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2016 Annual Minutes Locked Topic 0 G. Young FVIII/FIX/Rare Bleeding Disorders SSC Minutes Chairman: Guy Young Co-Chairs: Manuel Carcao, Gili Kenet, Johnny N. Mahlangu, Michael Makris, Danijela Mikovic, Elena Santagostino  Top Abstract Vincent Muczynski Dr. Muczynski presented the top abstract from the SSC abstracts relevant to our program. He described the development of a FVIII nanobody molecule which incorporates a nanobody that has a prolonged binding interaction with VWF thus prolonging the half-life of FVIII by approximately 2-fold.   Project updates Inhibitor Reporting Standardization in Previously Treated Patients Alfonso Iorio Dr. Iorio reported on the progress of his working group which developed guidelines for how reports on new inhibitors in PTPs should be reported in the literature. The project is complete and the manuscript regarding the recommendations are now published in JTH. Bleeding Score in Hemophilia Maria Elisa Mancuso Dr. Mancuso reported preliminary results on development of bleeding score in patients with hemophilia. Thus far 319 patient’s data have been collected however preliminary data suggest that there is some correlation based on ROC curves however more patients data needs to be collected to improve the score. A call was made to get more centers involved. When and how to start prophylaxis in boys with severe hemophilia without inhibitors Kathelijn Fischer Dr. Fischer reported that the working group completed its work and the recommendations from the group on when and how to start prophylaxis in boys with severe hemophilia A without inhibitors has now been published in JTH. Standardization of post-registration surveillance Flora Peyvandi Dr. Peyvandi described the mandate of the group which is to develop methods for long-term safety/efficacy of novel long-acting factor products (or even other products) for hemophilia. The project involved setting up a minimum dataset for what should be collected after licensure of new drugs. Currently, the dataset is posted on the ISTH website for comment. Consensus Definitions for Immune Tolerance Elena Santagostino Dr. Santagostino reported on the progress of her working group describing the definitions for achievement of immune tolerance. The work is nearly complete and a manuscript is in preparation. Working group on pharmacokinetics and population pharmacokinetics of factor concentrates Alfonso Iorio Dr. Iorio described the formation of this new working group whose goals are to describe the manner of reporting study results in the literature, interpretation of the results of PK and PopPK studies, assessment of PK/PopPK in clinical practice, and suggestion of study design for clinical trials that include PK/PopPK. Definitions in Acquired Hemophilia Andreas Tiede Dr. Tiede described the formation of this new working group whose aim is to develop a set of definitions for acquired hemophilia. His report demonstrated that previous registries report various definitions for success of treatment and time to success of treatment among other variables.   Gene Therapy Session  Definitions of Outcomes in Gene Therapy Alok Srivastava Dr. Srivastava described several issues for consideration with respect to reporting outcomes in gene therapy trials both for efficacy and safety. Efficacy should include peak and sustained factor levels and control of bleeding. Questions remain about what should be considered efficacious with respect to what levels should be achieved long-term for a product to be licensed. For safety, issues of insertional mutagenesis, immunotoxicity and horizontal and vertical transmission of vector. Dr. Srivastava suggested that a working group should be formed to discuss these issues with regulators, industry and academia involved. Regulatory guidance for gene therapy trials Annelise Hilger Dr. Hilger reviewed issues that are related to regulatory aspects of gene therapy in particular that the usual guidelines for approval of factor concentrates cannot apply to gene therapy trials. Gene therapy: From clinical trials to clinical practice Steve Pipe Dr. Pipe reviewed recent results from published clinical trials including abstracts and compared and contrasted what might be achieved from the current gene therapy trials versus the extended half-life drugs and other new compounds and speculated on where the role of gene therapy might be in the future with respect to other compounds in development.   