CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels.

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT's adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Case report: A successful clinical experience of transplantation of liver and kidney from a donor with myelodysplastic syndromes.

With a shortage of organs for transplant, the use of marginal donors can be an effective measure to meet the shortfall. Myelodysplastic syndromes (MDS) are considered an absolute contraindication for organ donation because of the high invasive potential. Currently, organ transplantation from donors with a past history of MDS has not been reported. In this paper, we report the successful clinical experience of one liver transplantation and two kidney transplantations, with organs donated by a 39-year-old patient diagnosed with a past history of MDS following intracranial hemorrhage. Four and a half years after transplantation, the three recipients are all doing well. However, it is still not clear to what extent organs donated by patients with a past history of MDS can be safely transplanted. This report provides support for the careful use of marginal donors. With effective treatment and full peer assessment, livers and kidneys from donors with a past history of MDS may be safely transplanted.

The role of leukocytes in myeloproliferative neoplasm thromboinflammation.

Classic myeloproliferative neoplasms lacking the Philadelphia chromosome are stem cell disorders characterized by the proliferation of myeloid cells in the bone marrow and increased counts of peripheral blood cells. The occurrence of thrombotic events is a common complication in myeloproliferative neoplasms. The heightened levels of cytokines play a substantial role in the morbidity and mortality of these patients, establishing a persistent proinflammatory condition that culminates in thrombosis. The etiology of thrombosis remains intricate and multifaceted, involving blood cells and endothelial dysfunction, the inflammatory state, and the coagulation cascade, leading to hypercoagulability. Leukocytes play a pivotal role in the thromboinflammatory process of myeloproliferative neoplasms by releasing various proinflammatory and prothrombotic factors as well as interacting with other cells, which contributes to the amplification of the clotting cascade and subsequent thrombosis. The correlation between increased leukocyte counts and thrombotic risk has been established. However, there is a need for an accurate biomarker to assess leukocyte activation. Lastly, tailored treatments to address the thrombotic risk in myeloproliferative neoplasms are needed. Therefore, this review aims to summarize the potential mechanisms of leukocyte involvement in myeloproliferative neoplasm thromboinflammation, propose potential biomarkers for leukocyte activation, and discuss promising treatment options for controlling myeloproliferative neoplasm thromboinflammation.

Detection and quantification of JAK2V617F copy number by droplet digital PCR versus real-time PCR.

Myeloproliferative neoplasm (MPN) usually has an adverse prognosis, progressing to acute leukemia or splanchnic vein thromboses (SVTs). Therefore, early diagnosis and intervention are significantly important. Clinically, the burden of JAK2V617F is a vital diagnostic basis, which can be detected during the early stage of MPN. Thus, an accurate and rapid detective technique is urgently required. In recent years, real-time quantitative PCR (qPCR) has primarily been applied to detect the copies of JAK2V617F, whereas droplet digital PCR (ddPCR), a novel and promising detective tool, can conduct precise and repeatable quantification of nucleic acid copies without relying on the standard curve. In our study, both qPCR and ddPCR are used to evaluate the mutation burden of JAK2V617F in a series of gradient diluted standards and clinical JAK2V617F-positive MPN patients' bone marrow samples collected, while using next-generation sequencing technology (NGS) as a contrast. With the help of statistical methods, our study concluded that ddPCR had a better performance in accuracy, sensitivity, and stability, especially in a low burden. Regarding the accuracy, ddPCR showed a better linearity (Pearson R2 = 0.9926; P < 0.0001) than qPCR (Pearson R2 = 0.9772; P < 0.0001). What is more, ddPCR showed lower intra-assay and inter-assay CVs and the limit of detection (LOD) for the series of diluted standards than qPCR, demonstrating better stability and lower LOD. In a nutshell, ddPCR is a more promising technique for the detection and quantification of JAK2V617F.

Pan-cancer analysis revealing that PTPN2 is an indicator of risk stratification for acute myeloid leukemia.

