Molecular pathology and computational profiling of Janus kinase 2 (JAK2) mutation in acute lymphoblastic leukemia: insights from a Pakistani cohort.

BACKGROUND: JAK2 mutation plays a clinically significant role in the pathogenesis of acute lymphoblastic leukemia (ALL) by enhancing its oncogenicity. The study aimed to characterize the molecular pathology and computational profile of the JAK2 mutation in an ALL cohort of Pakistani origin. METHODS: Ninety-three patients were enrolled in the current study. The disease diagnosis was confirmed via flow cytometry and karyotyping of bone marrow aspirate/blood. For the identification of causative gene variations and assessment of their potential impact, the JAK2 gene underwent direct sequencing and predictive computational and in silico structural analysis, respectively. RESULTS: JAK2 mutations were detected in 10 (11%) patients. All mutations were missense with 1 being frameshift. Most mutations showed a similar pattern to the wild type but p.N673H+p.V674L+p.C675W (AAD699), p.V674F (AAD704), and p.V674L (AAD705) exhibited statistically significant stability loss. The triple mutation displayed reduced stability both globally and locally. CONCLUSION: The pattern of gene defects in JAK2 in the studied cohort showed a disruption in proper folding behavior, evident from increased gyration values, resulting in the hypothesis that these mutations may cause structural alterations in the JAK2 protein that lead to disease progression.

Transcobalamin deficiency - a rare genetic defect in transportation of cobalamin; case report.

BACKGROUND: Vitamin B12 is primarily transported from plasma to cells by Transcobalamin. Deficiency of Transcobalamin is a rare autosomal recessive disorder that results in unavailability of cobalamin in cells and accumulation of homocysteine and methylmalonic acid. CASE REPORT: We report a case of a 2-year-old male child with persistent pancytopenia, recurrent infections, and megaloblastic anemia. Next-generation sequencing identified a novel variant in exon 8 of TCN2 gene. Substantial improvement has been observed following administration of high doses of parenteral methylcobalamin. CONCLUSION: In patients with unresolved pancytopenia and megaloblastic anemia, Transcobalamin deficiency should be investigated and treated promptly to prevent any irreversible and harmful outcome.

Exploring the perceptions and experiences of female's with ß-thalassemia major in a Tertiary Care Private Hospital in Pakistan.

INTRODUCTION: ß-Thalassemia, a genetic condition which influences both the physical and emotional facets of individuals specially females while also exerting substantial financial strain on families and healthcare systems. Recognizing the pivotal influence of social support, particularly on mental well-being, this study endeavors to delve into the shared psychosocial challenges experienced by females grappling with ß-thalassemia major. METHODS: Employing a qualitative-descriptive methodology and purposive sampling, this study conducted in-depth interviews with forty-two young girls, 18.64 ± 4.27, diagnosed with ß-thalassemia major and representing diverse academic backgrounds, using a semi-structured in-depth interview guide. NVIVO-12 software facilitated extended data analysis, encompassing coding, categorization, theme development, and mind-mapping techniques to unravel nuanced insights from the collected data. FINDINGS: This groundbreaking study delves into the psychosocial factors that impact the well-being of female ß-thalassemia patients. The research highlights the key factors that positively contribute to their quality of life by conducting thorough inductive content analysis. These include receiving an education, having robust family support, experiencing overall life satisfaction, and making meaningful societal contributions. The findings of this study can be used to improve the lives of female ß-thalassemia patients and enhance their overall well-being. Contrariwise, notable impediments encompassed depression, social isolation, limited access to insurance services, challenges in educational and employment spheres, as well as difficulties in nurturing social relationships. These findings underscore the multifaceted influences shaping the quality of life for girls navigating ß-thalassemia, shedding light on empowering and challenging elements within their experiences. CONCLUSION: In conclusion, psychosocial factors supporting or hindering the well-being of young girls with ß-thalassemia major in Pakistan include an inductive environment at homes, organizations, education, institutions with adequate knowledge of thalassemia disease among patients, and society. More research is needed to understand their needs and advocate for societal support and acceptance. Family and friends support are crucial for improving their quality of life, necessitating focused efforts to provide understanding and aid within the community.

