Molecular analysis in Burkitt's lymphoma

Background: The t(8;14) translocation in Burkitt’s lymphoma (BL) was the first non-random cytogenetic lesion to be described in lymphoproliferative disorders. This lesion occurs in 75-85% of all BL cases. However, the breakpoints in this cytogenetic lesion are very variable and far apart such that the t(8;14) translocation is not always amenable to standard polymerase chain reaction analysis. This is mainly due to the inability of the Thermus aquaticus (Taq) polymerase enzyme to synthesize long DNA products. Long range polymerase chain reaction (LD-PCR) with a high fidelity polymerase enzyme mix capable of longer PCR product synthesis has recently become available. In early studies, LD-PCR appeared to be capable of amplifying the t(8;14) translocation in the majority of published sporadic Burkitt’s lymphoma analyses. The utility of t(8;14) translocation LD-PCR for routine use in the diagnosis of BL in our setting has not yet been studied. The aim of this study was to establish and optimize the t(8;14) LD-PCR technique and to apply it in the retrospective analysis of all BL diagnosed in the University of the Witwatersrand teaching hospitals in a ten year period from January 1994 to December 2003. Materials and methods: High molecular weight non-degraded DNA was extracted from control cell lines as well as stored, unstained bone marrow slides remaining after routine diagnostic workup of previously identified Burkitt’s lymphoma patients. Three hundred nanograms of patient and control DNA were amplified with the LD-PCR high fidelity polymerase enzyme mix under reaction conditions which were optimized using the tissue plasminogen activator (tPA) gene as well as known Burkitt’s lymphoma cell lines as controls. Each control and patient DNA sample was amplified with tPA primers as well as four pairs of MYC/IgH primer sets. The resulting amplicons were size fractionated on an agarose gel and visualized with ethidium bromide under ultraviolet (UV) light. The fractionated DNA fragment sizes were compared to those of the t(8;14) translocation positive controls, tPA controls and known DNA molecular weight markers. Results: One hundred and ten Burkitt’s lymphoma diagnoses were made in the three teaching hospitals of the University of the Witwatersrand from January 1994 to December 2003. Bone marrow involvement by BL was present in 84 of these cases. Archival bone marrow slides were available in 74 of the 84 BL patients. Intact high molecular DNA on which the t(8;14) LD-PCR analysis could be performed was present in 41 of the 74 BL patients. In the presence of appropriate controls, an t(8;14) translocation specific product was demonstrable by t(8;14) LD-PCR analysis in only 6 of 41 BL patients. Conclusion: In this t(8;14) LD-PCR retrospective analysis of a large number known Burkitt’s lymphomas, the diagnostic yield in carefully selected patients was extremely poor. With five primer pairs required per BL sample analysis, this technique was found too labour intensive and costly in our hands making it unsuitable for routine diagnostic use. The reasons for the poor diagnostic yield remains unclear and may need to be explored in future studies. Emerging alternative techniques for the diagnosis of BL such as fluorescence in situ hybridization and microarray gene expression analyses may prove to be better diagnostic tools than LD-PCR in its current form

A cohort study of the relationship between anaemia, mean corpuscular volume and mortality among a CKD population in South Africa

Background: The burden of chronic kidney disease is increasing globally and prompt identification, coupled with improved management of CKD patients have increased the population of pre-dialysis patients. We, therefore, aimed to evaluate the predictors of survival among pre-dialysis CKD patients in South Africa. Methods: We conducted a cohort study of 256 consecutive consenting Black non-dialysis requiring CKD patients attending the renal outpatient clinic of a tertiary Hospital in South Africa from 1st June 2016 to 1st December 2016. Socio-demographic and clinical information of the participants were obtained. Descriptive statistics, Kaplan-Meier curves and Cox proportional hazard regression analyses were conducted to evaluate factors affecting the survival of the participants. Results: The mean age of the participants was 52.8±14.3 years and 48.0% were females, 52% were males. The death rate increased with worsening haemoglobin level from 0.96 among patients with mild anaemia to 4.29 per 100-person years among patients with severe anaemia. Anaemic patients with GFR < 30mls/min had significantly increased risk of death (HR 11.51, 95% CI 1.62–78.32, P < 0.001). Conclusion: Mortality in pre-dialysis CKD patients was associated with anaemia and hyperphosphatemia. Clinical interventions targeted at preventing these conditions may improve outcomes among this group of CKD patients. Keywords: Chronic kidney disease; mortality anaemia; outcomes, survival

Once-weekly prophylaxis with 40 IU/kg nonacog beta pegol (N9-GP) achieves trough levels of >15% in patients with haemophilia B: Pooled data from the paradigm™ trials.