The non-receptor protein tyrosine phosphatases gene family (PTPNs) is involved in the tumorigenesis and development of many cancers, but the role of PTPNs in acute myeloid leukemia (AML) remains unclear. After a comprehensive evaluation on the expression patterns and immunological effects of PTPNs using a pan-cancer analysis based on RNA sequencing data obtained from The Cancer Genome Atlas, the most valuable gene PTPN2 was discovered. Further investigation of the expression patterns of PTPN2 in different tissues and cells showed a robust correlation with AML. PTPN2 was then systematically correlated with immunological signatures in the AML tumor microenvironment and its differential expression was verified using clinical samples. In addition, a prediction model, being validated and compared with other models, was developed in our research. The systematic analysis of PTPN family reveals that the effect of PTPNs on cancer may be correlated to mediating cell cycle-related pathways. It was then found that PTPN2 was highly expressed in hematologic diseases and bone marrow tissues, and its differential expression in AML patients and normal humans was verified by clinical samples. Based on its correlation with immune infiltrates, immunomodulators, and immune checkpoint, PTPN2 was found to be a reliable biomarker in the immunotherapy cohort and a prognostic predictor of AML. And PTPN2'riskscore can accurately predict the prognosis and response of cancer immunotherapy. These findings revealed the correlation between PTPNs and immunophenotype, which may be related to cell cycle. PTPN2 was differentially expressed between clinical AML patients and normal people. It is a diagnostic biomarker and potentially therapeutic target, providing targeted guidance for clinical treatment.

Improving the diagnosis of myelodysplastic syndrome by red blood cell parameters

Purpose To investigate the value of red blood cell parameters in Myelodysplastic syndrome (MDS) diagnosis and their relations to MDS subtypes and risk groups. Methods The red blood cell parameter [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red cell distribution width (RDW)] levels [203 MDS, 99 aplastic anemia (AA), 145 megaloblastic anemia (MA)] were collected from a single-center retrospective cohort. The cut-off values, area under the receiver operating characteristic curve (ROC) curve (AUC), sensitivity and specificity of the four parameters were calculated from the ROC. Furthermore, Kruskal–Wallis test and Dunn’s Test were performed to determine erythrocyte parameters in different subtypes and prognostic risks MDS. Results There are significant statistic differences in RDW (P < 0.001), MCH (P = 0.036) and MCHC (P < 0.001) (MDS vs AA); RDW (P = 0.009), MCV (P < 0.001), MCH (P < 0.001) and MCHC (P = 0.001) (MDS vs MA); MCV (P = 0.011) and MCH (P = 0.008) (higher-risk MDS vs lower-risk MDS). Between MDS and MA, the area under the receiver operating characteristic curve (ROC) curve (AUC) values of MCV, MCH, MCHC, RDW were 0.846, 0.855, 0.617, and 0.593. Between MDS and AA, the AUC values of MCH, MCHC, RDW were 0.609, 0.671, and 0.662, respectively. Conclusions The red blood cell parameters contribute to the differential diagnosis of MDS, AA and MA and are related to MDS subtypes and risk groups (AU)

Improving the diagnosis of myelodysplastic syndrome by red blood cell parameters.

PURPOSE: To investigate the value of red blood cell parameters in Myelodysplastic syndrome (MDS) diagnosis and their relations to MDS subtypes and risk groups. METHODS: The red blood cell parameter [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red cell distribution width (RDW)] levels [203 MDS, 99 aplastic anemia (AA), 145 megaloblastic anemia (MA)] were collected from a single-center retrospective cohort. The cut-off values, area under the receiver operating characteristic curve (ROC) curve (AUC), sensitivity and specificity of the four parameters were calculated from the ROC. Furthermore, Kruskal-Wallis test and Dunn's Test were performed to determine erythrocyte parameters in different subtypes and prognostic risks MDS. RESULTS: There are significant statistic differences in RDW (P < 0.001), MCH (P = 0.036) and MCHC (P < 0.001) (MDS vs AA); RDW (P = 0.009), MCV (P < 0.001), MCH (P < 0.001) and MCHC (P = 0.001) (MDS vs MA); MCV (P = 0.011) and MCH (P = 0.008) (higher-risk MDS vs lower-risk MDS). Between MDS and MA, the area under the receiver operating characteristic curve (ROC) curve (AUC) values of MCV, MCH, MCHC, RDW were 0.846, 0.855, 0.617, and 0.593. Between MDS and AA, the AUC values of MCH, MCHC, RDW were 0.609, 0.671, and 0.662, respectively. CONCLUSIONS: The red blood cell parameters contribute to the differential diagnosis of MDS, AA and MA and are related to MDS subtypes and risk groups.

