Factor VIII and Factor IX
30 June and 1 July 2006
Oslo Kongressenter , Norway
Chair: K. Mertens, The Netherlands
Co-chairs: J.C. Gill, USA; C. Lee, UK; J. Oldenburg, Germany; F. Peyvandi, Italy; J.M. Saint-Remy, Belgium;
A. Srivastava, India; H.M. van den Berg, The Netherlands
The Chairman opened the Subcommittee meeting at 14.15 for an audience of approximately 170 attendants. He announced a few amendments to the final program and provided the timing details of the various sections in the agenda.
S ection 1: FACTOR VIII CLINICAL ISSUES
Co-chairs: J.M. Saint-Remy and H.M. van den Berg
Phenotypic heterogeneity in severe haemophilia A and B: A. Srivastava
Though it has been recognized that 10-15% of patients with severe haemophilia have clinically mild disease in terms of frequency of bleeds and extent of arthropathy, the basis for this phenomenon has not been fully understood. We hypothesized that the levels of various pro- and anti-coagulant factors and functional polymorphisms in the coagulation protein genes as well as genes of cytokines involved in the inflammatory response may modulate the development of hemophilic arthropathy. A total of 114 minimally treated patients (50-150 IU/kg/year) with hemophilia A (n=94) and hemophilia B (n=19) diagnosed were evaluated as outlined above. They were categorized as ‘mild’ ( <1 affected joint and < 5 bleeds in the preceding year, n=15) or ‘severe’ (>1 affected joint and >5bleeds, n=99). Early data suggests that polymorphisms in TNFα-308 AA/AG (pro-inflammatory) (RR-3.4, p=0.037, 95% CI, 1.07-10.7), TGFβ Codon 10 CC/CT (pro-inflammatory) (RR-2.8, p=0.07, 95% CI, 0.91-8.3) are associated with clinically severe disease while MDM2 GG (RR-0.3, p=0.038, 95% CI, 0.1-0.93) tended to ameliorate severity. These data suggest that the clinical phenotype of severe haemophilia could be influenced by interactions between a variety of hemostatic factors as well as some inflammatory response proteins.
Factor VIII inhibitors and continuous infusion: I. Scharrer
Dr. Scharrer presented data also on behalf of Drs. v. Auer and Oldenburg and the German Hemophilia Society University Hospitals Mainz and Bonn . They have conducted a retrospective study to investigate the development of inhibitors after continuous infusion of F VIII in Germany . 42 hemophilia centers were questioned. 19 of these conducted 200 continuous infusions in 128 patients. 14 patients developed inhibitors (10 HR, 4 LR). 5 of these patients were suffering from severe, 1 from moderate and 8 from mild hemophilia (age between 7 months and 73 years, 11 PTT’s, 3 PUP’s). The infused amount ranged between 4300 and > 100 000 U before inhibitor development. Exposure days ranged from 4 to > 100. Regarding the genotype only 2 intron -22- inversions have been found. In Dr. Scharrer’s center no inhibitors had been found in 81 patients with major orthopedic surgery and bolus infusions compared to 2 inhibitors in 8 patients with similar surgery. In conclusion the inhibitors developed very often in mild hemophiliacs without the typical gene mutations for inhibitor development.
Feedback from EMEA Workshop on Inhibitors: R. Seitz
Prof. Seitz reported that in a CHMP class review of recombinant FVIII products and inhibitor development the data received were heterogeneous and not comparable between products. An expert meeting was convened at the EMEA in London on 28.02. – 02.03.2006. He presented a brief feedback on this workshop; a detailed report is currently prepared. A letter has been sent to the Chair of the FVIII&IX Subcommittee, Prof. Mertens. CHMP notes the ISTH SSC sub-committee on FVIII and FIX initiatives regarding the improvement of the FVIII inhibitor assay and the establishment of the global PTP inhibitor surveillance. These will be complementary to CHMP’s ongoing work in revising guidelines on clinical investigation of plasma-derived and recombinant FVIII products. CHMP would appreciate to be informed about the outcome of the ISTH SSC sub-committee on FVIII and FIX discussions on the above topics. In the discussion dr. Mertens indicated that several of the issues are already ongoing actions of the Subcommittee. He proposed that Working Parties could be initiated to address these issues whenever appropriate.