SIPPET session Presentation of SIPPET data Flora Peyvandi Dr. Peyvandi presented the results of the SIPPET study which coincidentally was published in the New England Journal of Medicine the day before the session. Debate on the SIPPET study Manuel Carcao and Kathelijn Fischer Drs. Carcao and Fischer debated whether or not PUPs should be started on recombinant or plasma-derived factor products. This was followed by a discussion between the audience and the speakers. It was a robust debate the suggested the need for a “Debate-type” paper in JTH. Stakeholders discussion on SIPPET results Brian O’Mahony (patient), Marijke van den Berg (WFH), Stephanie Seremetis (Industry-recombinant), Garrett Bergman (Industry-plasma), Annelise Hilger (Regulatory) The above each discussed the SIPPET results (those from the abstract from ASH) from the perspective of the groups they were representing. This was followed by a discussion between the audience and the speakers.   Rare Bleeding Disorders European Network on Rare Bleeding Disorders Report on FXIII Flora Peyvandi Dr. Peyvandi describe the work of this network describing the risk for bleeding as related to the FXIII level in patients with FXIII deficiency. The research demonstrated that patients with severe FXIII deficiency are generally diagnosed at 5 years of age however often have their first bleeding symptom in the first year of life. In addition, severe bleeding is noted for patients with levels below 20%. Next Generation Testing for Rare Bleeding Disorders Diane Nugent Dr. Nugent reported her work on next generation sequencing for rare bleeding disorders. She stated that with this method, she identified a number of previously unreported mutations. In addition, this method allows for the identification of co-inherited mutations that could influence the bleeding phenotype. Plasminogen deficiency Amy Shapiro Dr. Shapiro described the background of plasminogen deficiency as well as provided an update on clinical trials of plasminogen concentrates and plasminogen eye drops for ligneous conjunctivitis, the major manifestation of severe plasminogen deficiency. Extended Half-life Factor Products Laboratory issues with the extended half-life products Steve Kitchen Dr. Kitchen described the publications and abstracts regarding the measurement of recovered factor from various extended half-life products demonstrating that there are inaccuracies in measuring recovered factor depending on the product and PTT reagent combination. He also demonstrated that much of these errors can be averted if the chromogenic factor assays are adopted. The discussion led to the suggestion to form a working group to develop an SSC recommendation regarding the chromogenic assay. Debate on the use of extended half-life FVIII therapies Amy Dunn Gerry Dolan Drs. Dunn and Dolan debated the merits of switching patients from standard half-life factor VIII products to extended half-life products. The debate was followed by a vigorous discussion. Debate on the use of extended half-life FIX therapies Johnny Mahlangu Charlie Hay Drs. Mahlangu and Hay debated the merits of whether all patients with FIX deficiency who require factor should switch to extended half-life factor IX products. The debate was followed by a vigorous discussion.   Inhibitor session Framing the inhibitor problem Donna Di Michele Dr. Di Michele discussed several issues with regards to inhibitors including inhibitor development, prediction as well as treatment and prevention of bleeding. Her discussion set up the rest of the session by framing the major issues facing inhibitor patients. SSC Inhibitor Collaborative Study on the South Mimms Inhibitor Assay Sanj Raut Dr. Raut presented data on a novel method for the detection of inhibitors in patients with hemophilia. His goal is to develop a collaborative study to further validate this assay. He presented the protocol and will solicit other participants from the community. Alternative treatment options using current therapies Gili Kenet Dr. Kenet described alternative approaches to treat bleeding in inhibitor patients including using sequential/combined therapy with rFVIIa and FEIBA as well as using FVIII along with bypassing agents. Future therapies for patients with inhibitors David Lillicrap Dr. Lillicrap provided an overview of 3 new products in clinical development that are in current human trials: ACE910 (emicizumab), ALN-AT (fitusiran) and the anti-TFPI product concizumab. Inhibitor Management in 2020 Guy Young Dr. Young moderated a session discussing how inhibitor patients may be treated in 2020 with the assumption that novel agents such as ACE910 (emicizumab), ALN-AT (fitusiran) and anti-TFPI products. The discussion revolved around management of PUPs, immune tolerance induction, and treatment/prevention of bleeding.