Antibiotic Stewardship: A Handshaking Strategy Among Physicians and Pharmacists to Improve therapeutic Outcomes in Hematology-Oncology.

Background: Infections are highly susceptible in patients with hematological malignancies due to immune suppression, immunosuppressive therapies and disease progression. Rational use of antibiotics following Antimicrobial Stewardship (AMS) guidelines in early detection and response to infection is significant to improve patient care. Objectives: The present study was conducted to determine the impact of clinical pharmacists' interventions (PIs) on antibiotics usage in hematology-oncology set up in Karachi, Pakistan. Methodology: An observational prospective study was conducted for a period of 4 months in a well-known 75-bed teaching hospital, specializing in bone marrow transplantation in Karachi, Pakistan without a structured Antimicrobial stewardship programs (ASPs). The information was gathered from patient medical histories, laboratory, and microbiological records. Results: A total of 876 PIs (1 to 5 per patient) were implemented. Dose modifications or interval changes accounted for the major interventions (n = 190, 21.6%). The majority of all recommendations were related to antipseudomonal ß-lactams, aminoglycosides, sulfamethoxazole-trimethoprim and vancomycin. Overall, 94.3% (n = 876) of the 928 PIs were accepted. Conclusion: The PIs and the high physician acceptance rate may be useful for improving the safe use of antibiotics, lowering their toxicity, lowering the need for special-vigilance medications and potentially improving patient care.

Haematopoietic Stem Cell Transplant Trends in Pakistan: Activity Survey from Pakistan Bone Marrow Transplant Group.

Pakistan is the fifth most populous country with a population of 225 million and has health expenditure accounting for only 2.8 percent of gross domestic product (GDP). Accordingly, there are a limited number of haematology-oncology and transplant centers in the country. The Pakistan Blood and Marrow Transplant (PBMT) group was established in 2020, and this report is the first activity survey from January 2021 to December 2022 focusing on the trends of matched-related donor, haploidentical, and autologous transplants in a developing country. A total of 12 transplant centers contributed data on the modified PBMT survey form retrospectively and 806 haematopoietic stem cell transplants (HSCTs) were carried out during the study duration. Allogeneic HSCT constituted 595 (73.8%) of all the transplants; this is in stark contrast to Western data, where autologous HSCT accounts for the majority of transplants. ß-thalassemia major and aplastic anemia were the commonest indications for allogeneic HSCT, in contrast to Western data, where acute leukemia is the leading transplant indication. Autologous transplants were more frequently performed for Hodgkin's lymphoma as compared to non-Hodgkin's lymphoma and multiple myeloma. The use of peripheral and bone marrow stem cells was comparable. A myeloablative conditioning regimen was routinely used in patients with acute leukemia. This report provides an insight of HSCT trends in Pakistan which are different from those of Western centers contributing to transplant data from South Asia.

Development of a Thalassemia International Prognostic Scoring System (TIPSS).

A prognostic scoring system that can differentiate ß-thalassemia patients based on mortality risk is lacking. We analysed data from 3145 ß-thalassemia patients followed through a retrospective cohort design for the outcome of death. An a priori list of prognostic variables was collected. ß Coefficients from a multivariate cox regression model were used from a development dataset (n = 2516) to construct a formula for a Thalassemia International Prognostic Scoring System (TIPSS) which was subsequently applied to a validation dataset (n = 629). The median duration of observation was 10.0 years. The TIPSS score formula was constructed as exp (1.4 × heart disease + 0.9 × liver disease + 0.9 × diabetes + 0.9 × sepsis + 0.6 × alanine aminotransferase ≥42 IU/L + 0.6 × hemoglobin ≤9 g/dL + 0.4 × serum ferritin ≥1850 ng/mL). TIPSS score thresholds of greatest differentiation were assigned as <2.0 (low-risk), 2.0 to <5.0 (intermediate-risk), and ≥5.0 (high-risk). The TIPSS score was a good predictor for the outcome of death in the validation dataset (AUC: 0.722, 95%CI: 0.641-0.804) and survival was significantly different between patients in the three risk categories (P < 0.001). Compared to low-risk patients, the hazard ratio for death was 2.778 (95%CI: 1.335-5.780) in patients with intermediate-risk and 6.431 (95%CI: 3.151-13.128) in patients with high-risk. This study provides a novel tool to support mortality risk categorization for patients with ß-thalassemia that could help management and research decisions.

