Naresh Gupta

Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A

This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had 3 treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared recombinant FVIII (rFVIII) and rFVIIIFc pharmacokinetics. End points included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 hours) was extended 1.5-fold vs rFVIII (12.4 hours; P < .001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with 1 injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1 to 2 times per week.

Long-acting recombinant factor VIII Fc fusion protein (rFVIIIFc) for perioperative haemostatic management in severe haemophilia A

The Phase 3 A-LONG and Kids A-LONG studies demonstrated the prolonged half-life of rFVIIIFc compared with rFVIII, and the safety and efficacy of rFVIIIFc in subjects with severe haemophilia A. Eligible subjects from A-LONG and Kids A-LONG continued rFVIIIFc treatment by enrolling in ASPIRE, an ongoing extension study. Based on combined data from the primary studies and ASPIRE interim data, the safety and efficacy of rFVIIIFc in subjects requiring surgery were evaluated. Perioperative dosing regimens were determined by investigators with guidance based on pharmacokinetic data and recommendations from a clinical dosing committee. In addition to dosing frequency, factor consumption, blood loss, transfusions, bleeding episodes, and haemostatic response were assessed. Across studies, 21 subjects underwent 23 evaluable major surgeries, including 19 orthopaedic surgeries; 41 subjects underwent 52 minor surgeries, including 30 dental procedures. No major and 10 minor surgeries were performed in paediatric subjects. Of the major (n = 22) and minor (n = 32) surgeries assessed for haemostatic response, all were rated as excellent or good by the investigator/surgeon. During most major surgeries (95.7 %), haemostasis was maintained with one rFVIIIFc infusion. Blood loss in major surgeries was consistent with similar surgeries in subjects without haemophilia. Across studies, rFVIIIFc was well tolerated; no subject developed an inhibitor.

Evans syndrome: a study of six cases with review of literature

Abstract Evans syndrome is an uncommon condition characterised by simultaneous or sequential development of immune thrombocytopenia (ITP) and autoimmune haemolytic anaemia (AIHA) with a positive direct antiglobulin test (DAT) in the absence of a known underlying aetiology. The great majority of patients with Evans syndrome have a chronic relapsing course despite treatment, which is associated with significant morbidity and mortality. We reviewed the clinical and laboratory features of six patients with Evans syndrome. All patients had thrombocytopenia, bleeding symptoms and haemolytic anaemia with positive direct Coombs test at presentation. We discuss the aetiopathogenic, clinical, therapeutic and natural history of Evans syndrome.

Epigenetic Silencing of DAPK1 Gene is Associated with Faster Disease Progression in India Populations with Chronic Myeloid Leukemia

Background: One of the major epigenetic changes in human cancer is DNA methylation of tumour suppressor genes which leads to silencing of gene leading to disease progression. Therefore, DNA methylation status of such genes may serve as the epigenetic biomarker for prognosis of human Chronic Myeloid Leukemia. Material and methods: We used MSP-PCR technique for the analysis of aberrant promoter DAPK1 methylation on 200 CML venous blood samples. Stastical analysis was done for evaluating differences between different parameters using SPSS 16.0 version. Results: We could detect 91/200 promoter methylation (45.5%) in CML patients. Percentage of methylation detected was seen higher in blast phase (63.07%) and in accelerated phase (48.1%) than in chronic phase (29.6%). A significant correlation was seen between CML stages and DAPK1 aberrant methylation. We also found a significant association of DAPK1 methylation in gender and in haematological resistance CML patients. However no correlation was found between DAPK1 promoter methylation and other clinical parameters like age, BCR-ABL type and Thrombocytopenia. Conclusion: In summary we concluded that methylation status of DAPK1 gene is associated with advanced phase of CML and may be related to disease progression in chronic myeloid leukemia. Further study on a more number of patients is needed to explore the role of DAPK1 methylation in the prognosis of CML.

