Mutations in subunit interface and B-cell epitopes improve antileukemic activities of Escherichia coli asparaginase-II: evaluation of immunogenicity in mice.

L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared with WT-EcA. Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagine and also down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.

Prospective identification of potential factors influencing stem cell mobilization and the necessity for plerixafor use in newly diagnosed multiple myeloma patients undergoing autologous stem cell transplantation.

INTRODUCTION: To study the efficacy and safety of single large volume leukapheresis by using generic G-CSF or G-CSF plus Plerixafor in achieving adequate stem cell yield and various factors influencing thereof in newly diagnosed multiple myeloma patients undergoing autologous stem cell transplant . METHOD: This prospective study was undertaken among 55 newly diagnosed multiple myeloma patients undergoing autologous stem cell transplant and aged between 18 and 75 years. Mobilization and harvesting of stem cells were performed by using GCSF or GCSF plus Plerixafor and large volume leukapheresis, respectively. A stem cell yield of ≥2×106kg-1 and the number of apheresis procedures were primary efficacy endpoints, while the ideal stem cells yield >5×106kg-1, the engraftment day and D100 response/graft sustainability were secondary endpoints. RESULT: The primary endpoint was achieved in all cases in both the groups by using a single LVL leukapheresis procedure. Fulfillment of all the secondary endpoints was satisfactory and comparable in both the groups. Age, pre-apheresis CD34+ count and number of interruptions during the LVL were significant factors influencing the stem cell yield (p<0.05). Adverse drug reactions during the apheresis and post-ASCT period were manageable. CONCLUSION: The LVL is safe and cost-effective in attaining a minimum of CD34+ cells in a single procedure with manageable adverse reactions. Judicious intervention during the procedure may be helpful in ensuring the adequate yield.

Extraovarian primary peritoneal papillary serous carcinoma.

OBJECTIVE: To identify an unusual histologic entity, extraovarian primary peritoneal carcinoma (EOPPC) along with a review of the recent literature. METHODS: A thorough clinical examination along with detail laboratory parameters was studied in a 56-year-old female who presented with ascites and an elevated serum CA-125. Multiple microscopic sections were studied from the surgical specimen received comprising of total hysterectomy, bilateral salpingo-oophorectomy and omentectomy. RESULTS: A diagnosis of EOPPC was made after a thorough study. CONCLUSION: A correct diagnosis and timely management of this unusual histologic entity can result in long-term disease-free survival of the patient.

Beneficial Effect of Low Fixed Dose of Hydroxyurea in Vaso-occlusive Crisis and Transfusion Requirements in Adult HbSS Patients: A Prospective Study in a Tertiary Care Center.

Significant reduction in morbidity and mortality have been documented in patients with sickle cell disease (HbSS) by most of the studies using hydroxyurea at a dose of 25-35 mg/kg/day or maximum tolerated dose. But toxicities, need for frequent monitoring, compliance and cost are important hurdles particularly in Indian set up. We undertook this study to find out the efficacy, safety compliance rate of low fixed dose of hydroxyurea (10 mg/kg/day) in patients presenting to our hospital and its impact on clinical profile and laboratory parameters. A cohort of 128 (82 males, 46 females) confirmed HbSS cases (each >18 years age, vaso-occlusive crisis >2/years and/ or rate of transfusion 1-2 units/month) with no disease related end organ damage were assessed prospectively between 2013 and 2016. They were started on 10 mg/kg/day hydroxyurea along with other supportive care and followed up monthly for 1 year. Clinical and laboratory parameters before and after therapy were reviewed and compared. In 92% of cases presenting with repeated vaso-occlusive crisis, VOC disappeared completely during follow up and in 8% we found significant reduction in severity as well as frequency of attacks (p < 0.01). Again in 87%, no further transfusion was required during follow up and in 13%, it further reduced the rate of transfusion (p < 0.01). The median time of response for VOC was 3 months and in transfusion requirement was 5 months. There was also significant reduction in S.Billirubin, S.LDH, disease related complications and rate of hospitalisation with significant improvement in Hb, MCV, and MCH. There is insignificant increase in HbF with median (1.5-2.4)% and in 5 cases >5%. We did not find any remarkable adverse effect of the drug during the study period. Low fixed dose hydroxyurea (10 mg/kg/day) is beneficial in reducing the vaso-occlusive crisis and transfusion requirement in adult HbSS Patients (Arab-Indian Haplotype). It is safe, suitable and is a effective mode of treatment in resource poor setting like India.