The Combination of JAK2V617F Allele Burden and WT1 Expression can Be Helpful in Distinguishing the Subtype of MPN Patients.

INTRODUCTION: Classical Philadelphia-negative myeloproliferative neoplasm (MPN) includes Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF). The JAK2V617F mutation is part of the major criteria for diagnosis of MPN. WT1 is reported to be highly overexpressed in most hematological malignancy. Our aim was to explore the combination value of JAK2V617F allele burden and WT1 expression in distinguishing the subtype of MPN patients. METHODS: Allele specific real-time quantitative fluorescence PCR (AS-qPCR) was conducted to detect JAK2V617F allele burden. WT1 expression was assessed by RQ-PCR. Our study is a retrospective study. RESULTS: JAK2V617F allele burden and WT1 expression were different in MPN subgroups. The expression of WT1 in PMF and PV is higher than in ET. JAK2V617F allele burden in PMF and PV is also higher than in ET. ROC analysis indicated that combination of JAK2V617F allele burden and WT1 expression to discriminate ET and PV, ET and PMF, PV and PMF is 0.956, 0.871, 0.737 respectively. Furthermore, their ability to distinguish ET patients with high Hb levels from PV patients with high platelet counts is 0.891. CONCLUSIONS: Our data revealed that combination of JAK2V617F allele burden and WT1 expression is useful in distinguishing the subtype of MPN patients.

Serum proteomics screening intercellular adhesion molecule-2 improves intermediate-risk stratification in acute myeloid leukemia.

Background: The clinical risk classification of acute myelocytic leukemia (AML) is largely based on cytogenetic and molecular genetic detection. However, the optimal treatment for intermediate-risk AML patients remains uncertain. Further refinement and improvement of prognostic stratification are therefore necessary. Objectives: The aim of this study was to identify serum protein biomarkers to refine risk stratification in AML patients. Design: This study is a retrospective study. Methods: Label-free proteomics was used to identify the differential abundance of serum proteins in AML patients. Transcriptomic data were combined to identify key altered markers that could indicate the risk rank of AML patients. The survival status was assessed by Kaplan-Meier and multivariate Cox regression analyses. Results: We delineated serum protein expression in a population of AML patients. Many biological processes were influenced by the identified differentially expressed proteins. Association analysis of transcriptome data showed that intercellular adhesion molecule-2 (ICAM2) had a higher survival prediction value in the intermediate-risk AML group. ICAM2 was detrimental for intermediate-risk AML, regardless of whether patients received bone marrow transplantation. ICAM2 well distinguishes the intermediate group of patients, whose probability of survival is comparable to that of patients with the ELN-2017 according to the reference classification. In addition, newly established stratified clinical features were associated with leukemia stem cell scores. Conclusion: The inclusion of ICAM2 expression into the AML risk classification according to ELN-2017 was a good way to transfer patients from three to two groups. Thus, providing more information for clinical decision-making to improve intermediate-risk stratification in AML patients.

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma.

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain κ or λ. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

Hemophagocytic Lymphohistiocytosis Associated with Synergistic Defects of AP3B1 and ATM Genes: A Case Report and Literature Review.

