the 3' untranslated region of the BCL2 gene. In luciferase assays, H22954 expression inhibited BCL2 expression. In transfected K562 cells and mouse xenograft tumors, H22954 overexpression reduced BCL-2 protein levels and promoted cell death. In AML patients, H22954 expression inversely correlated with BCL-2 protein levels in bone marrow cells, blast cell numbers and disease prognosis. These results indicate that H22954 is a novel regulator of BCL-2 and that reduced H22954 expression may play an important role in the pathogenesis of AML.