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Core data set on safety, efficacy, and durability of hemophilia gene therapy for a global registry: Communication from the SSC of the ISTH

BackgroundGene therapy for people with hemophilia (PWH) will soon become available outside current clinical trials. The World Federation of Hemophilia (WFH), in collaboration with International Society of Thrombosis and Hemostasis Scientific and Standardization Committee (ISTH SSC), the European Haemophilia Consortium (EHC), the US National Hemophilia Foundation (NHF), the American Thrombosis and Hemostasis Network (ATHN), industry gene therapy development partners and Regulatory liaisons have developed the Gene Therapy Registry (GTR), designed to collect long- term data on all PWH who receive hemophilia gene therapy.ObjectiveThe objectives of the GTR are to record the long- term safety and efficacy data post gene therapy infusion and to assess the changes in quality of life and burden of disease post- gene- therapy infusion.MethodsThe GTR is a prospective, observational, and longitudinal registry developed under the guidance of a multi- stakeholder GTR Steering Committee (GTR SC), composed of health care professionals, patient advocates, industry representatives, and regulatory agency liaisons. All PWH who receive gene therapy by clinical trial or commercial product will be invited to enrol in the registry through their hemophilia treatment centers (HTCs). The registry aims to recruit 100% of eligible post gene therapy PWH globally. Through an iterative process, and following the guidance of the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA), the GTR SC has developed a core set of data to be collected on all patients post gene therapy.ResultsThe core data set includes demographic information, vector infusion details, safety, efficacy, quality of life and burden of disease.ConclusionsThe GTR is a global effort to ensure that long term safety and efficacy outcomes are recorded and analysed and rare adverse events, in a small patient population, are identified. Many unknowns on the long- term safety and efficacy of gene therapy for hemophilia may also be addressed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163440/2/jth15023.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163440/1/jth15023_am.pd

Efficacy and safety of long-acting recombinant fusion protein linking factor IX with albumin in haemophilia B patients undergoing surgery.

IntroductionRecombinant factor IX fusion protein (rIX‐FP) has been developed to improve the pharmacokinetic (PK) profile of factor IX (FIX), allowing maintenance of desired FIX activity between injections at extended intervals, ultimately optimizing haemophilia B treatment.AimTo determine the efficacy and safety of rIX‐FP in the perioperative setting.MethodsSubjects were adult and paediatric patients with severe to moderately severe haemophilia B (FIX ≤ 2%) participating in three Phase III clinical trials and undergoing a surgical procedure. PK profiles were established prior to surgery for each patient. Haemostatic efficacy was assessed by the investigator for up to 72 h after surgery. Safety measurements during the study included adverse events and inhibitors to FIX. FIX activity was monitored during and after surgery to determine if repeat dosing was required.ResultsTwenty‐one, both major and minor, surgeries were performed in 19 patients. Haemostatic efficacy was rated as excellent (n = 17) or good (n = 4) in all surgeries. A single preoperative dose maintained intraoperative haemostasis in 20 of 21 surgeries. Nine major orthopaedic surgeries were conducted in eight patients with a mean of 7 (range: 6–12) rIX‐FP injections during surgery and the 14‐day postoperative period. Median rIX‐FP consumption for orthopaedic surgeries was 87 IU kg−1 preoperatively and 375 IU kg−1 overall. No subject developed inhibitors to FIX or antibodies to rIX‐FP.ConclusionRecombinant factor IX fusion protein was well tolerated and effectively maintained haemostasis during and after surgery. Stable FIX activity was achieved with a prolonged dosing interval and reduced consumption compared to conventional or currently available long‐acting recombinant FIX