Evaluation of the combination therapy of hydroxyurea and thalidomide in ß-thalassemia.

Transfusion-related complications and lack of resources in low-to-middle-income countries have led to a search for novel therapies to reduce the need for blood transfusions in patients with ß-thalassemia. Hydroxyurea (HU) has demonstrated promising outcomes; additionally, thalidomide has also shown improvement in hemoglobin (Hb) levels for patients with ß-thalassemia in some studies. This study presents the findings of a single-arm nonrandomized trial to evaluate the efficacy of combination therapy of HU and thalidomide in children with ß-thalassemia. A total of 135 patients (median age, 6 [interquartile range, 3-10] years), 77 (57%) males and 58 (43%) females, were followed first using HU alone, for 6 months, and then using the combination of HU and thalidomide for another 6 months. The primary outcome was a response to therapy, as measured by the number of transfusions required and Hb levels, for patients while receiving HU alone and then while using the combination therapy. Study findings showed a significant decline in blood transfusion volume (P < .001) and a significant increase in median Hb levels within 3 and 6 months of the combination therapy (P < .001). Eighty-nine (65.93%) participants were good responders, 16 (11.85%) were responders, and 30 (22.22%) were nonresponders, whereas the responders had variable genetic mutations. A total of 38 adverse events were reported that resolved on supportive treatment or temporary hold of the intervention. The combination therapy demonstrated promising results and could be considered for a diverse patient population with ß-thalassemia. This trial was registered at www.clinicaltrials.gov as #NCT05132270.

A Pragmatic Scoring Tool to Predict Hydroxyurea Response Among ß-Thalassemia Major Patients in Pakistan.

Despite high prevalence and incidence of ß-thalassemia in Pakistan, there is very limited work on the use of hydroxyurea (HU) in thalassemia patients in the country. This is the first insight regarding genetic profiling of BCL11A and HU responses in Pakistani ß-thalassemia. It correlates single-nucleotide polymorphisms on BCL11A (rs4671393, rs766432) and HBG2 (XmnI), age at first transfusion, and ß-globin mutations with HU response in ß-thalassemia major (BTM). Of 272 patients treated with HU, 98 were complete responders, 55 partial responders, and 119 nonresponders. Our analysis shows that HU response was significantly associated with patients having IVSI-1 or CD 30 mutation (P<0.001), age at first transfusion >1 year (P<0.001), and with the presence of XmnI polymorphism (P<0.001). The single-nucleotide polymorphisms of BCL11A were more prevalent among responders, but could not show significant association with HU response (P>0.05). Cumulative effect of all 5 predicting factors through simple binary scoring indicates that the likelihood of HU response increases with the number of primary and secondary genetic modifiers (P<0.001). Predictors scoring is a pragmatic tool to foresee HU response in patients with BTM. The authors recommend a score of ≥2 for starting HU therapy in Pakistani patients with BTM.

Primary HBB gene mutation severity and long-term outcomes in a global cohort of ß-thalassaemia.