Rectourethral fistula after injection sclerotherapy for haemorrhoids, a rare complication

Injection sclerotherapy is widely used for grade 1 haemorrhoids. We report possibly the first case of rectourethral fistula following its use. A 35-year-old male attended as an emergency, complaining of perineal pain and foul-smelling discharge from the anal orifice for 4 days. There was a history of haemorrhoidal bleeding for 5 months, for which he had received injection sclerotherapy (5% phenol in almond oil) a week previously. On digital rectal examination and proctoscopy, there was a small, irregular mucosal defect with indurated edges at 12 o’clock about 6 cm from the anal verge. The haemoglobin was 9.4 g ⁄ dl, the total leukocyte count 12 600 (polymorphs 90%), the urea 16 mg% and blood sugar 98 mg ⁄ dl. Microscopic examination of the urine showed three or four red blood cells and eight to 10 pus cells per high-power field. An indwelling catheter was inserted, but to our surprise, it came out of the anal orifice, thus confirming the diagnosis of rectourethral fistula (Fig. 1). A suprapubic cystostomy was carried out, and the patient was started on ceftriaxone 1 g i.v. every 12 h and metronidazole 500 mg i.v. every 8 h. The patient responded well to this treatment, and at 6 weeks contrast radiology was normal. The suprapubic cystostomy was removed, and the patient was well thereafter.

Isolated CNS Blast Crises in Chronic Myeloid Leukaemia Presenting as Hypertrophic Pachymeningitis and Bilateral Optic Neuritis: A Case Report.

Extramedullary blast crises of Chronic Myeloid Leukaemia (CML) involving CNS is rare and usually accompanies systemic relapse. Isolated CNS blast relapse is an extremely uncommon event. A 35-year-old male was diagnosed with chronic phase (CP) CML two years back at our hospital and was started on imatinib 400 mg daily. Patient achieved haematological and cytogenetic remission at three and 12 months respectively but was non-compliant with medications thereafter. He presented to our emergency with headache and bilateral visual loss. CNS examination revealed neck rigidity and fundoscopy revealed disc edema with retinal vein dilatation and haemorrhages. Cerebrospinal fluid (CSF) analysis revealed lymphocytic pleocytosis and a positive cytospin for myeloid blasts. MRI brain suggested pachymeningeal enhancement involving falx cerebri and tentorium along with bilateral optic nerve thickening. Patient maintained cytogenetic remission at current presentation. A diagnosis of isolated CNS blast crises with pachymeningitis and bilateral optic nerve involvement was made and two doses of intrathecal chemotherapy were administered. However, patient died due to a rapidly downhill course. A previously unreported finding of pachymeningitis with bilateral optic neuritis has been highlighted in this case, along with a brief review of this rare condition.

Simple multiplex RT-PCR for identifying common fusion BCR-ABL transcript types and evaluation of molecular response of the a2b2 and a2b3 transcripts to Imatinib resistance in north Indian chronic myeloid leukemia patients.

INTRODUCTION Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome, an abnormally shortened chromosome 22. It is the result of a reciprocal translocation of chromosomes 9 and 22, creating BCR-ABL fusion transcripts, b3a2, b2a2, and e1a2. The aim of our study was to determine the type of BCR-ABL fusion transcripts for molecular diagnosis and investigate the frequency of BCR-ABL fusion transcripts in CML patients by multiplex RT-PCR in CML. MATERIALS AND METHODS A single reaction with multiple primers multiplex PCR was used to detect and investigate the type and frequency in 200 CML patients among which 116, 33, and 51 were in CP, AP, and BC phase, respectively. RESULTS The study included 200 CML patients, among whom breakpoints in b3a2, b2a2 transcripts were detected in 68% and 24%, respectively, while 8% of the patients showed both b3a2/b2a2. A statistically significant difference was seen between frequency of BCR-ABL fusion transcripts and gender (P = 0.03), molecular response (P = 0.04), and hematological response (P = 0.05). However, there was no correlation found between frequencies of BCR-/ABL fusion transcripts and other clinicopathological parameters like age, type of therapy, thrombocytopenia, and white blood cell count. CONCLUSION Multiplex reverse transcriptase-polymerase chain reaction is useful and saves time in the detection of BCR-ABL variants; the occurrence of these transcripts associated with CML can assist in prognosis and treatment of disease.

