Unveiling IL6R and MYC as Targeting Biomarkers in Imatinib-Resistant Chronic Myeloid Leukemia through Advanced Non-Invasive Apoptosis Detection Sensor Version 2 Detection.

The management of chronic myelogenous leukemia (CML) has seen significant progress with the introduction of tyrosine kinase inhibitors (TKIs), particularly Imatinib. However, a notable proportion of CML patients develop resistance to Imatinib, often due to the persistence of leukemia stem cells and resistance mechanisms independent of BCR::ABL1 This study investigates the roles of IL6R, IL7R, and MYC in Imatinib resistance by employing CRISPR/Cas9 for gene editing and the Non-Invasive Apoptosis Detection Sensor version 2 (NIADS v2) for apoptosis assessment. The results indicate that Imatinib-resistant K562 cells (K562-IR) predominantly express IL6R, IL7R, and MYC, with IL6R and MYC playing crucial roles in cell survival and sensitivity to Imatinib. Conversely, IL7R does not significantly impact cytotoxicity, either alone or in combination with Imatinib. Further genetic editing experiments confirm the protective functions of IL6R and MYC in K562-IR cells, suggesting their potential as therapeutic targets for overcoming Imatinib resistance in CML. This study contributes to understanding the mechanisms of Imatinib resistance in CML, proposing IL6R and MYC as pivotal targets for therapeutic strategies. Moreover, the utilization of NIADS v2 enhances our capability to analyze apoptosis and drug responses, contributing to a deeper understanding of CML pathogenesis and treatment options.

Association Between Leg Muscle Thickness and Walking Test with the Haemophilia-Specific Functional Parameters.

OBJECTIVE: To evaluate the muscle thickness and walking test in people with haemophilia A (PWH) and their correlation to joint health and functional impairments. DESIGN: Cross-sectional study. RESULTS: 29 severe/moderate PWH were enrolled. Muscle thickness of quadriceps and medial gastrocnemius were measured using ultrasound. Joint health and functional capacity were assessed using Haemophilia Joint Health Score (HJHS), Haemophilia Early Arthropathy Detection with Ultrasound (HEAD-US), 6-Minute Walking test (6MWT), Haemophilia Quality of Life Questionnaire for Adults (Haem-A-QoL), and Haemophilia Activities List (HAL). Quadriceps muscle thickness significantly correlated with HJHS knee, HEAD-US knee, and HAL. Calf muscle thickness significantly correlated with the HJHS ankle. After adjusted age and BMI, calf muscle thickness was inversely associated with the HJHS ankle. 6MWT was found to significantly correlate with HJHS total, HEAD-US total, Haem-A-QoL, and HAL. CONCLUSION: Muscle thickness and the distance of 6MWT were linked to assessment of joint health, quality of life and activity participation in PWH. Ultrasound measurement of muscle thickness and walking test appear to be useful tools for the assessment of joint health and functional status in PWH.

Recapitulating the immune system of hemophilia A patients with inhibitors using immunodeficient mice.

BACKGROUND AND AIM: Treating hemophilia A patients who develop inhibitors remains a clinical challenge. A mouse model of hemophilia A can be used to test the efficacy of strategies for inhibitor suppression, but the differences in the immune systems of mice and humans limit its utility. To address this shortcoming, we established a humanized NOD/SCID-IL2rγnull hemophilia A (hu-NSG-HA) mouse model with a severely deficient mouse immune system presenting a patient's adapted immune cells. METHODS AND RESULTS: Through intrasplenic injection with patient inhibitor-positive peripheral blood mononuclear cells (PBMCs), utilizing an adeno-associated viral delivery system expressing human BLyS, and regular FVIII challenge, human C19+ B cells were expanded in vivo to secrete anti-FVIII antibodies. Both the inhibitor and the human anti-FVIII IgG, including the predominant subclasses (IgG1 and IgG4) present in the majority of inhibitor patients, were detected in the mouse model. We further segregated and expanded the different clones of human anti-FVIII-secreting cells through subsequent transplantation of splenocytes derived from hu-NSG-HA mice into another NSG-HA mouse. By transplanting a patient's PBMCs into the NSG-HA mouse model, we demonstrated the success of reintroducing a strong anti-FVIII immune response for a short period in mice with the immune systems of inhibitor-positive patients. CONCLUSION: Our results demonstrate a potential tool for directly obtaining functional human-derived antigen-specific antibodies and antibody-secreting cells, which may have therapeutic value for testing patient-specific immune responses to treatment options to assist in clinical decisions.