by G. Young
Wednesday, June 8, 2016
2015 Annual Minutes Locked Topic 0 L. Schmeidler Chairman: Guy Young Co-Chairs:  Manuel Carcao, Michael Makris, Danijela Mikovic, Flora Peyvandi, Steven W. Pipe, Elena Santagostino   ​Project Reports 1.      Standardization of post-registration surveillance: F. Peyvandi  (IT) Dr. Peyvandi updated the SSC on her project on post-registration surveillance for efficacy and safety of novel factor products. She described all of the registries in the various world regions that are collecting data. The goal of the group is to recommend standards for the data collection within these registries such that data can be compared across those databases. 2.      Prophylaxis in patients without inhibitors: V. Blanchette (CA) and K. Fischer (NL) Dr. Fischer discussed the results of her project on prophylaxis in patients without inhibitors describing when and how to start prophylaxis in FVIII and FIX deficiency in children. The manuscript is nearly complete and will put up on the SSC website for comment in the coming weeks. 3.      Prophylaxis in patients with inhibitors: C. Escuriola (DE) Dr. Nadia Ewing (in place of Dr. Escuriola) presented the working group’s progress on recommendations for prophylaxis in inhibitor patients. The group has nearly completed its work and will develop a manuscript over the summer. 4.      Bleeding Score in Hemophilia: E. Mancuso (IT) Dr. Mancuso presented her working groups progress on this project. Of note, 37 centers have agreed to participate in a data collection project to validate the bleeding score that was developed. So, far 99 patients with hemophilia and B have been enrolled.  5.      ITI definitions: E. Santagostino (IT) Dr. Santagostino presented an update of her group’s project. She reviewed the recommended definitions for successful ITI based on both pharmacokinetic as well as clinical endpoints. The group is working on developing a manuscript which is planned for completion by next year. 6.      Mild hemophilia definitions: M. Makris (GB) Dr. Makris discussed the final results of his group’s work on defining mild hemophilia. Specifically, he discussed modifications of the existing SSC definitions of mild hemophilia including defining some patients with >40% levels of factor VIII as having hemophilia if they have a DNA change consistent with hemophilia and a family member with a level of <40%. He also discussed issues with measuring factor levels with the one-stage versus choromogenic assay that could lead to missing the diagnosis in some patients. 7.      Inhibitor reporting standardization in previously treated patients: A. Iorio (CA) Dr. Iorio provided an interim report of his group’s work on reporting of results from trials and other studies on the inhibitor rate in PTPs. The group has developed recommendations regarding how to report inhibitors in PTPs in the literature. 8.      Standardization of genetic assays for diagnosis of hemophilia: V. Jenkins (IE) Dr. Jenkins reported on his group’s work on standardization and quality assurance as it applies to genetic testing in hemophilia. First, a survey was sent out to various laboratories to determine the types of tests that are done, the logistics of test performance, the methods of determining the genotype. The group will use this data to develop recommendations for standardization and quality assurance for genetic testing in hemophilia. Education Program 1.      From Alexie Romanov to Stephen Christmas to 2015: What is different about factor IX deficiency: E. Santagostino (IT) Dr. Santagostino provided a review of the differences between FIX and FVIII deficiency focusing on whether or not FIX deficiency is a less severe bleeding disorder. The presentation evaluated the literature on factor VIII and IX deficiency with respect to the frequency of prophylaxis, hemophilic arthropathy, and bleeding rates, and in general as opposed to FVIII deficiency, patients with FIX deficiency were less likely to be on prophylaxis, had less hemophilic arthropathy, and had the first joint bleed at a later age, however it was not clear if this is a real difference in terms of disease severity or epidemiologic findings that may be due to the notion that FIX deficiency is less severe. 2.      What is the “true” incidence of inhibitors in haemophilia: M. Soucie (US) Dr. Soucie provided an epidemiological overview of inhibitor incidence including a detailed discussion on the definitions of incidence and prevalence and elaborating on the limitations of the statistical methods for detection of inhibitors in a low prevalence disease. Rare Bleeding Disorders 1.      Update on afibrinogenemia and dysfibrinogenemia: P. de Mooerloose and A. Casini (CH) Dr. Casini provided an overview of fibrinogen disorders including a detailed discussion on genotype-phenotype correlations. Dr. Casini discussed congenital afibrinogenemia, congenital hypofibrinogenemia, and congenital dysfibrinogenemia. All 3 conditions are associated with a bleeding disorder with congenital afibrinogenemia clearly being the most severe of the three. 2.      PRO-RBDD Project: Prospective evaluation of the intensity of bleeding episodes in patients with fibrinogen and FXIII deficiency: F. Peyvandi (IT) Dr. Peyvandi updated the SSC on her project of these two rare bleeding disorders including data from a multisite registry study on bleeding events in these two rare disorders as well as treatments given by the institutions that participated. Specifically for FXIII deficiency, she discussed that patients with a mild deficiency exhibit bleeding symptoms as well including mucus membrane bleeding and menorrhagia. Treatments varied throughout the world, however most developed nations now have factor concentrates available for treatment of both and many patients with FXIII deficiency are on prophylaxis.   