Global PTP inhibitor surveillance study: update : D.M. DiMichele
The impetus for this project was derived from regulatory issues identified by the FDA and EMEA associated with potential immunogenicity of new FVIII products studied in pre-licensure PTP clinical trials. The need for harmonized international data collection on the natural history of inhibitors in PTP populations was reiterated at both the FDA (11/03) and EMEA (2/06) workshops, although no consensus definition of the PTP yet exists. International guidelines for PMS studies were reviewed as were the pros and cons of conducting this project through the ISTH FVIII/IX subcommittee. The history of this project in this committee was reviewed. Within this context, this report focused on the feasibility of national PTP database harmonization with respect to a data collection tool. A preliminary effort to do this by two established databases in the US and UK was reported on behalf of its contributors (CRM Hay, chair of the UKHCDO in the UK and Dr. Mike Soucie and the CDC in the US . Dr. DiMichele concluded that based on this effort, such harmonized data collection was feasible. This harmonization project between the US and UK will be further pursued. Furthermore, the proposed schema for collaborative data collection will be shared with other countries planning or engaged in such prospective data collection. Finally, as per the formal request to this subcommittee by the EMEA, the Chair will request approval for this project to proceed a sanctioned FVIII/ IX Subcommittee activity and a formal Working Party will be established to continue this work on an international scale.
Treatment-related inhibitors in PUPs with severe hemophilia: Rodin study : H.M. van den Berg
The Rodin study has been developed to determine treatment related risk factors in Pups with severe hemophilia A. The Rodin study will be performed as a sattelite study of the Pednet registry and it has been extended to the Rodin study group which consist of other major hemophilia centres from Europe, Israel and Canada which do not participate in the PedNet/ Rodin study. The study started to include data of full cohorts of patients with severe hemophilia A born from 1-1-2000. The aim is to collect upto 1-1- 2008 a total of 400 patients in the Pednet/Rodin study and another 400 patients through the Rodin study group. Data will be collected on all first 75 exposure days, which will include every reason for treatment, the dosage, peak treatment, surgery. Besides extensive data on patients characteristics. Further information can be obtained from the websites; www.pednet.nl and www.rodinstudy.nl.
Low-titre inhibitors in Canal study and Recombinant studies : E. Santagostino
Dr. Santagostino indicated that our knowledge on the natural history of low-titer inhibitors is mainly based on the data provided by the studies carried out in PUPs with hemophilia A treated with recombinant FVIII. Overall, almost half of the inhibitors detected during these studies showed titers of 5BU/mL or less and remained below this level although FVIII exposure was repeated. More than two third of these low-titer inhibitors were transient and disappeared spontaneously continuing FVIII treatment, while immunetolerance induction was started in the remaining cases. Transient inhibitors have also been reported in hemophiliacs with titers always maintained between 5 and 10 BU/mL. Transient inhibitors may represent a model for understanding the mechanisms of immunetolerance so that immunological studies in this specific setting should be implemented. Many aspects concerning the clinical management of hemophiliacs with low-titer inhibitors are still empirical because few data are available on the behaviour of inhibitor titers, FVIII recoveries and half-lives over time. In particular, the need for immune tolerance induction treatment remains controversial and should be better defined.
New methods for the assessment of FVIII inhibitors : J.M. Saint-Remy
Factor VIII immunogenicity is not limited to the detection of inhibitors but its evaluation should take into account all antibodies formed towards FVIII. Current ELISAs can easily detect FVIII antibodies but the use of such assays is far too limited. Identifying the precise epitopes recognized by anti-FVIII antibodies is important to understand the reasons/mechanisms by which such antibodies are formed. It could in addition provide some insight into possible new therapies for inhibitors. The combined transcription-translation method using a library of FVIII fragments is well suited for this purpose. The immune memory compartment of the anti-FVIII immune response can also be evaluated at both B and T cell levels. Methods such as the ELISPOT can be used on a routine basis to count the number of memory B and T cells and thereby evaluate the risk of producing an inhibitor. Such methods are also appropriate to compare the efficacy of therapies aiming at the eradication of inhibitor antibodies.