In ß-thalassaemia, the severity of inherited ß-globin gene mutations determines the severity of the clinical phenotype at presentation and subsequent transfusion requirements. However, data on associated long-term outcomes remain limited. We analysed data from 2109 ß-thalassaemia patients with available genotypes in a global database. Genotype severity was grouped as ß0 /ß0 , ß0 /ß+ , ß+ /ß+ , ß0 /ß++ , ß+ /ß++ , and ß++ /ß++ . Patients were followed from birth until death or loss to follow-up. The median follow-up time was 34·1 years. Mortality and multiple morbidity outcomes were analyzed through five different stratification models of genotype severity groups. Interestingly, ß0 and ß+ mutations showed similar risk profiles. Upon adjustment for demographics and receipt of conventional therapy, patients with ß0 /ß0 , ß0 /ß+ , or ß+ /ß+ had a 2·104-increased risk of death [95% confidence interval (CI): 1·176-3·763, P = 0·011] and 2·956-increased odds of multiple morbidity (95% CI: 2·310-3·784, P < 0·001) compared to patients in lower genotype severity groups. Cumulative survival estimates by age 65 years were 36·8% for this subgroup compared with 90·2% for patients in lower genotype severity groups (P < 0·001). Our study identified mortality and morbidity risk estimates across various genotype severity groups in patients with ß-thalassaemia and suggests inclusion of both ß+ and ß0 mutations in strata of greatest severity.

Prenatal diagnosis of maternal serum from mothers carrying ß-thalassemic fetus.

BACKGROUND: This study focuses on the discovery of protein biomarkers from the maternal serum of ß-thalassemic trait mothers carrying the normal fetus and ß-thalassemic major fetus. METHODS: Serum samples from ß-thalassemic trait mothers carrying major (N = 5) and normal fetuses (N = 5) were studied. The IVS1-5 thalassemia mutation was common among ß-thalassemic trait mothers who were carrying a homozygous ß-thalassemic fetus (IVS1-5/ IVS1-5 mutation) or a normal fetus (no mutation). We employed two-dimensional gel electrophoresis and mass spectrometry analysis to explore differentially expressed maternal serum proteins from thalassemia carrier couples with the same ß-thalassemia mutation. Western blotting was performed for one of the identified proteins to validate our data. RESULTS: Ten proteins were identified in the maternal serum of ß-thalassemic trait mothers carrying the ß-thalassemic major fetus and normal fetus. Among these, serotransferrin, haptoglobin, α-1 anti-trypsin, apo-lipoprotein A1, and the fibrinogen-ß chain were found to be upregulated in mothers carrying major fetuses and are known to be associated with pregnancy-related disorders. The expression of α-1 anti-trypsin was validated through western blotting. CONCLUSIONS: Proteins identified in the current study from maternal serum are reported to contribute to hereditary disorders. We suggest that these can serve as putative screening markers for non-invasive prenatal diagnosis in ß-thalassemic pregnancies.

Seasonal variations in hematological disorders: A 10-year single-center experience.

AIMS: To assess the seasonal variations in hematological disorders among patients diagnosed on the basis of bone marrow biopsy, who attended National Institute of Blood Diseases (NIBD) clinics during 2006 to 2015. METHODS: We retrospectively reviewed the 10-year records of hematological disorders among patients' NIBD clinics from year 2006 to 2015. All cases of aplastic anemia (AA), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), immune thrombocytopenic purpura (ITP), and acute promyelocytic leukemia (APML) were categorized on the basis of the seasons in which they were diagnosed such as winter (December-February), spring (March-May), southwest monsoon periods (June-September), and retreating monsoon period (October and November). Statistical analysis was performed by using SPSS and STATA. Inferential statistics were explored using the chi-square test for heterogeneity to evaluate seasonal variations. P-value <0.05 was taken as significant. RESULTS: A total of 1982 cases were reviewed. Men were predominantly higher (n = 1190, 60%) as compared to women (n = 792, 40%). Frequency of ALL was found to be higher (513, 25.9%), followed by ITP (504, 25.4%), AML (490, 24.7%), AA (396, 20%), while APML was observed in only 79 (4%) patients. Seasonal variations in the diagnosis of hematological disorders were observed (P-value < .001), except in APML diagnosis (P-value = .445). Significant seasonal variations were also detected in both genders in stratified analysis. CONCLUSION: The finding of this study has reported an increase in the hematological disorder during 2006 to 2015. Particularly, majority of the cases were reported in southwest monsoon period, whereas least cases were reported in retreating period. Significant seasonal and yearly variations were detected in all diagnosis except the APML.