Laboratory Issues 1 1.      Update on Nijmegen group’s high sensitivity inhibitor assay: B. Verbruggen (NL) Dr. Verbruggen described a novel increased sensitivity inhibitor assay based on the Nijmegen-modified Bethesda assay. He reviewed a small validation study that his team performed which did demonstrate that the assay correlates with the half-life, however the number of patients was small and further validation is warranted. 2.      Inhibitor measurement using SMIA: a new approach in inhibitor measurement: S. Raut (GB) Dr. Raut described a novel assay (South Mimms Inhibitor Assay) which is a modification of the Classical Bethesda Assay which is aimed mostly at reducing the inter-laboratory variation of the Bethesda assay and the Nijmegen-modified Bethesda assay. It may also improve the sensitivity. His next step is to do a multi-laboratory study to determine the inter-laboratory coefficient of variation. 3.      Establishment of the 5th International Standard for FIX: E. Gray (UK) Dr. Gray described the establishment of the 5th international plasma standard FIX and the 1st international standard for recombinant FIX. There is general agreement to have this new standard move towards endorsement by WHO. There is less agreement about the need for an international standard for recombinant FIX and plans for that have been deferred for now. 4.      Update on EAHAD Coagulation Factor Variant Genotype Database: K. Gomez (UK) Dr. Gomez presented the newly developed EAHAD supported database of mutations in FVIII, FIX, and VWF. He demonstrated the utility and ease of use of the database.  5.      IU vs %: Clearing up the confusion and a proposal for a position paper: S. Kitchen (GB) Dr. Kitchen described the issues and concerns with the use of the terms units versus IU versus %-age. He discussed that %-age is not always equal to IU and he felt that a position paper or recommendation from the FVIII/FIX/RBD subcommittee. Dr. Kitchen will send me a proposal for a position paper on this topic in the next 2 months for further discussion. 6.      WAPPS Project (Web-accessible population pharmacokinetics service): A. Iorio (CA) Dr. Iorio presented the development of his web-accessible pharmacokinetics service and reviewed how clinicians and researchers can access it and utilize it for clinical decisions or for developing research projects. Once the application is ready to go live, anyone will be able to access it as a free web-based application. Once this is available, he will let me know and we can put an announcement out on the SSC webpage.   Clinical Issues 1.      Moderate hemophilia update: K. Fischer (NL) Dr. Fischer provided an update on the clinical aspects of moderate hemophilia based on some recent observations and large studies mostly from The Netherlands. She demonstrated data from a large database in the Netherlands that a subset of patients with moderate hemophilia will develop joint disease if not treated aggressively as they act more like patients with severe hemophilia, and even those who don’t bleed like severe hemophilia are prone to developing severe bleeds on occasion. 2.      Clinical use of extended half-life factor products: G. Young (US) Dr. Young presented a series of questions regarding the use of extended half-life factor products to the audience and solicited audience opinions which led to an open forum discussion on this topic. Several audience members spoke of their opinions regarding the use of these drugs in PUPs indicating that they would not treat PUPs with these drugs until there was more data though one person indicated that she thought PUPs with their poor venous access would be the ideal patient group to initiate these drugs in. There was a general feeling that for patients with poor venous access or those who have not been on prophylaxis due to the frequency of the IV infusions are the best candidates for extended half-life drugs.   Laboratory Issues 2 1.      Extended half-life factors and laboratory assays--overview of the problem: S. Pipe (US) Dr. Pipe provided an overview of the issues surrounding laboratory analysis of the extended half-life factors demonstrating that this is a relevant clinical issue when treating patients with these agents as it relates to measuring levels of the infused factor. 2.      Overview of chromogenic assay basics: K. Friedman (US) Dr. Friedman provided an overview of the mechanism for the factor VIII and factor IX chromogenic assay. He described in detail the differences between the assays stating that the chromogenic assay has less variability and is more reliable as it truly focuses on the action of FVIII or FIX rather than evaluating the entire coagulation cascaded. 3.      Implementation of the chromogenic assay into the clinical lab: R. Ko (US) Dr. Ko described the implementation of the chromogenic assays for factor VIII and factor IX in his clinical coagulation laboratory. He described the steps that he went through to make this assay available in his institution. Briefly, the steps included making a request to the clinical laboratory, identifying vendors for the assays, choosing a vendor, performing laboratory calibration followed by validation, and then once ready, developing a mechanism for ordering the laboratory tests.