Efficacy, safety and cost of emicizumab prophylaxis in haemophilia A patients with inhibitors: A nationwide observational study in Taiwan.

INTRODUCTION: Emicizumab mimicking the cofactor function of activated factor VIII (FVIII) restores haemostasis. METHODS: This nationwide observational study aimed to retrospectively investigate efficacy, safety, and cost in 1 year before and up to 3 years after emicizumab prophylaxis for haemophilia A (HA) patients with FVIII inhibitors. RESULTS AND DISCUSSION: A total of 39 severe HA patients with a median age of 23.0 years were enrolled. The median historical peak FVIII inhibitor titre was 174.2 BU/mL with an interquartile range of 56.5-578.8 BU/mL. The median annualized bleeding rate reduced from 24 to 0 events in the first year after emicizumab prophylaxis (p < .01) and sustained in the second and third years. The median annualized joint bleeding rate reduced to 0 and maintained up to 3 years (p < .01). Twenty-seven patients (69.2%) had target joints before emicizumab prophylaxis and only seven patients (17.9%) of them had target joints after prophylaxis. Medical costs, including cost of haemostatic therapy, frequency of outpatient department visits, emergency room visits and hospital admission, were significantly reduced after emicizumab prophylaxis (p < .01). FVIII inhibitor titre decreased after emicizumab prophylaxis. Overall, three (7.7%) patients experienced 202 grade 1 drug-related adverse events after emicizumab prophylaxis. No serious adverse events were reported during emicizumab prophylaxis period. The adherence to emicizumab prophylaxis was 100% up to 3 years. CONCLUSIONS: HA patients with FVIII inhibitors treated with emicizumab prophylaxis resulted in a significant reduction in treated bleeds and associated costs. No new safety events were observed.

Feasibility and Toxicity of Interval-Compressed Chemotherapy in Asian Children and Young Adults with Sarcoma.

Twelve Asian patients with sarcoma received interval-compressed (ic-) chemotherapy scheduled every 14 days with a regimen of vincristine (2 mg/m2), doxorubicin (75 mg/m2), and cyclophosphamide (1200-2200 mg/m2) (VDC) alternating with a regimen of ifosfamide (9000 mg/m2) and etoposide (500 mg/m2) (IE), with filgrastim (5-10 mcg/kg/day) between cycles. Carboplatin (800 mg/m2) was added for CIC-rearranged sarcoma. The patients were treated with 129 cycles of ic-VDC/IE with a median interval of 19 days (interquartile range [IQR], 15-24 days. Median nadirs (IQR) were neutrophil count, 134 (30-396) × 106/L at day 11 (10-12), recovery by day 15 (14-17) and platelet count, 35 (23-83) × 109/L at day 11 (10-13), recovery by day 17 (14-21). Fever and bacteremia were observed in 36% and 8% of cycles, respectively. The diagnoses were Ewing sarcoma (6), rhabdomyosarcoma (3), myoepithelial carcinoma (1), malignant peripheral nerve sheath tumor (1), and CIC-DUX4 Sarcoma (1). Seven of the nine patients with measurable tumors responded (one CR and six PR). Interval-compressed chemotherapy is feasible in the treatment of Asian children and young adults with sarcomas.

Clinical impact of minimal residual disease and genetic subtypes on the prognosis of childhood acute lymphoblastic leukemia.