Inactivation of RIZ1 Gene by Promoter Hypermethylation is Associated with Disease Progression and Resistance to Imatinib in Indian Chronic Myelogenous Leukemia Patients, First Study from India

Background: The epigenetic impact of DNA methylation in chronic myelogenous leukemia (CML) is not completely understood. RIZ1 expression and activity are reduced in many cancers. In CML, blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located. RIZ1 is a PR domain methyltransferase that methylates histone H3 lysine 9, a modification important for transcriptional repression. In CML blast crisis cell lines RIZ1 represses insulin-like growth factor-1 expression and autocrine signaling. Together these observations suggest that RIZ1 may have a role in the chronic phase to blast crisis transition in CML. Methods: To examine whether promoter methylation is involved in the disease development and progression of CML, we investigated promoter methylation status of RIZ1 gene in 100 chronic myeloid leukemia’s (CML) patients and 50 controls by MSP method. Results: The RIZ1 methylation was studied in 100 CML patients, 9 were cases were methylation positive cases, six of nine were in blastic phase, 2 in chronic phase and one patient in accelerated phase. It was seen that RIZ1 methylation was increased significantly from early to advanced phase. The higher frequency of RIZ1 methylation was reported in haematologically resistant cases (42% vs 2%) and molecularly resistant cases (16.77% vs 1.92%) than the responders. The higher frequency of RIZ1 methylation was found in CML patients who were treated with interferon initially followed by imatinib treatment. Also RIZ1 hypermethylation was associated with faster disease progression p<0.003 than the non methylated cases. No correlation was found between RIZ1 gene methylation with age, thrombocytopenia, types of bcr/abl transcripts of CML patients. Conclusion: We conclude that epigenetic silencing of RIZ1 gene is associated with CML progression and imatinib resistance. Early detection of RIZ1 methylation could be a predictive marker for imatinib resistance and disease progression in CML.

A deletion polymorphism in the RIZ gene is associated with increased progression of imatinib treated chronic myeloid leukemia patients

Abstract RIZ1 encodes a retinoblastoma (Rb)-interacting zinc finger protein, is commonly lost or expressed at reduced levels in cancer cells. The RIZ1 gene locus commonly undergoes LOH in many cancers. Here, we analyzed Proline insertion–deletion polymorphism at amino acid position 704 in the RIZ1 gene and its association with CML. The RIZ1 pro-704 LOH genotypes were determined by AS-PCR in 100 CML patients among which 50 were in CP-CML, 25 in AP-CML, and 25 in BC-CML. Pro704 ins/del polymorphism (LOH) was detected in 27% CML patients. Proline ins–ins homozygosity, del–del homozygosity and ins–del heterozygosity was detected in 9%, 18%, and 73% CML patients compared with 3%, 3%, and 94% in healthy controls, respectively (p < .0003). A four-fold increased risk was found to be associated del-del genotype. We found a statistically significant association between RIZ1 LOH and stage (p > .01) and hematological resistance (p > .001). However, there were no correlations found with other clinical parameters like age, gender, thrombocytopia, type of BCR–ABL, and molecular response. Our findings suggest that proline 704 del–del homozygosity phenotype can play an important role in progression of CML.

Inactivation of P16 (INK4a) Gene by Promoter Hypermethylation is Associated with Disease Progression in Chronic Myelogenous Leukaemia

Background: Chronic Myelogenous Leukemia (CML) has a typical progressive course with transition from a chronic phase to a terminal blast crisis phase. The mechanisms that lead to disease progression remain to be elucidated. Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators in haematological malignancies. Therefore, aim of our study was to examine whether the methylation status of P16 (INK4a) gene is a useful biomarker in the development and progression of CML.Material and Methods: The methylation status of p16INK4A gene was evaluated by Methylation Specific Polymerase Chain Reaction (MSP) in 200 CML patients among which, 81 were in CP-CML, 54 in AP-CML and 65 in BC-CML.Results: The p16INK4A gene was hypermethylated in 84 of 200 (42%) of CML patients (P<0.0001). Among the three stages p16 (INK4A) promoter gene was methylated in 26% (CP-CML), 43% (AP-CML and 68% (BCCML) patient (P<0.0001). Methylation was more frequent in blastic and accelerated phase patients than in chronic phase. A significant correlation was found between p16INK4A methylation and loss of Imatinib response. Similarly higher frequency of p16INK4A methylation was reported in CML patients with haematological (P<0.02) and molecular resistances (P<0.04). Significantly higher (p<0.0001) frequency of p16INK4A promoter methylation was reported in patients with thrombocytopenia. However no correlation was found between p16INK4a hypermethylation and other clinic-pathological parameters like age, gender, BCR-ABL transcripts etc.Conclusion: Our results suggest that p16INK4a is a primary target for inactivation by promoter methylation in the disease progression of CML patients and that its detection is useful in the follow up of patients with a high risk of developing CML and resistance to Imatinib therapy.