Underdiagnosis of iron deficiency anaemia in HIV-infected individuals : a pilot study using soluble transferrin receptors and intensive bone marrow iron stores to improve the diagnosis

DATA AVAILABILITY STATEMENT : Data are available upon reasonable request.AIM : We compared soluble transferrin receptors (sTfR), serum ferritin, mean cell volume (MCV) of red cells and the sTfR-ferritin index with the intensive method bone marrow trephine (BMT) iron stores in the diagnosis of iron deficiency anaemia (IDA) in Human Immunodeficiency Virus (HIV)-positive hospitalised participants. METHODS : In this cross-sectional study, we recruited hospitalised HIV-positive and coronavirus of 2019 (COVID-19)-negative adults with anaemia who required a bone marrow examination as part of their diagnostic workup. We measured the full blood count, ferritin, sTfR and assessed iron using the intensive method in Haemotoxylin and Eosin (H&E)-stained BMT core biopsies of consenting participants. RESULTS : Of the 60 enrolled participants, 57 were evaluable. Thirteen (22.80%) had IDA on H&E BMT iron stores assessment, and 44 (77.19%) had anaemia of chronic diseases (ACD). The sTfR and the sTfR-ferritin index had sensitivities of 61.54% and 53.85%, respectively, for IDA diagnosis. The sensitivity and specificity of ferritin was 7.69% and 92.31%, respectively. The sTfR and sTfR-ferritin index’s diagnostic specificity was relatively low at 46.15% and 38.46%, respectively. CONCLUSION : In this pilot study in HIV-positive participants, the prevalence of iron deficiency using the BMT assessment was low. Both the sTfR and the sTfR-ferritin index had a better quantitative correlation to bone marrow iron stores when compared with the MCV and ferritin and, may be more accurate surrogate markers of IDA.https://jcp.bmj.comhj2023Anatomical Patholog

Outcomes in children with hemophilia A with inhibitors: Results from a noninterventional study

Background: Data regarding management of pediatric persons with hemophilia A (PwHA) with factor VIII (FVIII) inhibitors are limited. This prospective noninterventional study (NCT02476942) evaluated annualized bleeding rates (ABRs), safety, and health-related quality of life (HRQoL) in pediatric PwHA with FVIII inhibitors. Procedure: PwHA aged <12 years with current FVIII inhibitors and high-titer inhibitor history were enrolled. Participants remained on usual treatment; no interventions were applied. Outcomes included ABR, safety, and HRQoL. Results: Twenty-four PwHA aged 2-11 years (median 7.5) were enrolled and monitored for 8.7-44.1 weeks (median 23.4). In the episodic (n = 10) and prophylactic (n = 14) groups, respectively, 121 of 185 (65.4%) and 101 of 186 (54.3%) bleeds were treated using activated prothrombin complex concentrate (aPCC) and/or recombinant activated FVII (rFVIIa). ABRs (95% confidence interval) were 19.4 (13.2-28.4) and 18.5 (14.2-24.0) for treated bleeds, and 32.7 (20.5-52.2) and 33.1 (22.4-48.9) for all bleeds, respectively. Most prophylactic group participants (92.9%) were prescribed aPCC; 50% adhered to their prescribed treatment regimen. Adherence to prophylactic rFVIIa was not assessed. Serious adverse events included hemarthrosis (12.5%) and mouth hemorrhage (12.5%); the most common nonserious adverse event was viral upper respiratory tract infection (12.5%). HRQoL showed functional impairment at baseline; scores remained stable throughout, with little intergroup variation. Conclusions: ABRs remained high in pediatric PwHA with inhibitors receiving standard treatment. This study demonstrates the need for more effective treatments, with reduced treatment burden, to prevent bleeds, increase prophylaxis adherence, and improve patient outcomes.Was funded by F. Hoffmann-La Roche Ltd

World Federation of Hemophilia Gene Therapy Registry

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156126/2/hae14015_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156126/1/hae14015.pd