Antioxidant Activity and Oxidative Stress Status in Maternal Serum of ß-thalassemic Mothers Carrying ß-thalassemic Major and Normal Fetuses.

Objectiveß-thalassemia is a genetic disorder characterized by reduction or absence of ß-globin chain with mutations in both copies (ß-thalassemia major) or in one copy (ß-thalassemia minor). Pregnancies in ß- thalassemic carrier women are considered symptom free but have risk of inheriting ß-thalassemic fetuses. Current study was designed to compare oxidative stress and antioxidants status in maternal serum from ß-thalassemic minor mothers having ß-thalassemic major and normal fetuses. Method: We investigated paraoxonase (PON1) and arylesterase (ARE) activities along with malondialdehyde (MDA) and reactive oxygen species (ROS) in maternal serum of ß-thalassemic carrier women. Results: PON1 and ARE activities were found to be significantly decreased, whereas the concentration of MDA and ROS were significantly increased in ß-thalassemic minor mothers with ß-thalassemic major fetuses. Conclusion: The study concludes that redox imbalance in ß-thalassemic trait mothers carrying thalassemic fetuses is higher than in mothers carrying normal fetuses.

Incorporating a clinical oncology pharmacist into an ambulatory care pharmacy in pediatric hematology-oncology and transplant clinic: Assessment and significance.

BACKGROUND: Beta thalassemia patients, post-bone marrow transplant, and leukemia patients require long term therapy with an intense care follow-up especially for pediatric hematology-oncology origin. Emergence of side effects and noncompliance to therapy lead to reduced efficacy of medicines resulting in relapse of diseases. There is an increasing fact to support the incorporation of a pharmacist into clinical team due to their distinctive skills. Clinical oncology pharmacist with experience and specialized training in hematological cancers and bone marrow transplantation (BMT) patient care has in-depth knowledge and skills of chemotherapy regimens including drug information, monitoring parameters of cancer treatment, dose adjustment, drug-drug interactions, adverse effects, and patient counseling skills. AIM AND OBJECTIVES: The main objective of our study was to assess the significance of incorporation of clinical oncology pharmacist in ambulatory care in pediatric hematology-oncology and transplant clinic. MATERIAL AND METHOD: This study was conducted at National Institute of Blood Diseases and Bone Marrow Transplantation hospital with duration of five months from 17 March 2019 to 16 July 2019. In this study the clinical oncology pharmacist was made available at ambulatory clinic of hematology-oncology and transplantation. The activities performed by a clinical oncology pharmacist were observed by resident BMT clinical pharmacist during the visits of patients and their families in a clinic. The BMT pharmacist is a clinical oncology pharmacist with experience and specialized training in hematological cancers and BMT patient care. Only pediatrics patients with beta thalassemia major and those who were on chemotherapy treatment and post-transplant patient were included in this study. RESULTS: During the five months' tenure, there were 1820 pediatric patients' visits in total. The clinical oncology pharmacist performed 980 direct patient interviews and documented 1665 pharmacist interventions. The majority of the documented clinical oncology pharmacist interventions were review of medication histories (n: 404, 24%) and "deferiprone" dose adjustments (n:400, 24%). Genomic profiling interventions were also among the commonly reported activities by the clinical oncology pharmacist. For beta thalassemia patients undergoing hydroxyurea therapy, the genomic profiling was performed to assess whether the hydroxyurea treatment is clinically effective or not (n:396, 23%). CONCLUSION: The involvement of clinical oncology pharmacist into a specialized outpatient clinic of hematology-oncology and transplant clinic plays an integral role in minimizing the adverse effect and reduction in readmission into the hospital. This is new expansion of pharmacist's role especially in underdeveloped country, considering the relevant clinical participation of clinical oncology pharmacist into specialized clinic revealing through optimized therapy and future prospect of clinical oncology pharmacist in pediatric hematology.

A complication risk score to evaluate clinical severity of thalassaemia syndromes.