Hemophagocytic lymphohistiocytosis (HLH) is an overwhelming immune system activation that manifests as hyperinflammation and life-threatening multiple organ failure. However, the clinical manifestations of the systemic inflammatory response in sepsis and fulminant cytokine storm caused by HLH macrophage activation are very similar and difficult to distinguish. HLH triggered by two novel gene defects manifesting with multiorgan dysfunction syndrome (MODS) and distributive shock has not been reported. A 14-year-old male patient was hospitalized with a high fever, his condition deteriorated rapidly, accompanied by cytopenia, shock, and MODS, and he was subsequently transferred to our intensive care unit (ICU) for symptomatic and organ-supportive treatments. Laboratory indicators of cytopenia, hypofibrinogenemia, hypertriglyceridemia, hyperferritinemia, high soluble CD25, low natural killer (NK) cell cytotoxicity, and hemophagocytosis in the bone marrow confirmed the diagnosis of HLH. Molecular genetic analysis revealed that two novel heterozygous gene mutations in AP3B1 (c.3197 C > T) and ATM (c.8077 G > T) might have accounted for the onset. After treatment, the patient's condition successfully improved. This case report demonstrates the timely determination of underlying triggers and critical care supports (supportive and etiological treatment) of HLH related to the improved outcome.

WEE1 inhibition induces glutamine addiction in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemias (T-ALLs) are aggressive and heterogeneous hematologic tumors resulting from the malignant transformation of T-cell progenitors. The major challenges in the treatments of T-ALL are dose-limiting toxicities of chemotherapeutics and drug resistance. Despite important progress in deciphering the genomic landscape of T-ALL, translation of these findings into effective targeted therapies remains largely unsuccessful. New targeted agents with significant antileukemic efficacy and less toxicity are in urgent need. We herein report that the expression of WEE1, a nuclear tyrosine kinase involved in cell cycle G2-M checkpoint signaling, is significantly elevated in T-ALL. Mechanistically, oncogenic MYC directly binds to the WEE1 promoter and activates its transcription. T-ALL cells particularly rely on the elevated WEE1 for cell viability. Pharmacological inhibition of WEE1 elicits global metabolic reprogramming which results in a marked suppression of aerobic glycolysis in T-ALL cells, leading to an increased dependency on glutaminolysis for cell survival. As such, dual targeting of WEE1 and glutaminase (GLS1) induces synergistic lethality in multiple T-ALL cell lines and shows great efficacy in T-ALL patient-derived xenografts. These findings provide mechanistic insights in the regulation of WEE1 kinase in T-ALL and suggest an additional vulnerability during WEE1 inhibitor treatments. In aggregate, we highlight a promising combination strategy of dual inhibition of cell cycle kinase and metabolic enzymes for T-ALL therapeutics.

Tracing asymptomatic SARS-CoV-2 carriers among 3674 hospital staff:a cross-sectional survey.

BACKGROUND: Asymptomatic carriers were positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without developing symptoms, which might be a potential source of infection outbreak. Here, we aim to clarify the epidemiologic and influencing factors of asymptomatic carriers in the general population. METHODS: In our hospital, all hospital staff have received throat swab RT-PCR test, plasma COVID-19 IgM/IgG antibodies test and chest CT examination. We analyzed the correlation between infection rates and gender, age, job position, work place and COVID-19 knowledge training of the staff. After that, all asymptomatic staff were re-examined weekly for 3 weeks. FINDINGS: A total of 3764 hospital staff were included in this single-center cross-sectional study. Among them, 126 hospital staff had abnormal findings, and the proportion of asymptomatic infection accounted for 0.76% (28/3674). There were 26 staff with IgM+, 73 with IgG+, and 40 with ground glass shadow of chest CT. Of all staff with abnormal findings, the older they are, the more likely they are to be the staff with abnormal results, regardless of their gender. Of 3674 hospital staff, the positive rate of labor staff is obviously higher than that of health care workers (HCWs) and administrative staff (P<0.05). In the course of participating in the treatment of COVID-19, there was no statistically significant difference in positive rates between high-risk departments and low-risk departments (P>0.05). The positive rate of HCWs who participated in the COVID-19 knowledge training was lower than those did not participate in early training (P <0.01). Importantly, it was found that there was no statistical difference between the titers of IgM antibody of asymptomatic infections and confirmed patients with COVID-19 in recovery period (P>0.05). During 3 weeks follow-up, all asymptomatic patients did not present the development of clinical symptoms or radiographic abnormalities after active intervention in isolation point. INTERPRETATION: To ensure the safety of resumption of work, institutions should conduct COVID-19 prevention training for staff and screening for asymptomatic patients, and take quarantine measures as soon as possible in areas with high density of population. FUNDING: The Key Project for Anti-2019 novel Coronavirus Pneumonia from the Ministry of Science and Technology, China; Wuhan Emergency Technology Project of COVID-19 epidemic, China.

