Efficacy of emicizumab in von Willebrand disease (VWD) patients with and without alloantibodies to von Willebrand factor (VWF): Report of two cases and review of literature.

INTRODUCTION: von Willebrand disease (VWD) is the common bleeding disorder with a clinically relevant bleeding prevalence of 1:10,000. von Willebrand disease patients lack both von Willebrand factor (VWF) and factor VIII (FVIII), which are critical for normal haemostasis. The conventional treatment for VWD includes desmopressin and replacement therapy with plasma derived FVIII with VWF concentrates or recombinant VWF. Development of alloantibodies is a rare occurrence, there is a paucity in the literature of treatment modalities in these patients. Not many reports are available in literature on the efficacy of emicizumab in VWD patients with or without alloantibodies to VWF. AIM: To do systematic review of literature on emicizumab in VWD and report our experience of emicizumab in two patients of VWD METHODS: We used electronic search engines till May 2021 in 'Google scholar' and 'PubMed', to collect the case reports or case series on use of emicizumab for management of VWD. Two of our severe VWD patients were successfully treated with emicizumab. A systematic review was performed and the results discussed. RESULTS: The electronic search revealed six case reports using emicizumab for treatment of VWD. Two were in vitro studies and four in patients with VWD type 3 disease. In vitro studies and in VWD patients on emicizumab, showed improvement in thrombin generation and fibrin formation. Among four patients, three had alloantibodies to VWD and one was negative. All these patients were treated with emicizumab for 6-12 m. After starting emicizumab, none of them had spontaneous bleeding requiring treatment. During treatment with emicizumab, one patient had trauma-associated soft tissue hematoma, which was treated with rFVIIa and another patient had bleeding following dental exfoliation treated with Humate P. We treated two of our VWD patients one with and one without inhibitors with emicizumab after failure of other therapies. Both the patients showed marked improvement and continued to remain well and free of bleeding episodes. None of the patients had any thrombosis or thrombotic microangiopathy (TMA) during treatment with emicizumab. CONCLUSION: In conclusion, this review supports the safety and efficacy of emicizumab in type 3 VWD patients with or without alloantibodies. Further large studies are required to confirm the safety and efficacy of emicizumab in VWD.

Myeloproliferative hypereosinophilic syndrome presenting as cardiac failure and response to imatinib.

Idiopathic hypereosinophilic syndrome was first defined by Chursid et al in 1975; however, following the advances in molecular biology, the World Health Organization has proposed a classification in 2008. Hypereosinophilic syndrome is a heterogeneous group of uncommon disorders characterized by marked peripheral eosinophilia and end-organ manifestation. The authors describe a case of sudden-onset cardiac failure in a young individual who had marked peripheral eosinophilia and detection of FIP1L1/PDGFRA fusion gene. A diagnosis of myeloproliferative neoplasm with eosinophilia and eosinophilic endocarditis was made. His clinical and laboratory parameters showed a dramatic response to imatinib and prednisone.

Unusual clinical presentations of familial hemophagocytic lymphohistiocytosis type-2.

BACKGROUND: Mutations of PRF1 gene have been identified in familial hemophagocytic lymphohistiocytosis type-2 (FHL-2) patients, and it has been reported as the commonest gene defect causing FHL. Patients with severe perforin deficiency usually present within first 1 year of life and with severe clinical manifestations. OBSERVATION: We report 4 cases of severe perforin deficiency presenting with delayed onset and unusual clinical presentations viz., B-cell acute lymphoblastic leukemia, the Hodgkin lymphoma, tuberculosis, and the Still disease. Three of these 4 cases showed a common heterozygous missense mutation (p.Trp129Ser). Two of these patients expired because of uncontrolled hemophagocytic lymphohistiocytosis, one patient had 3 relapses while on therapy and one patient was in remission on maintenance therapy. CONCLUSION: This study shows variety of clinical manifestations of perforin deficiency and although the onset of hemophagocytic lymphohistiocytosis is delayed in these patients, the outcome remains poor as in classical severe perforin deficiency patients.

FLT3 and NPM-1 mutations in a cohort of acute promyelocytic leukemia patients from India.