BACKGROUND: This study analyzed data from two consecutive protocols for children newly diagnosed with acute lymphoblastic leukemia (ALL) to determine the clinical impact of minimal/measurable residual disease (MRD) and recently identified tumor genetic subtypes. METHODS: Genetic subtypes were determined by sequential approaches including DNA indexing, reverse transcriptase-polymerase chain reaction, multiplex ligation-dependent probe amplification, and RNA-sequencing. MRD was assessed by flow cytometry. The Taiwan Pediatric Oncology Group TPOG-ALL-2013 study enrolled patients who received MRD-directed therapy. RESULTS: The 5-year event-free survival (EFS) and overall survival rates in the 2013 cohort were 77.8% and 86.9% compared to those of the 2002 cohort, which were 62.4% and 76.5%. Among patients treated with MRD-guided therapy, those with ETV6-RUNX1 fusion and high hyperdiploidy had the highest 5-year EFS (91.4% and 89.6%, respectively). The addition of dasatinib improved outcomes in patients with BCR-ABL1 ALL. Recently identified subtypes like DUX4-rearranged, ZNF384-rearranged, MEF2D-rearranged, and PAX5alt subtypes were frequently positive for MRD after remission induction, and these patients consequently received intensified chemotherapy. Treatment intensification according to the MRD improved the outcomes of patients presenting DUX4 rearrangements. In high-risk or very-high-risk subtypes, the TPOG-ALL-2013 regimen did not confer significant improvements compared to TPOG-ALL-2002, and the outcomes of BCR-ABL1-like, MEF2D-rearranged, and KMT2A-rearranged ALL subtypes (in addition to those of T-cell ALL) were not sufficiently good. Novel agents or approaches are needed to improve the outcomes for these patients. CONCLUSIONS: The TPOG-ALL-2013 study yielded outcomes superior to those of patients treated in the preceding TPOG-ALL-2002 study. This study provides important data to inform the design of future clinical trials in Taiwan. PLAIN LANGUAGE SUMMARY: MRD-directed therapy improved the outcomes for pediatric ALL, especially standard-risk patients. Genomic analyses and MRD might be used together for risk-directed therapy of childhood ALL. Our work provides important data to inform the design of future clinical trials in Taiwan.

Sequential Approach to Improve the Molecular Classification of Childhood Acute Lymphoblastic Leukemia.

Identification of specific leukemia subtypes is a key to successful risk-directed therapy in childhood acute lymphoblastic leukemia (ALL). Although RNA sequencing (RNA-seq) is the best approach to identify virtually all specific leukemia subtypes, the routine use of this method is too costly for patients in resource-limited countries. This study enrolled 295 patients with pediatric ALL from 2010 to 2020. Routine screening could identify major cytogenetic alterations in approximately 69% of B-cell ALL (B-ALL) cases by RT-PCR, DNA index, and multiplex ligation-dependent probe amplification. STIL-TAL1 was present in 33% of T-cell ALL (T-ALL) cases. The remaining samples were submitted for RNA-seq. More than 96% of B-ALL cases and 74% of T-ALL cases could be identified based on the current molecular classification using this sequential approach. Patients with Philadelphia chromosome-like ALL constituted only 2.4% of the entire cohort, a rate even lower than those with ZNF384-rearranged (4.8%), DUX4-rearranged (6%), and Philadelphia chromosome-positive (4.4%) ALL. Patients with ETV6-RUNX1, high hyperdiploidy, PAX5 alteration, and DUX4 rearrangement had favorable prognosis, whereas those with hypodiploid and KMT2A and MEF2D rearrangement ALL had unfavorable outcomes. With the use of multiplex ligation-dependent probe amplification, DNA index, and RT-PCR in B-ALL and RT-PCR in T-ALL followed by RNA-seq, childhood ALL can be better classified to improve clinical assessments.

Clinical significance and intestinal microbiota composition in immunocompromised children with norovirus gastroenteritis.