Section 2: FACTOR VIII ASSAYS AND STANDARDISATION ISSUES
Co-chairs: A. Srivastava, K. Mertens
Factor VIII inhibitor assays: technical issues: B. Verbruggen
FVIII inhibitor tests still are assays that show a very high interlaboratory variation in international survey programs despite recent described modifications. There are a number of variables that influence test outcome: pH stability in incubation mixtures, type of control sample, type of used deficient plasma, liquid handling, incubation time, FVIII content of normal pool plasma, type of Factor VIII assay / type of reagent. It is advised to use a FVIII deficient plasma that contains von Willebrand factor as substrate plasma in the assay of residual factor VIII assay in the test and control mixture. With respect to the assay of type II inhibitors the following items are important:
To increase sensitivity of inhibitor detection we have developed a FVIII inhibitor test that is ca. 20 times more sensitive than the Nijmegen-Bethesda assay. The test is independent of residual FVIII in the test sample and has a cut-off value of at least 0,04 NBu/ml, but probably better. There is a strong correlation between a decreased half life of infused FVIII and a low inhibitor titre and low titre inhibitors may therefore be of clinical importance. The putative clinical significance of low-titre inhibitors has to be validated in a bigger cohort of patients.
Collaborative Study on 1 st International Standard for FVIII inhibitors : S. Raut
The international need for a reference FVIII inhibitor standard was established at the FVIII/IX Subcommittee in 2001, and the project ratified by the Subcommittee in 2003. Sanj Raut presented a progress report on this ongoing project. The aim of this study was to develop a definitive WHO International Reference Preparation for measurement of FVIII Inhibitors in plasma. 5 candidate preparations (rabbit FVIII PAb, 2 humanised MAbs I & II, human low (X) and high (Y) titre inhibitor plasmas) were assessed against 2 human inhibitor patient plasmas. Inhibitor methods and the FVIII assays were also assessed. Samples were distributed to 25 expert laboratories. Data were received from 21 labs. For all samples, results without a reference standard gave CVs between 17-34%. For the overall mean inhibitor titres, comparisons of the Nijmegen vs Bethesda assays, and comparisons of one-stage vs chromogenic methods, the candidate with lowest CVs was preparation Y. On assessing the patients’ inhibitor plasma relative to the 5 candidate preparation, once again preparation Y gave the lowest CVs of 17 & 18%. It was interesting that no marked improving in CVs were observed for the Nijmegen method compared to the Bethesda method, but a marked improvement in the CVs was observed for the chromogenic assay compared to the one-stage assay. Overall preparation Y (05/206) was the most suitable candidate material for the proposed 1st IS FVIII Inhibitor Plasma standard with an overall Bethesda Titre of 8.2 BU/vial and an overall GCV of 17.5%. Preliminary stability studies show a predicted loss of inhibitor titre potency of < 0.01% per year at
Collaborative Study SSC 8: evaluation of result: M. Lee
This presentation was intended to provide a basic statistical analysis of the results obtained from the most recent worldwide collaborative study of Factor VIII assays sponsored by the ISTH Factor VIII/IX subcommittee. In this study, four different Factor VIII preparations (three recombinant and one plasma-derived) were evaluated by 31 laboratories using their local assay (primarily chromogenic or one-stage) and pre-diluent (deficient plasma or buffer). Using analysis of variance techniques, buffer pre-diluent typically provides lower potencies for recombinant preparations, but the reverse is true for the plasma-derived material. The chromogenic assay gives a significantly higher result for full-chain recombinant Factor VIII products, but not for the B-domain deleted preparation. The combination of pre-diluent and assay is an important source of variation, particularly for B-domain deleted Factor VIII. For the plasma-derived product only the assay method really matters. This information has been used to help design the next collaborative study, SSC9, where the components of variation for the Factor VIII assay will be examined in greater detail.