by L. Schmeidler
Sunday, June 21, 2015
2014 Annual Minutes Locked Topic 0 F. Peyvandi Factor VIII, Factor IX & Rare Coagulation Disorders Chairman: Flora Peyvandi (Italy) Co-Chairmen: Michael Makris (UK), Danijela Mikovic (Serbia), Steven Pipe (USA), Elena Santagostino (Italy), Midori Shima (Japan), Guy Young (USA) Session 1: Wednesday, 25 June (8:00-12:00)Session 2: Thursday, 26 June (8:45-12:45)  BUSINESS SESSION, FIRST PART June, 25th 2013 Dr. Peyvandi opened the session welcoming the audience. Dr Peyvandi opened the SSC business session by thanking all co-chairs of this subcommittee for the work done in the last year.    She reported about the state of the art of the SSC: 1.    Recommendations published as Official SSC Communications on JTHConsensus definitions in rare bleeding disorders (Peyvandi et al, JTH 2012:10)Pharmacokinetics (Bjorkman and Collins, JTH 2013:11)Potency labelling of clotting factor concentrates (Hubbard et al, JTH 2013;11:988-9) -          Standardization of methods for performing the clot wave form analysis (Shima et al, JTH 2013;11:1417-20)Standardization of methods for performing the thromboelastogram (Chitlur et al, JTH 2014;12:103-6) 2.    Consensus definitions in hemophilia (Chair: V. Blanchette) – 2009/2011. Recommendation accepted as Official SSC Communications to JTH 3.    Clinical trial design for hemophilia (Chair: D. DiMichele) – 2011/2013. Recommendation will be soon submitted  as Official SSC Communications to JTH     Dr. Peyvandi presented the on-going projects:Bleeding score in hemophilia: a prognostic tool for clinical outcome  (Chairs: E.M. Mancuso and A. Tosetto) – 2012/2014The definition of mild Hemophilia A (Chair: M. Makris) – 2012/2014Factor V deficiency, clinical heterogeneity and treatment(Chair: D. Mikovic) – 2013/2015Standardization and quality management of genetic assays for diagnosis of hemophilia (Chair: V. Jenkins) – 2013/2015Prophylaxis in patients with inhibitors (Chair: C. Escuriola) – 2013/2015Prophylaxis in patients without inhibitors (Chair: V. Blanchette) – 2013/2015Consensus definitions and recommendations for immune tolerance induction (ITI) in hemophilia with inhibitors (Chair: E. Santagostino) – 2013/2015Standardization of anti-FVIII inhibitor assays (Chair: K. Moertens) – 2013/2015IRS-PTPs: inhibitor reporting standardization in previously treated patient (Chair: A. Iorio) – 2013/2015Standardisation of post-registration surveillance(Chair: F. Peyvandi) – 2014/2016Evaluation of hemostatic efficacy of novel  FVIII / FIX concentrates and FVIII-inhibitor by-passing agents (Chairs: A. Lawrie and A. Tripodi) – temporarily stopped    CLINICAL TRIALS DESIGN AND SURVEILLANCE Session Chairpersons: Donna Di Michele (USA) and Frits R. Rosendaal (The Netherlands)    Report on the SSC project:  Definitions in Hemophilia. Victor Blanchette (Canada) on behalf of of the ISTH SSC on Factor VIII, IX & Rare Coagulation Disorders Project Group on Consensus definitions in hemophilia (Nigel Key, MD, Rolf Ljung, MD, Marilyn Manco-Johnson, MD, Marijke van den Berg, MD and Alok Srivastava, MD)  Evaluation of the safety and efficacy of novel hemostatic agents and of different prophylaxis regimens in individuals with inherited bleeding disorders (focus the hemophilias) requires consistency in definitions of commonly used end points.  The mandate of the Project Group was to perform a critical review of relevant information and to prepare definitions of: classification; inhibitors; regular replacement therapy (prophylaxis); bleeding (and re-bleeding) into joints and muscles; target joints; and response to therapy including surgical haemostasis. Reaching a consensus proved to be more challenging for some definitions such as the cut-off between normal and a positive inhibitor level for which a value of ≥ 0.6 Nijmegen-Bethesda Units was considered a positive test result.  Inhibitors were considered transient if they disappeared within six months of first appearance despite antigenic re-challenge.  Another contentious area is the consensus definition of primary prophylaxis.  The current proposed definition is "regular continuous” replacement therapy  started in the absence of documented joint disease as determined by physical examination and/or imaging studies, and before both the second clinically evident joint bleed and  age 3 years. Finally, the chosen definition of a joint bleed, a commonly used end point in clinical trials of new factor concentrates or prophylaxis regimens, was "an unusual sensation ("aura”) in the joint, in combination with any of the following: increasing swelling or warmth of the skin over the joint; increasing pain; progressive loss of range of motion, or difficulty in using the limb as compared to baseline. In infants and young children reluctance to use the limb alone may be indicative of a joint/muscle bleed…” Response to treatment of joint /muscle bleeds should  be assessed  at  defined times following infusion of clotting factor concentrates; in the case of surgery assessment of response should be performed by a surgeon/ anesthetist involved with the procedure . The proposed definitions have been accepted for publication by the Journal of Thrombosis and Haemostasis.    