BACKGROUND: Acute promyelocytic leukemia (APL) with t (15;17) is a distinct category of acute myeloid leukemia (AML) and is reported to show better response to anthracyclin based chemotherapy. A favorable overall prognosis over other subtypes of AML has been reported for APL patients but still about 15% patients relapse. METHODS: This study evaluated the presence of Famus like tyrosine kinase-3 (FLT3) and nucleophosmin-1 (NPM1) gene mutations in a cohort of 40 APL patients. Bone marrow/peripheral blood samples from patients at the time of diagnosis and follow-up were processed for immunophenotyping, cytogenetic markers and isolation of DNA and RNA. Samples were screened for the presence of mutations in FLT3 and NPM1 genes using polymerase chain reaction followed by sequencing. RESULTS: Frequency of FLT3/internal tandem duplication and FLT3/tyrosine kinase domain was found to be 25% and 7% respectively. We observed a high frequency of NPM1 mutation (45%) in the present population of APL patients.

Low-dose emicizumab prophylaxis in patients with severe hemophilia A: a retrospective study bringing new hope for our patients.

BACKGROUND: Low-dose emicizumab can potentially offer a cost-effective treatment option in persons with hemophilia A, especially in developing countries. OBJECTIVES: To compare the efficacy and safety of low-dose emicizumab with those on low-dose factor (F)VIII prophylaxis via chart review. METHODS: After ethics approval, chart data of 2 groups of patients were reviewed: group 1 (low-dose emicizumab, n = 10; 3 mg/kg monthly without a loading dose) and group 2 (low-dose FVIII prophylaxis, n = 10; 10-20 IU/kg of FVIII concentrates twice a week). Outcomes were target joints, annual bleeding rate, annual joint bleeding rate, Hemophilia Joint Health Score, nonactivated thromboelastometry-rotational thromboelastometry clotting time, plasma emicizumab levels, and direct costs of treatment. RESULTS: All outcome measures were significantly better in the low-dose emicizumab group than in the low-dose FVIII prophylaxis group. For nonactivated thromboelastometry-rotational thromboelastometry, median values after 6 months in the low-dose emicizumab group were comparable with values seen in patients with mild hemophilia, while the values in the low-dose FVIII prophylaxis group were similar to those of patients with moderate hemophilia. The direct cost of low-dose emicizumab was found to be approximately US $6000 and that for low-dose recombinant FVIII prophylaxis used in our study was US $6282 (the cost may range from US $3432 to $7920 depending on the type of factor) when compared to approximately US $15 000 for standard-dose emicizumab. CONCLUSION: Low-dose emicizumab offers a cost-effective treatment option and can improve access in developing countries. These findings need to be confirmed in a larger and better-controlled study.

Evaluation of danazol, cyclosporine, and prednisolone as single agent or in combination for paroxysmal nocturnal hemoglobinuria.

OBJECTIVE: The responses of 32 patients with paroxysmal nocturnal hemoglobinuria (PNH) were assessed after the patients were put on various combinations of danazol, prednisolone, and cyclosporine. MATERIALS AND METHODS: Nineteen males and 13 females aged between 14 and 60 years with confirmed diagnosis of PNH were treated with danazol (4), danazol + cyclosporine (7), cyclosporine (1), and prednisolone + danazol (20). Response to these interventions was assessed regularly. Danazol was added to cyclosporine in patients with aplastic bone marrow after 3 months of cyclocporine use only unless the former therapy was successful. Four patients with aplastic marrow received only danazol because they had renal insufficiency at presentation. Patients were evaluated with regular complete blood count and routine liver and renal function tests. RESULTS: One patient responded to cyclosporine only. Thirteen of 32 patients (40%) had complete response, 12/32 patients (37%) had partial response leading to freedom from red cell transfusion, and 2/32 (7%) had no response. Five patients (16%) died due to thrombosis or hemorrhage within 3 months of therapy before their response to therapy could be assessed. The median period of review of the cases was 4 years and 6 months. CONCLUSION: Danazol is a useful addition to PNH therapy both in combination with cyclosporine for hypoplastic PNH and with prednisolone for other forms of PNH, and this therapy could be a good alternative where eculizumab and anti-lymphocyte globulin cannot be used for various reasons. CONFLICT OF INTEREST: None declared.