BACKGROUND: Norovirus (NoV) infection is common in pediatric patients with immunodeficiency and is more likely to cause severe disease. Objective Our study aims to figure out the clinical differences and distribution of intestinal microbiota in immunocompromised children with NoV gastroenteritis. METHODS: Pediatric patients admitted to Shang-Ho Hospital with diagnosis of acute gastroenteritis including different immune status were enrolled and their medical records were reviewed. NoV gastroenteritis was validated using RT-PCR molecular methods. Viral shedding period was determined by real-time RT-PCR assays. Intestinal microbiota enrichment analysis was carried out by next generation sequencing after fecal DNA extraction and subsequent Linear Discriminant Analysis (LDA) Effect Size (LEfSe) method. RESULTS: Significantly higher frequency of diarrhea [mean, (IQR), 3.8 (3-5) /day] and longer viral shedding time [mean, IQR, 8.5 (5-13) days] was found in immunocompromised NoV infections than in immunocompetent patients without NoV infections (p = 0.013*) and immunocompetent patients with NoV infections (p = 0.030**). The fever prevalence was significantly lower in immunocompromised NoV infections than in different immune or infection status. Intestinal microbiota metagenomics analysis showed no significant community richness difference while the LEfSe analysis showed a significant difference in commensal richness at the phylum level, the family level, and the genus level in patients under different immune status. CONCLUSION: We evaluated the clinical significances and microbiota composition in immunocompromised children with norovirus gastroenteritis. This will further facilitate studies of the interaction between the intestinal microbiota in such patients with precise determination of their bacterial infection control and probiotic supplements strategy.

Influence of blood group and von Willebrand factor on population pharmacokinetics and dose individualization of recombinant factor VIII in Taiwanese patients with haemophilia A.

INTRODUCTION: The large interpatient variability in the pharmacokinetic (PK) parameters of recombinant Factor VIII (rFVIII) observed in haemophilia A hinders efficient and cost-beneficial prophylactic regimen initiation. Identification of factors influencing the PK of rFVIII may shed more light on personalised treatment. AIM: This study aimed to develop a population PK model in the Taiwanese haemophilia A and evaluate the current national health insurance (NHI) reimbursement guidelines of Taiwan for haemophilia treatment. METHODS: A population PK analysis was established based on 69 Taiwanese with moderate or severe haemophilia A. A nonlinear mixed-effects modelling (NONMEM® ) was used to estimate PK parameters and their variabilities. A Monte Carlo simulation was performed to evaluate different prophylactic regimens. RESULTS: A two-compartment model with first-order elimination best described the rFVIII data. Weight-based allometric scaling was related to clearance and central volume of distribution. Blood type and baseline von Willebrand factor (VWF) were significant covariates for clearance. For single dose simulations, a time achieving target level (> 1 IU/dL) was associated with increasing rFVIII dose and VWF level. The multiple dose simulations showed that > 96.4% of patients with high VWF level (> 200%) had predicted trough level > 1 IU/dL for all dosing regimens (15-40 IU/kg, two to three times weekly). However, for twice weekly dosing, lower percentage (47.62-62.20%) of patients with blood group O and low VWF level (< 50%) achieved a predicted trough level > 1 IU/dL. CONCLUSION: The population PK of rFVIII was successfully developed. Dose adjustment based on blood type and VWF level should be considered.

Platelet-Derived Growth Factor Receptor-α Subunit Targeting Suppresses Metastasis in Advanced Thyroid Cancer In Vitro and In Vivo.

Thyroid cancer is the most common endocrine malignancy. Patients with well-differentiated thyroid cancers, such as papillary and follicular cancers, have a favorable prognosis. However, poorly differentiated thyroid cancers, such as medullary, squamous and anaplastic advanced thyroid cancers, are very aggressive and insensitive to radioiodine treatment. Thus, novel therapies that attenuate metastasis are urgently needed. We found that both PDGFC and PDGFRA are predominantly expressed in thyroid cancers and that the survival rate is significantly lower in patients with high PDGFRA expression. This finding indicates the important role of PDGF/PDGFR signaling in thyroid cancer development. Next, we established a SW579 squamous thyroid cancer cell line with 95.6% PDGFRA gene insertion and deletions (indels) through CRISPR/Cas9. Protein and invasion analysis showed a dramatic loss in EMT marker expression and metastatic ability. Furthermore, xenograft tumors derived from PDGFRA geneedited SW579 cells exhibited a minor decrease in tumor growth. However, distant lung metastasis was completely abolished upon PDGFRA gene editing, implying that PDGFRA could be an effective target to inhibit distant metastasis in advanced thyroid cancers. To translate this finding to the clinic, we used the most relevant multikinase inhibitor, imatinib, to inhibit PDGFRA signaling. The results showed that imatinib significantly suppressed cell growth, induced cell cycle arrest and cell death in SW579 cells. Our developed noninvasive apoptosis detection sensor (NIADS) indicated that imatinib induced cell apoptosis through caspase-3 activation. In conclusion, we believe that developing a specific and selective targeted therapy for PDGFRA would effectively suppress PDGFRA-mediated cancer aggressiveness in advanced thyroid cancers.