Report on the SSC project: Clinical trial design for hemophilia. Donna Di Michele (USA) The mandate for the Project Group on Clinical Trials for New Products in Hemophilia (CTPG)  arose not only from pragmatic concerns about the feasibility of populating and conducting multiple simultaneous new clotting factor trials, but also from the perspective of harmonization of these requirements based on common principles. This led to the question of whether innovative and evidence-based approaches to trial simplification might increase feasibility without compromising assessment of product safety and efficacy. Based on this rationale, the CTPG was tasked with exploring alternative approaches for the pre- and post- licensure study of so called ‘me-too’ and novel biologic replacement products for hemophilia A and B. The group was constituted on the basis of member expertise in clinical care and investigation, immunology, clinical trial design and statistics, and included regulatory science representatives from the Food and Drug Administration (FDA), USA, and the European Medicinal Agency (EMA). Input from critical stakeholders was solicited at international and FVIII/IX SSC Subcommittee meetings; from the relevant industry; and from selected experts in clinical trial design methodology. The CTPG ultimately narrowed the project scope to encompass recommendations for an alternative approach to statistical analysis and the clinical design and statistical analysis of pre-authorization trials for both ‘me-too’ and novel factor VIII (FVIII) concentrates in previously treated patents (PTPs). This approach is rooted in the combined agency regulatory goals for pre-licensure studies, and incorporates both current immunological theories of neoantigenicity and consensus clinical efficacy endpoint definitions. Innovative approaches to the clinical design of new product safety (immunogenicity) trials were considered and based on the known epidemiology and immunology of FVIII inhibitor development in congenital hemophilia A. Recognizing that the optimal design of pre-authorization clinical trials remains hampered by poor understanding of the precise nature of the interactions between the therapeutic FVIII products and the recipient’s immune system, the CTPG recommended a systematic and harmonized collection of clinical and biological data from subjects entering pre- and post- authorization new product studies. The CTPG also advocated for  future exploration of 1) the feasibility of international harmonized post-authorization studies using existing national and international database infrastructure and consensus standardized minimum datasets, and 2) eventual EMA/FDA harmonization in critical areas such as the evolving landscape of inhibitor assays and the disparate approaches to product authorization in children.    Too many PUPs studies, not enough PUPs. Guy Young (USA) From the mid-90s to the late 2000s, few new medications/studies were developed for patients with hemophilia. Recently, however, with the advent of modified factor products as well as renewed interest in studying the immunology of inhibitor development, several new PUP trials are underway or will soon be underway. With the recent approval of rFIXFc and the imminent approval of rFVIIIFc as well as other modified agents in various stages of clinical development, there will suddenly be a need for many PUPs to participate in clinical trials as part of post-licensing agreements. In addition, investigator-initiated PUP trials are ongoing (HIPS and SIPPET) and additional such studies will be developed (INHIBIT study pending funding). Keeping in mind that the total number of PUPs born in the USA and Europe per year is 400 for FVIII and 100 for FIX and 500 and 125, respectively, and considering that many of these PUPs won’t end up at sites that open the trial, many of these patients will be needed to participate in clinical trials. This creates a variety of concerns for patients, investigators, and sponsors. For patients, the main concerns are the safety of participating in trials with novel modified factors for which the risk for inhibitor development (and perhaps other adverse events that may be unique to PUPs) is unknown. For investigators, the main concerns will be the time commitment required to participate in trials as well as ethical concerns in enrolling PUPs in trials with new agents. In addition, investigators may feel pressured by sponsors to participate in trials or worse may pressure patients to participate given financial incentives and other considerations. Furthermore, investigators who open more than one PUP trial face the prospect of deciding which patients should participate in which trials. For sponsors, the major concern will be identifying enough sites and subjects to complete the studies in a timely fashion. There are no easy solutions to any of these issues and an ongoing dialog between the scientific community and the sponsors is the best way to minimize potential problems.    