Comparison of in-vitro and in-vivo response to fetal hemoglobin production and γ-mRNA expression by hydroxyurea in Hemoglobinopathies.

BACKGROUND: Hydroxyurea, which induces Fetal hemoglobin (HbF) synthesis, is the only drug widely used in different hemoglobinopathies; however, the response is very variable. We compared the efficacy of hydroxyurea in-vitro in erythroid cultures and in-vivo in the same patients with different hemoglobinopathies to induce HbF production and enhance γ-messenger RNA expression. MATERIALS AND METHODS: A total of 24-patients with different Hemoglobinopathies were given hydroxyurea and their response was studied in-vivo and in-vitro on mononuclear cells collected from them simultaneously. RESULTS: A total of 57.7% of patients (responders) showed no further crisis or transfusion requirements after hydroxyurea therapy with a mean increase in fetal cells (F-cells) of 63.8 ± 59.1% and γ-mRNA expression of 205.5 ± 120.8%. In-vitro results also showed a mean increase in F-cells of 27.2 ± 24.7% and γ-mRNA expression of 119.6% ± 65.4% among the treated cells. Nearly 19.0% of the partial-responders reduced their transfusion requirements by 50% with a mean increase in F-cells of 61.2 ± 25.0% and 28.4 ± 25.3% and γ-mRNA-expression of 21.0% ± 1.4% and 80.0% ± 14.1% in-vivo and in-vitro respectively. The non-responders (15.3%) showed no change in their clinical status and there was no significant increase in F-cells levels and γ-mRNA expression in-vivo or in-vitro. CONCLUSION: Thus, this method may help to predict the in-vivo response to hydroxyurea therapy; however, a much larger study is required.

Frequency and pattern of chromosomal abnormalities in acute myeloid leukemia from Western India: A retrospective study.

Context: Chromosomal abnormalities play an important role in diagnosis and prognosis of hematological diseases. Aims: The aim of the present study was to study the pattern and frequency of chromosomal aberrations in acute myeloid leukemia (AML) subgroups from western India. Settings and Design: A retrospective study was conducted through evaluating laboratory proforma which were filled during 2005 to 2014 for diagnosis and treatment of AML subjects. Methods and Material: We have studied chromosomal aberrations in 282 subjects with AML from western India. AML patients were sub-grouped according to FAB classification. Cytogenetic study using conventional cytogenetics (GTG-banding) and Fluorescence in situ hybridization (FISH) was carried out using FISH probes (AML1/ETO, PML/RARA, CBFB). Statistical analysis: Student's t test for continuous variables and Pearson's Chi-squared test for categorical variables were used to identify the relationship between variables. Results: Cytomorphological study revealed AML- M3 as most frequent (32.3%) group followed by AML-M2 (25.2%) and AML-M4 (19.9%). Chromosomal abnormalities were identified in 145 (51.42%) of the total AML cases. A high frequency (38.6%) of chromosomal abnormalities was identified in AML-M3 subgroup as compared to AML-M2 (31%) and AML-M4 (20.6%). Conclusions: Cytogenetic study is important for the diagnosis and management of the AML patients. Our study identified chromosomal abnormalities in AML subgroups with varied frequencies. It is important in diagnosis and monitoring of the disease. As younger AML patients were more affected in our study, etiological factors such as environmental factors need to be studied. Combination of conventional cytogenetics and FISH has an advantage of identifying high frequency of chromosomal aberrations in AML patients.