Philadelphia chromosome-negative B-cell acute lymphoblastic leukaemia with kinase fusions in Taiwan.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukaemia (ALL), a high-risk subtype characterised by genomic alterations that activate cytokine receptor and kinase signalling, is associated with inferior outcomes in most childhood ALL clinical trials. Half of the patients with Ph-like ALL have kinase rearrangements or fusions. We examined the frequency and spectrum of these fusions using a retrospective cohort of 212 newly diagnosed patients with childhood B-cell ALL. Samples without known chromosomal alterations were subject to multiplex reverse transcription polymerase chain reaction to identify known Ph-like kinase fusions. Immunoglobulin heavy chain locus (IGH) capture and kinase capture were applied to samples without known kinase fusions. We detected known kinase fusions in five of 212 patients, comprising EBF1-PDGFRB, ETV6-ABL1, ZC3HAV1-ABL2, EPOR-IGH, and CNTRL-ABL1. Two patients with P2RY8-CRLF2 were identified. Patients with non-Ph kinase fusions had inferior 5-year event-free survival and overall survival compared with patients with other common genetic alterations. The prevalence of non-Ph kinase fusions in our Taiwanese cohort was lower than that reported in Caucasian populations. Future clinical trials with tyrosine kinase inhibitors may be indicated in Taiwan because of the inferior outcomes for B-cell ALL with kinase fusions.

Treatment outcomes of pediatric acute myeloid leukemia: a retrospective analysis from 1996 to 2019 in Taiwan.

Improvement in outcomes of children with acute myeloid leukemia (AML) is attributed to several refinements in clinical management. We evaluated treatment outcomes of Taiwanese pediatric AML patients in the past 20 years. Overall, 860 de novo AML patients aged 0-18 years and registered in the Childhood Cancer Foundation of R.O.C during January 1996-December 2019 were included. Survival analysis was performed to identify factors that improved treatment outcomes. Regardless of treatment modalities used, patients during 2008-2019 had better 5-year event-free survival (EFS) and overall survival (OS) rates than patients during 1996-2007. For patients received the TPOG-AML-97A treatment, only 5-year OS rates were significantly different between patients diagnosed before and after 2008. Patients with RUNX1-RUNX1T1 had similar relapse-free survival rates, but 5-year OS rates were better during 2008-2019. However, the survival of patients who received hematopoietic stem-cell transplantations (HSCT) did not differ significantly before and after 2008. For patients without relapse, the 5-year OS improved during 2008-2019. Non-relapse mortality decreased annually, and cumulative relapse rates were similar. In conclusion, 5-year EFS and OS rates improved during 2008-2019, though intensities of chemotherapy treatments were similar before and after 2008. Non-relapse mortality decreased gradually. Further treatment strategies including more intensive chemotherapy, novel agents' use, identification of high-risk patients using genotyping and minimal residual disease, early intervention of HSCT, and antibiotic prophylaxis can be considered for future clinical protocol designs in Taiwan.

Classic Ehlers-Dalnos syndrome presenting as atypical chronic haematoma: a case report with novel frameshift mutation in COL5A1.