Variability of Iron Load in Patients of Sickle Cell Anaemia (HbSS): A study from Eastern India.

INTRODUCTION: Sickle Cell Anaemia (SCA) is one of the commonest haemoglobinopathies due to a point mutation (A→T) of the ß-globin gene. Out of five haplotypes, the Arab-Indian haplotype present in India is one of the least severe phenotype and least studied also. It is characterized by lifelong haemolytic anaemia requiring red cell transfusion leading to iron overload. In contrast, there is very high incidence of deficiency of iron, folic acid and vitamin B12. AIM: Our objective was to access the Iron status of SCA patients and to find its correlation with various parameters like red cell transfusion, haemolysis and serum hepcidin. MATERIALS AND METHODS: This was a cross-sectional study conducted on 208 patients for a period of five years. Complete Blood Count (CBC), iron profile, haemolytic parameters and transfusion requirement were studied and data compared with 52 healthy controls. RESULTS: Few patients (9.6%) revealed significant iron overload (Serum ferritin > 1000 ng/ml). In majority (80.8%) it was either normal or border line raised (300 to 1000 ng/ml) or iron deficiency was noted in a small fraction (9.6%). Frequency of transfusion is the principal factor which positively correlated with level of iron load (p<0.001) while parameters of haemolysis and serum hepcidin level play an insignificant role in this context (p= 0.0634). CONCLUSION: This study supports the notion that the presentation of SCA patients in India is of "Viscosity - Vaso-Occlusive Crisis (VOC) phenotype" with high incidence of VOC, low haemolytic rate and transfusion requirement. Iron deficiency may be present in SCA patients requiring Iron supplementation. We suggest further studies to establish the role of hepcidin, ferroportin and other factors that control iron absorption in these patients.

Prospective identification of potential factors influencing stem cell mobilization and the necessity for plerixafor use in newly diagnosed multiple myeloma patients undergoing autologous stem cell transplantation

ABSTRACT Introduction: To study the efficacy and safety of single large volume leukapheresis by using generic G-CSF or G-CSF plus Plerixafor in achieving adequate stem cell yield and various factors influencing thereof in newly diagnosed multiple myeloma patients undergoing autologous stem cell transplant . Method: This prospective study was undertaken among 55 newly diagnosed multiple myeloma patients undergoing autologous stem cell transplant and aged between 18 and 75 years. Mobilization and harvesting of stem cells were performed by using GCSF or GCSF plus Plerixafor and large volume leukapheresis, respectively. A stem cell yield of ≥2 × 106 kg-1 and the number of apheresis procedures were primary efficacy endpoints, while the ideal stem cells yield >5 × 106 kg-1, the engraftment day and D100 response/graft sustainability were secondary endpoints. Result: The primary endpoint was achieved in all cases in both the groups by using a single LVL leukapheresis procedure. Fulfillment of all the secondary endpoints was satisfactory and comparable in both the groups. Age, pre-apheresis CD34+ count and number of interruptions during the LVL were significant factors influencing the stem cell yield (p < 0.05). Adverse drug reactions during the apheresis and post-ASCT period were manageable. Conclusion: The LVL is safe and cost-effective in attaining a minimum of CD34+ cells in a single procedure with manageable adverse reactions. Judicious intervention during the procedure may be helpful in ensuring the adequate yield.

Idiopathic Atypical Hemolytic Uremic Syndrome (aHUS) with Trilineage Myelodysplasia.

Atypical hemolytic uremic syndrome (HUS) is a heterogeneous group of disorders, with an unexplained pathogenesis. We report here with an interesting case of a 6 years old male child presenting with atypical feature of HUS and bone marrow trilineage myelodysplasia.