Comprehensive analysis of genetic factors predicting overall survival in Myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clonal hematological disease with high risk of progression to AML. Accurate risk stratification is of importance for the proper management of MDS. Genetic lesions (Cytogenetic and Molecular mutations) are known to help in prognosticating the MDS patients. We have studied 152 MDS patients using cytogenetics and next generation sequencing (NGS). These patients were evaluated and as per cytogenetic prognostic group, majority (92.1%) of the patients classified as good (81.6%) and intermediate (10.5%) group. The NGS identified 38 different gene mutations in our cohort. Among 111 MDS patients with mutations, the most frequent mutated genes were SF3B1 (25.2%), SRSF2 (19%) U2AF1 (14.4%) ASXL1 (9.9%) RUNX1 (9.9%) TET2 (9%), TP53 (9%), ATM (6.3%), NRAS (5.4%) and JAK2/3 (5.4%). The survival analysis revealed that the mutations in TP53, JAK2/3, KRAS, NRAS and ASXL1 were significantly (P < 0.05) associated with poor survival of the patients. The univariate cox and multivariate cox analysis of our study suggested that the age, marrow morphology, cytogenetic and gene mutations with IPSS-R should be considered for prognosticating the MDS patients. We have proposed M-IPSS-R which changed the risk stratification i.e. 66.3% patients had decreased risk whereas 33.75% showed increased risk compared to IPSS-R. The survival analysis also showed that the M-IPSS-R were more significant in separating the patients as per their risk than the IPSS-R alone. The change in risk stratification could help in proper strategy for the treatment planning.

The Spectrum of Clinical, Immunological, and Molecular Findings in Familial Hemophagocytic Lymphohistiocytosis: Experience From India.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of immune dysregulation characterized by hyperactivation of the immune system, excessive cytokine secretion and severe systemic inflammation. HLH is classified as familial (FHL) when associated with mutations in PRF1, UNC13D, STX11, and STXBP2 genes. There is limited information available about the clinical and mutational spectrum of FHL patients in Indian population. This study is a retrospective analysis of 101 molecularly characterized FHL patients over the last 10 years from 20 different referral centers in India. FHL2 and FHL3 together accounted for 84% of cases of FHL in our cohort. Patients belonging to different FHL subtypes were indistinguishable based on clinical and biochemical parameters. However, flow cytometry-based assays viz. perforin expression and degranulation assay were found to be specific and sensitive in diagnosis and classification of FHL patients. Molecular characterization of respective genes revealed 76 different disease-causing mutations including 39 (51%) novel mutations in PRF1, UNC13D, STX11, and STXBP2 genes. Overall, survival was poor (28%) irrespective of the age of onset or the type of mutation in our cohort. Altogether, this article sheds light on the current scenario of FHL in India. Our data reveal a wide genetic heterogeneity of FHL in the Indian population and confirms the poor prognosis of FHL. This study also emphasizes that though mutational analysis is important for diagnostic confirmation of FHL, flow cytometry based assays help significantly in rapid diagnosis and functional validation of novel variants identified.

Chromosomal breakage study in children suspected with Fanconi anemia in the Indian population.

Chromosomal breakage investigation using diepoxybutane induction was carried out in 195 pediatric patients suspected with Fanconi anemia (FA). Chromosomal breakage evaluation showed 33 (17%) patients with classical FA, 9 (4%) with somatic mosaicism FA, (when at least 50% of the metaphases showed chromosomal breakage and radial figures), 25 (13%) with FA with high frequency of chromosomal breakage and without clinical features, and 128 (66%) with suspected FA but had no chromosomal breakage and clinical features of FA. Chromosomal breakage investigation is an important diagnostic tool for differentiating FA from idiopathic aplastic anemia.

Hydroxyurea in sickle cell disease--a study of clinico-pharmacological efficacy in the Indian haplotype.

There is clinical variability in the presentation of sickle cell disease among Indians. Vaso-occlusive crisis is common among non-tribal patients. Hydroxyurea, induces fetal hemoglobin (HbF) synthesis and reduces the clinical severity of sickle cell disease but individual patients have a variable response. This study was undertaken to investigate the efficacy and safety of hydroxyurea in Indians with severe manifestations where the beta(s) gene is linked to the Arab-Indian haplotype and is associated with higher HbF levels. Seventy-seven patients (29 adult sickle homozygous, 25 pediatric sickle homozygous, 23 adult sickle beta-thalassemia) selected for hydroxyurea therapy were evaluated for clinical, hematological, biochemical and genetic parameters and were followed for 24 months. Ninety-eight point seven percent of the sickle chromosomes were linked to the Arab-Indian haplotype, 27% of patients had associated alpha thalassemia and 65% were Xmn I +/+. Seventy-eight percent of the patients had no further crises after starting hydroxyurea. This effect was accompanied by a significant increase in HbF (p<0.001), but this increase was variable in individual cases. There was also an increase in gamma gene mRNA expression in the few cases so studied. Hemoglobin levels increased significantly (p<0.001) resulting in the cessation of blood transfusions. Leucopoenia was observed in one patient. Hydroxyurea was effective in reducing the clinical severity in Indian patients who initially had higher HbF levels and the presence of ameliorating factors, such as alpha-thalassemia and the Xmn I polymorphism. Hydroxyurea therapy with careful monitoring can thus change the quality of life of Indians with sickle cell disease.