Wilms' tumor 1 gene in hematopoietic malignancies: clinical implications and future directions.

The Wilms' tumor 1 (WT1) gene is an important regulatory molecule that plays a vital role in cell growth and development. Initially, knowledge of WT1 was mostly limited to Wilms' tumor. Over the past years, numerous studies have shown that WT1 is aberrant expressed or mutated in hematopoietic malignancies, including acute leukemia (AL), myelodysplastic syndrome (MDS) and chronic myelogenous leukemia (CML). Currently, many studies focus on exploring the role of WT1 in hematopoietic malignancies. Such studies improve the understanding of hematopoietic malignancies, and the collection of data about WT1 expression or mutation in hematopoietic malignancies over the past years can facilitate the risk stratification of hematopoietic malignancies. In this review, we highlight the important role of WT1 in hematopoietic malignancies, discuss its potential clinical applications as a minimal residual disease (MRD) and prognostic biomarker, and evaluate the possible therapy target of WT1 in hematopoietic malignancies.

SF3B1 Mutation but Not Ring Sideroblasts Identifies a Specific Group of Myelodysplastic Syndrome-Refractory Cytopenia With Multilineage Dysplasia.

BACKGROUND: Integrating the proportion of ring sideroblasts and SF3B1 mutation status is required for diagnosis of sideroblastic subgroups in myelodysplastic syndrome (MDS) as proposed by the World Health Organization 2016 classification. However, the clinical implications of SF3B1 mutation and ring sideroblasts in MDS-refractory cytopenia with multilineage dysplasia (MDS-RCMD) remain unclear. PATIENTS AND METHODS: Clinical and laboratory features in 238 MDS-RCMD patients were retrospectively analyzed, and the prognostic significance of SF3B1 mutation and ring sideroblasts on overall survival and leukemia-free survival in total MDS-RCMD patients and different subgroups stratified by the percentage of ring sideroblasts or SF3B1 mutation status were evaluated. RESULTS: MDS-RCMD patients with ring sideroblasts ≥ 15% showed a significantly higher prevalence of SF3B1 mutation compared to ring sideroblasts 5%-14% or ring sideroblasts < 5% (75.6% vs. 15.1% vs. 6.4%, P < .001). In multivariate analysis, SF3B1 mutation was associated with a significantly prolonged survival (hazard ratio [HR] = 0.430, P = .013) and reduced leukemic transformation (HR = 0.174, P = .021) in total MDS-RCMD patients, while ring sideroblasts showed no independent effect on either survival or leukemic transformation. There were no significant differences in clinical characteristics or survival between MDS-RCMD patients with ring sideroblasts ≥ 15% and ring sideroblasts 5%-14% in the presence of SF3B1 mutation. Furthermore, SF3B1 mutation showed an independent prognostic effect on overall survival in MDS-RCMD patients with ring sideroblasts 5%-14% (HR = 0.195, P = .046). CONCLUSION: SF3B1 mutation, not the presence of ring sideroblasts, identifies a distinct subtype and showed independent prognostic value on survival and leukemia transformation in MDS-RCMD patients.

Current Status and Perspective Biomarkers in AFP Negative HCC: Towards Screening for and Diagnosing Hepatocellular Carcinoma at an Earlier Stage.

Hepatocellular carcinoma (HCC) is one of the most malignant cancer with high morbidity and mortality which lead to a serious burden to society. AFP (alpha-fetoprotein) is the most widely used serum biomarker to detect HCC worldwide. However, no AFP elevation have been found in many HCC and AFP analysis can't be used to screen HCC in these cases. Currently, many studies have been carried out to find reliable biomarker in diagnosing AFP-negative HCC. Such biomarker would help the diagnosis of AFP-negative HCC, ensuring the timely initiation of treatment. In this review, we highlight the important role of biomarkers that can differentiate AFP-negative HCCs, and discuss their potential clinical applications as biomarkers for the diagnosis of AFP-negative HCC.