Collaborative Study SSC 9: preliminary report: S. Raut
Dr. Raut reported on the Phase II SSC9 “Controlled” study following on from the Phase I (SSC8) “Field” study carried and reported last year. The aim of this study was to investigate the differences and variability observed in the Phase I study and further identify any potential sources of variability, by following strict protocol instructions. Furthermore, it tested the ISTH/SSC recommendation by studying the chromogenic method and pre-diluting in FVIII deficient plasma (containing normal levels of VWF) provided. The effect of different operators and different day set-ups were also assessed. The same 4 samples used in the SSC8 study (3 recombinants and 1 plasma-derived) were distributed to 25 participants. Data were received from 21 labs. The results obtained gave a marked improvement in inter-lab variability with GCVs between 4-11% (compared to 14-19% in the SSC8 study). For the 3 recombinant concentrates both the chromogenic method and predilution in FVIII deficient plasma containing normal levels of VWF were particularly important. For the plasma concentrate, predilution in FVIII deficient plasma was not as important. Variability appeared not to be influenced by different operators, assays on different days or by local standards. This unique data set will be further analysed in more detail (components of variation analysis) by Martin Lee and will be reported in 2007. In the discussion Dr. Mertens noted that the results were much better than in previous studies, where other methods than the SSC recommended method were also included.
Genetic reference materials for Haemophilia A: A. Hubbard
On behalf of Dr. E. Gray, Dr. Hubbard announced a study that will involve reference materials for the detection of the intron 22 inversion. DNA samples are being prepared, and include an affected male, a carrier female, and the appropriate normal controls. The study will start in November 2006. Those who are interested should contact Dr. Gray (egray@nibsc.ac.uk).
Calibrated measurement of thrombin generation in hemophilia: P. Giesen
Dr. Giesen (Thrombinoscope,The Netherlands) reported that with the calibrated automated thrombogram™ (CAT) plasma is divided in two samples, in one sample thrombin generation is triggered and to the other sample a calibrated amount of thrombin calibrator is added. Both samples receive fluorogenic substrate and the signal is followed in a 96-well plate fluorometer. This allows proper calculation of thrombin in time such that the measured signal is corrected for donor-to-donor differences in plasma color as well as for non-linearities of the signal. It is shown that the same amount of factor VIII in different donors gives substantial different thrombin generation. At the same time a perfect relationship exists between factor VIII concentration and thrombin generation measured in one plasma. Therefore individual calibration in combination with individual measurement of factor VIII are the basis for efficient monitoring of factor VIII treatment.
Thrombin Generation Assay: Application for measuring the haemostatic efficacy of FVIII: K. Varadi
Dr. Varadi presented assay principles and technical details of a thrombin generation assay (TGA) have been presented. Thrombin generation is triggered by tissue factor-phospholipid complex (TF-PL) and CaCl 2 in the presence of a fluorogenic thrombin substrate. The changes in fluorescence signal are converted to thrombin concentrations using a reference curve, where thrombin is added instead of plasma. The TGA is sensitive to factor VIII levels even below the detection limit of factor activity assays, demonstrated by in vitro spiking experiments and in ex vivo samples obtained after FVIII administration. The ability to measure the haemostatic effect of factor VIII down to 0.002 U/ml may help to predict the possible risk of spontaneous bleeding episodes and to tailor the prophylactic therapy. Individual plasma calibration could be used in the assay, however it is not essential, and therefore the TGA is easily applicable to any fluorescence reader having the appropriate excitation/emission filters for the AMC-fluorophore. The assay is commercially available from Technoclone ( Vienna , Austria ).
Thrombin generation in hemophilia: proposal for collaborative study: E. Gray
Dr. Gray presented a brief overview of the collaborative study on thrombin generation as performed in the Subcommittee on plasma coagulation inhibitors (see minutes of that Subcommittee for summary). The next stage could be to prepare a reference plasma in order to assist in reducing interlaboratory variation. In the next collaborative study Dr. Gray is willing to include plasma samples of thrombophilia and haemophilia, the latter in collaboration with the FVIII&IX subcommittee. In the discussion several attendees stressed the potential of thrombin generation for haemophilia. The view was expressed that substitution therapy might be more appropriately expressed in terms of thrombin generation than in factor VIII levels, and that the Subcommittee should focus on this point.