Paroxysmal nocturnal haemoglobinuria: diagnostic tests, advantages, & limitations.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal disorder of haematopoietic stem cells. The molecular defect in PNH is mutation in the phosphotidylinositol glycan complementation class A (PIGA gene) causing defect in glycosylphosphatidylinositol anchored proteins (Cell, 73, 1993, 703). The deficiency of these GPI-anchored proteins on the membranes of haematopoietic cells lead to the various clinical manifestations of PNH. Clinically PNH is classified into classic PNH, PNH in the setting of another specified bone marrow disorder and sub clinical PNH. Size of the PNH clone differs in these different subtypes. The management of PNH has been revolutionized by the advent of monoclonal antibody, eculizumab. Thus, today it is important to have sensitive tests to diagnose and monitor the clone size in patients of PNH. Before 1990, diagnosis of PNH was made using complement based tests. However in the last decade, flowcytometry has become the gold standard diagnostic test as it has increased sensitivity to detect small clones, ability to measure clone size and is not affected by blood transfusions. This review is aimed to focus mainly on the different methods available for the detection of PNH clone and the recent advances and recommendations for the flowcytometric diagnosis of PNH.

Cytogenetic study of myelodysplastic syndrome from India.

BACKGROUND & OBJECTIVE: Myelodysplastic syndrome (MDS) represents a group of clonal haematological disorders characterized by progressive cytopenia reflecting defects in erythroid, myeloid and megakaryocytic maturation. The incidence of MDS is more in older age groups and frequent chromosome abnormalities reported to be monosomies 5 and 7. However, the data on cytogenetic changes in Indian MDS patients are scanty. The present study was therefore undertaken to study the aetiology and frequency of chromosomal changes in MDS patients, attending a tertiary care hospital in Maharashtra, India. METHODS: The study was carried out in 145 MDS patients for six years (2001-2006) at National Institute of Immunohaematology (ICMR), and KEM Hospital, Mumbai, India. The patients were diagnosed according to FAB and WHO classification. Cytogenetic study was carried out using GTG-banding and fluorescence in situ hybridization (FISH) methods. Statistical analysis was done with Chi(2) and Fisher's exact test. RESULTS: Chromosomal abnormalities, including novel chromosome aberrations were detected in 54.48 per cent MDS patients and frequency of chromosomal aberrations increased with increase in age (> or = 30 yr). Among occupational exposure factors, chromosomal aberrations significantly (P<0.05) associated with pesticides exposure. INTERPRETATION & CONCLUSION: Our findings showed 54.48 per cent chromosome abnormalities including novel chromosome aberrations in MDS patients and these chromosome aberrations were increased with advancing age. In our series a high frequency of younger population (53%) developed MDS, a detailed molecular genetics and aetiological factors need to be studied.

Mutation profile in Indian primary myelofibrosis patients and its clinical implications.

BACKGROUND: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by abnormal proliferation of megakaryocytes, bone marrow fibrosis, and extramedullary hematopoiesis. We did mutation profile of 50 patients of PMF and tried to correlate it with initial clinical presentation of these patients. MATERIALS AND METHODS: All new and follow up patients who were diagnosed as PMF based on WHO 2016 definition of PMF were included. Mutation profile of these patients including JAK2 V617F, JAK2 exon 12, CALR and MPL mutations was done and all clinical, demographic and laboratory details were recorded. RESULTS: Total 50 patients were enrolled out of which 29 were males and 21 were females. Out of these patients, 32 (64%) were JAK2 positive, 13 (26%) were CALR positive, 1 (2%) were MPL positive and 4 (8%) were triple negative. As compared to JAK2+ve patients and triple negative group, CALR positive patients were younger, had lower total leucocyte count, larger spleen size, lower dynamic international prognostic scoring system (DIPSS) score and higher grade of fibrosis of marrow. CONCLUSION: This study depicts that incidence of JAK2 and CALR mutations in Indian PMF patients is fairly similar to that in rest of the world. CALR positive patients have better clinical parameters at presentation and have better prognosis as compared to JAK2 positive patients.