The thalassaemia syndromes (TS) show different phenotype severity. Developing a reliable, practical and global tool to determine disease severity and tailor treatment would be of great value. Overall, 7910 patients were analysed with the aim of constructing a complication risk score (CoRS) to evaluate the probability of developing one or more complications. Nine independent variables were included in the investigation as predictors. Logistic regression models were used for Group A [transfusion-dependent thalassaemia (TDT)], Group B [transfused non-TDT (NTDT)] and Group C (non-transfused NTDT). Statistically significant predictors included age (years), haemoglobin levels, hepatic transaminases [alanine aminotransferase (ALT) and aspartate aminotransferase] and left-ventricular ejection fraction (LVEF) for Group A; age (years), age at first chelation (months), ALT and LVEF for Group B; and age (years), mean serum ferritin (SF) levels and LVEF for Group C. The area under the receiver operating characteristic curve was 84·5%, 82·1% and 80·0% for Groups A, Group B and Group C respectively, suggesting the models had good discrimination. Finally, the CoRS for each group was categorised into four risk classes (low, intermediate, high, and very high) using the centiles of its distribution. In conclusion, we have developed a CoRS for TS that can assist physicians in prospectively tailoring patients' treatment.

Chromosomal Breakage in Fanconi Anemia and Consanguineous Marriages: A Social Dilemma for Developing Countries.

Introduction A clear picture of the prevalence of Fanconi anemia is not known due to limited studies and research of the subject. This study will detect the frequency of positive chromosomal breakage in pediatric aplastic patients and provide the evidence-based guidelines which help in consideration of appropriate treatment and awareness to the society. Methods A total of 104 aplastic anemia patients were recruited of age <18 years whose samples were tested for chromosomal breakage with mitomycin C (MMC). History of consanguinity between parents were documented for all the patients referred to us. Result Out of 104 diagnosed aplastic anemia patients, 35 (33.7%) patients were found to be Fanconi positive. Mean age of all hypoplastic patients for aplastic anemia and Fanconi anemia was 10.7 ± 4.5 and 10.6 ± 3.5, respectively. Male preponderance was found to be higher (64, 61.5%) as compared to females (40, 38.5%) in aplastic patients. The male to female ratio was observed as 2.5:1 in Fanconi patients while 1.3:1 in non-Fanconi aplastic patients. Parental consanguinity was observed in 33 (94.2%) with Fanconi anemia. Conclusion Fanconi anemia accounts for significant number of patients with hypoplastic bone marrow, therefore consanguineous marriages should be avoided through mass education in Pakistan.

Peripheral Blood Stem Cell Harvest HPC Count Is an Effective Surrogate Marker for CD34+ Cell Count in Allogeneic Stem Cell Transplant Setting.

OBJECTIVE: We assessed the predictive potential of XN-HPC for CD34+ cell count as obtained through Sysmex automated hematology analyzers (XN-1000). METHODS: This study was conducted at the National Institute of Blood Diseases and Bone Marrow Transplantation in 84 donors between December 2012 and December 2017 in the first phase and later validated in 112 donors between December 2017 and December 2018. Sysmex XN-1000 and BD FACS Calibur estimated XN-HPC and CD34+ cells of peripheral blood apheresis product, respectively. Spearman's correlation was assessed between XN-HPC and CD34+ cell count followed by receiver operating characteristic curve calculation to determine the XN-HPC cutoff for a CD34+ count of ≥2 million cells/kg of recipient's body weight RESULTS: There is a moderately positive correlation (P value = .003) between XN-HPC and CD34+ count. Receiver operating characteristic curve analyses demonstrated that a cutoff value for XN-HPC of ≥1·845×106cells/kg of recipient's body weight has a specificity and sensitivity of 100% and 78·2%, respectively, for predicting the CD34+ count of ≥2 million cells/kg of recipient's body weight. This cutoff value of XN-HPC was prospectively validated in 112 donors. The positive predictive value was found to be 100%, while negative predictive value was 17%. CONCLUSION: XN-HPC has a highly promising potential to serve as a cost-effective and time-saving surrogate for CD34+ cell count.