BACKGROUND: Ehlers-Danlos syndrome (EDS) is a group of inherited connective tissue disorders characterized by skin hyperextensibility, joint hypermobility and soft tissue vulnerable to blunt injury. Early recognition and diagnosis are crucial to patients to provide appropriate treatment, as well as to screen for life-threatening conditions such as aortic dissection and hollow organ perforation. The diagnosis of EDS is made based on clinical presentations, skin biopsy, and electron microscopy findings. To date, mutations in at least 20 genes have been found to cause the Ehlers-Danlos syndromes. However, EDS is still underestimated due to lack of awareness of its variable clinical presentations. Here we reported an EDS case with atypical initial presentation and a novel genetic mutation. CASE PRESENTATION: This 4-year-old Taiwanese male patient presented with easy bruising, multiple ecchymoses, joint hypermobility, hyperextensible skin, and prolonged pretibial haematoma. He was initially suspected of a bleeding tendency due to coagulation disorders. The coagulation test results were normal. DNA sequencing was performed for molecular diagnosis. Subsequently, the diagnosis of classical EDS was made by identifying a novel frameshift mutation in COL5A1 [NM_000093.4:c.4211_4212delAG, p.Gln1404Arg]. This mutation in the type V collagen gene COL5A1 contributes to the phenotype of classical EDS. This novel frameshift mutation may disturb the structural stability of collagen V and interfere with its heparin binding capacity, explaining the chronic haematoma. CONCLUSION: The reported case showed the unusual features of chronic haematoma. This novel frameshift mutation and its phenotype correlation can provide useful information for practitioners about early recognition in Ehlers-Danlos syndrome.

The impact of shared decision making with patient decision aids on the rotavirus vaccination rate in children: A randomized controlled trial.

Rotavirus vaccination reduces the incidence and severity of acute gastroenteritis due to rotavirus infection. However, because of a lack of understanding and private payment for the rotavirus vaccine, the rotavirus vaccination rate is still low in some countries. We intended to assess the impact of shared decision-making (SDM) with the assistance of patient decision aids (PDAs) on the rotavirus vaccination rate, and the knowledge, confidence, and congruence of value among baby's parents when decision-making. The study was a two-group, outcome assessor-blind, randomized, controlled trial. The families of 1-month-old infants for routine vaccination were enrolled; they were divided randomly into non-SDM and SDM groups. The influence of SDM on the acceptance of rotavirus vaccination was assessed when their infants were 2 months old. Outcome measures were decisional conflict, decision-making difficulties, and rotavirus vaccine knowledge, and the overall rotavirus vaccination rate. The study enrolled 180 participants. SDM, parents' education level, and rotavirus vaccination of a previous child were variables that influenced acceptance of rotavirus vaccination. The SDM group scored significantly higher for understanding the information on the oral rotavirus vaccine than the non-SDM group, which helped them to decide whether to vaccinate the baby against rotavirus. The rotavirus vaccination rate was 16.7% higher in the SDM group than the non-SDM group. SDM assisted with PDAs gives more information and helps infants' families understand what they need, reduces their decision conflict, and increases their baby's vaccination against rotavirus, which promotes public health. The clinical trial is registered at ClinicalTrials.gov (NCT03804489).

FPGS relapse-specific mutations in relapsed childhood acute lymphoblastic leukemia.

Although the cure rate for childhood acute lymphoblastic leukemia (ALL) has exceeded 80% with contemporary therapy, relapsed ALL remains a leading cause of cancer-related death in children. Relapse-specific mutations can be identified by comprehensive genome sequencing and might have clinical significance. Applying whole-exome sequencing to eight triplicate samples, we identified in one patient relapse-specific mutations in the folylpolyglutamate synthetase (FPGS) gene, whose product catalyzes the addition of multiple glutamate residues (polyglutamation) to methotrexate upon their entry into the cells. To determine the prevalence of mutations of the FPGS mutations, and those of two important genes in the thiopurine pathway, NT5C2 and PRPS1, we studied 299 diagnostic and 73 relapsed samples in 372 patients. Three more FPGS mutants were identified in two patients, NT5C2 mutations in six patients, and PRPS1 mutants in two patients. One patient had both NT5C2 and PRPS1 mutants. None of these alterations were detected at diagnosis with a sequencing depth of 1000X, suggesting that treatment pressure led to increased prevalence of mutations during therapy. Functional characterization of the FPGS mutants showed that they directly resulted in decreased enzymatic activity, leading to significant reduction in methotrexate polyglutamation, and therefore likely contributed to drug resistance and relapse in these cases. Thus, besides genomic alterations in thiopurine metabolizing enzymes, the relapse-specific mutations of FPGS represent another critical mechanism of acquired antimetabolite drug resistance in relapsed childhood ALL.