Twin pregnancy in a patient of chronic myeloid leukemia on imatinib therapy.

Imatinib is a tyrosine kinase inhibitor and is now used regularly in chronic myeloid leukaemia therapy in chronic phase with great success. This drug due its very nature of action is suspected to be teratogenic hence the patients are counseled not to get pregnant while on this drug. However in world literature few normal pregnancies have been reported in patients on Imatinib therapy, though no twin pregnancy has been reported on this medication. We report here the birth of normal mono-ovular mono-chorionic twin while the patient is on imatinib during conception and early pregnancy for chronic myeloid leukaemia.

Spontaneous resolution of severe aplastic anemia following thymic hemorrhage.

Over the last 7 years we have seen more than 200 severe aplastic anemia patients at this centre. Three of them developed an unusual complication in the form of thymic hemorrhage. Following this complication, all 3 patients recovered partially from their aplastic anemia, without any need for further immunosuppression. These cases show possible ways to manipulate the thymus gland as a management strategy for this disease.

Wiskott-Aldrich Syndrome Presenting with JMML-Like Blood Picture and Normal Sized Platelets.

Objective. The aim of this paper is to report the case of Wiskott-Aldrich syndrome (WAS) that presented with unusual laboratory features. Clinical Presentation and Intervention. Male neonate admitted with symptoms related to thrombocytopenia, whose initial diagnosis was considered as neonatal alloimmune thrombocytopenia and JMML (juvenile myelomonocytic leukemia) but subsequently diagnosis was confirmed as WAS. Conclusion. This case shows that a suspicion of WAS is warranted in the setting of neonatal thrombocytopenia with JMML-like blood picture and normal sized platelets.

Safety and efficacy of indigenous equine antithymocyte globulin along with cyclosporine in subjects with acquired aplastic anemia.

To confirm the safety and efficacy of an indigenous equine antithymocyte globulin (eATG) along with cyclosporine in Indian subjects with acquired aplastic anaemia. Subjects >2 years old with acquired aplastic anaemia were enrolled at six hospitals between April 2011 and February 2013, after approval from respective Ethics Committees. Equine ATG at a dose of 40 mg/kg/day was infused for 4 days. Efficacy analysis defined a priori, was in subjects, who had completed eATG treatment and followed-up on day 90 and/or 180. Complete response (CR) was defined as-transfusion independent, haemoglobin ≥11 g/dL, absolute neutrophil count (ANC) >1.5 10(9)/L and platelet ≥150 10(9)/L; partial response (PR) was transfusion independent, haemoglobin ≥8 g/dL, ANC >0.5 10(9)/L and platelet ≥20 10(9)/L; non responders were transfusion dependent. Lymphocyte subsets (CD 2, 3, 4 and 8) in the blood were tested on days 0 (pre eATG infusion), 3, 5, 7, 14 and 21 after eATG. Of the 30 subjects (two children <12 years old) enrolled, 19 completed day 90 and 18 completed day 180 visit. Of the remaining 11 subjects, two died on days 12 and 45 due to septicaemia and pneumonia, one was withdrawn after the first dose of eATG due to jaundice and eight were lost to follow-up. The median age was 30 (9-58) years and weight was 57 (26-84) kg. On day 90, 12 of 30 subjects responded (CR 1, PR 11) and 15 of 30 (CR 2, PR 13) on day 180. The most common adverse event was fever related to eATG infusion. There were two serious adverse events (acute renal failure, febrile neutropenia) and both recovered with treatment. There were no unusual adverse events noted during the study period. Blood T lymphocytes showed a mean decrease of 91 % from baseline that recovered by day 21. We conclude that eATG is safe and in combination with cyclosporine showed overall response in 50 % of enrolled subjects. The trial was registered with the clinical trial registry-india (Registration no. CTRI/2012/03/002498).