General discussion: future directions
In the general discussion Dr. Mertens raised the issue how to proceed from here. There was consensus within the audience that a Working Party should be established within the FVIII&IX Subcommittee to focus specifically on haemophilia and low factor levels. This group should work in close collaboration with Dr. Gray in order to prevent any overlap with activities ongoing elsewhere. Dr. Ingerslev suggested that it might be appropriate to also include methods based on thromboelastography in this Working Party.
Section 3: FACTOR IX
Co-chairs: J. Oldenburg, K. Mertens
Factor IX gene transfer: current status : A. Nathwani
Dr. Nathwani reported also on behalf of Drs. Tuddenham, Pasi, Kay, Nienhuis and Davidoff on new technology that may raise new hope for gene therapy of haemophilia A. They proposed a distinct approach to gene therapy of haemophilia B which addresses many of the limitations of previous gene therapy trials. Firstly vectors based on adeno-associated virus (AAV) serotype 8 will be used because of the lower prevalence of pre-existing humoral immunity to this virus in humans. Secondly the genome in our vector will have a self complementary ( scAAV2/8-LP1-hFIXco ) format to enable efficient therapeutic gene transfer with lower titres of vector. Finally, scAAV particles will be administered into a peripheral vein, which is safer and more convenient for patients with a bleeding diathesis. In nonhuman primates their approach has consistently resulted in mean stable plasma human FIX levels of 26% of normal without any toxicity even in animals with pre-existing immunity to AAV. The plan is to establish the safety and efficacy of peripheral vein administration of scAAV2/8-LP1-hFIXco in adults with severe HB. The protocol has received regulatory approval in the UK with plans to commence recruitment in 2007. Further details of this approach were presented and discussed.
Factor IX inhibitors: update J. Lusher
Dr. Jeanne Lusher gave a brief, near final report on Dr. Warrier's registry concerning FIX inhibitors accompanied by anaphylaxis (or severe allergic reactions) in persons with severe hemophilia B. Fifty-six subjects have been reported (29 from U.S. and 27 from other countries), and a few more were verbally reported to Dr. Warrier at the Vancouver meeting in May 2006, with the promise that registry forms would be completed. The median number of exposure days was 11; med. age at inhibitor detection 19.5 mos. Among those genotyped, null mutations appear to confer a significantly increased risk. Of those with inhibitors to FIX, roughly one-half also had one or more severe allergic reactions. Of those who were desensitized to FIX and then put on an ITI regimen, success was quite poor (12%), and 1/3 developed nephrotic syndrome within 8-9 months after ITI regimen was begun.
Factor IX standardisation; replacement of 3rd International Standard: E. Gray
Dr. Gray mentioned that the 3 rd international standard needs replacement. This will be done in parallel with the EP working standard replacement. Manufacturers are invited to submit materials for the standard, and participants who are willing to perform the calibration study should contact Dr. Gray (egray@nibsc.ac.uk).
Section 4: ISTH-SSC Working Party on RARE BLEEDING DISORDERS (RBDs)
Chair: F. Peyvandi
Co-chair: C. Lee
The next goal of this working group will be to prepare a specific data collection tool which could be used as a homogeneous questionnaire by a majority of the countries around the world. This will contain the most important clinical and therapeutic data useful to answer the missing information that remains unanswered in the field of RBDs. In addition, specific subcommittees will be organized with the aim to prepare evidence-based guidelines using data collected in either RBDD or other available National Registries.
RBDs in the USA ( US working group on RBDs): A. Shapiro:
The rare bleeding disorder group in the United States will be developing a resource center hosted on-line by the National Hemophilia Foundation that will contain information on many rare plasma protein deficiencies. Links to existing websites, registries, and organizations related to these disorders will be included. The material developed for this resource center will be submitted for publication. In addition, the development of a national database in the United States is presently underway and a uniform platform has been adopted. Development of a database for rare bleeding disorders will be included in this national database effort. The rare bleeding disorder group in the US wishes to collaborate on an international level to further both development of international data collection and research efforts, including expansion of treatment options.
Glanzmann thrombasthenia: World distribution, mutations and founder effects : U. Seligsohn
A preliminary survey was performed in order to estimate the minimal prevalence of the disease in different populations, the mutations so far detected and founder effects. Data were extracted from reports involving ³ 9 unrelated patients, national and reference center registries, a database of mutations (by Debra French and Alan Nurden) or personal communications. The minimal prevalence ranged from 1:80,000 in Jordanian, 1:143,000 in Israelis and Palestinians to 1:2,800,000 in Indians, with an average global prevalence of 1:1.1x10 6. As of May 2006, 159 mutations have been reported in peer-reviewed journals, 69 in b 3 and 90 in a IIb. 133 were identified in individual patients and 26 in 2 or more unrelated individuals. For 7 of the 26 mutations, a founder effect was discerned by haplotype analysis: 2 in Iraqi Jews, 1 in Palestinians, 1 in Jordanians, 2 in Indians and 1 in Manouce Gypsies in France . This preliminary survey is a rough estimate of the global problem related to this severe hemostatic abnormality, but is somewhat limited because of lack of data from China , Africa , Latin America , Eastern Europe and densely populated countries like Pakistan , Bangladesh , etc.
Menorrhagia in women affected by bleeding disorders- Proposal for an International study: F. Peyvandi
A significant number of women affected by blood coagulation diseases present with complications such as menorrhagia and post-partum hemorrhage. Previous studies on this issue are not very informative on this subject due to the heterogeneous group of patients enrolled and the different methodologies used to analyze the data. We therefore propose a multicentre prospective study which enroll a large number of female patients affected by vWD,rare bleeding disorders and carriers of hemophilia.Based on a specific questionnarie designed to collect clinical data in a homogeneous format.In future it could be available online (www.rbdd.org) for the participating centers.The results obtained will be examined by a scientific committee and then published on the space assigned to on-line studies within the URL: www.rbdd.org. The results of this project will provide information on the incidence of bleeding complications and to assess whether hormone therapy, anti-fibrinolytic and replacement therapy could have beneficial effects on bleeding symptoms and on quality of life of these patients.
Long term prophylaxis in afibrinogenemic patients: a rational based on results from single dose PK; T. Waegemans
FIBRINOGENE T-I (LFB) is a new concentrate of human plasma fibrinogen. It is derived from cryoprecipitate and its manufacturing process includes three major biological safety steps. A single dose PK study performed in 5 afibrinogenemic patients showed homogeneous results within a one-compartment pharmacokinetic model with IV infusion and first order elimination. This model was applied to simulate PK profiles at steady-state after repeated infusions. A selection of targeted parameters will allow investigators to determine individualized dosage regimens before initiating a long term prophylaxis in afibrinogenemic patients. A design of PK study will be presented, aiming at comparing the values predicted by the simulation with those observed during the prospective follow-up of patients.
Getting product to patients: Industry Options: J. Lloyd.
2 years ago, BPL identified market access, regulatory requirement and clinical trial design as constraints to manufacturing products for RBD. Over the past two years there have been some advances made. Registries and databases of rare bleeding disorders have made it easier to establish patient numbers and treatment regimes to support orphan drug designation. Further development and support of these registries is required to aid industry in deciding whether to develop a product. Orphan drug designation and the parallel scientific advice available from the FDA and EMEA have potentially reduced the need for multiple dossiers and trials. Clinical trial design and support remains an issue, which is overcome by protocol assistance from regulators. There are still some major hurdles for industry to overcome. Further ongoing cooperation between regulatory agencies is required. The maintenance of parallel scientific advice provided by the EMEA and FDA is vital
General discussion and concluding remarks
In the discussion Dr. Peyvandi raised the issue how to proceed with the Rare Bleeding Disorders now her outside funding for this project expires. The Chairman indicated that SSC activities in general would benefit from some level of funding by ISTH. This particularly includes Working Groups or Working Parties that have formally been endorsed by SSC. This issue was discussed in more detail in the audience. It was believed that ISTH, by virtue of its established authority, could drive related national or regional projects under the same umbrella. The general feeling was that ISTH could show leadership in providing funding for those SSC activities that are considered to have appropriate priority. The Chairman mentioned that this issue has been brought to the attention of the SSC Chairman. He concluded the meeting thanking all speakers for their presentations and the audience for attending and contributing to the discussions.