New SSC project: IRS-PTPs: inhibitor reporting standardization in previously treated patient.  Alfonso Iorio (Canada) on behalf of the ISTH SSC on Factor VIII, IX & Rare Coagulation Disorders Project Group (Bernardi F, Lillicrap D, Lip G, Makris M, Peyvandi F, Rosendaal F) Background: The development of inhibitors in previously treated hemophilia A patients (PTPs, i.e. patients having received =>150 days of treatment with factor VIII) is a rare event, estimated at about 3 per 1000 patient years of observation1. For this reason, the ISTH SSC identified PTPs as the most suitable population for assessing antigenicity of new factor VIII concentrates2. Inhibitor rates in PTPs have subsequently being used to perform direct 3, 4 or indirect1, 5 comparisons of the immunogenicity of different factor concentrates. Critical yet missing pieces in the picture are the understandings of the potential triggers for inhibitor development and their subsequent natural history. Indeed, inhibitors may occur in PTPs in absence of switching 6. Aims: 1) To define a checklist of patient and inhibitor characteristics to be included (either in the main text or as online-only appendix) when submitting for publication studies about the incidence of inhibitors in PTPs. 2)To produce a SSC recommendation to authors and journal editors to adopt the checklist. Methods: We performed a systematic review of the evidence to assess: a) which details are usually reported or not reported about published inhibitor cases b) which guidelines do exists informing the optimal reporting of clinical cases and c) which indications for reporting clinical cases are provided by the instructions for the authors of major journals. The results of the systematic review will inform the preparation of a draft checklist and recommendation, which will be refined in a Delphi process involving of hemophilia experts. Interim results:  We have collected information for 34/43 cases of inhibitors reported in the literature as occurring in cohorts of PTPs (results being presented at the ISTH meeting in Milwaukee). In a start-up conference call of the group held on April of 2014, we have defined a comprehensive set of information of potential interest and defined the work plan. Work plan: Develop an online survey of to gain further insight on the perceived value of the information in object. Analyze the retrieved reporting guidelines to inform preparation of the recommendation. Both documents will be presented for final approval at the ISTH SSC in 2015. References: (1) Xi M et al. J. Thromb. Haemost 2013; 11(9):1655–62.  (2) White GC, R. Thromb. Haemost 2001; 85:560. (3) Kempton CL, J. Thromb. Haemost 2006; 4(12):2576–81. (4) Makris M et al. Thromb. Res. 2011; 127(Suppl 2):S22–25. (5) Aledort LM et al. J. Thromb. Haemost 2011; 9(11):2180–92. (6) Iorio A et al, Blood. 2012; 120(4):720–7.    New SSC project: Standardization of post-registration surveillance.  Flora Peyvandi (Italy) In the recent years, many promising strategies are emerging to enhance especially the half-life of therapeutic proteins. These bioengineering strategies have been employed to manufacture novel coagulation factors that prolong the bioavailability with increased potency and resistance to inactivation and potentially reduced immunogenicity. These novel drugs have the potential to dramatically transform the treatment of hemophilia by substantially reducing the frequency of injections. Promising data from the pre-authorization phase of clinical trials have been presented. Nevertheless, a prolonged period of surveillance will be required to collect more accurate data on the safety and efficacy and additional data to ensure consistency in the long-term of novel drugs. Moreover, the available data on the novel long acting drugs are limited by the relatively few number of patients enrolled in the pre-authorization phase, and for this reason, large-scale post-marketing pharmacovigilance studies are necessary to gain sufficient numbers of patients for statistically valid assessments. A project group assembled by the Factor VIII, Factor IX and Rare Coagulation Disorders SSC Subcommittee aims to establishing the requirements for an international harmonized post-authorization studies using existing national and international database infrastructure and consensus standardized minimum datasets. These data would complement limited pre-authorization data on product safety and haemostatic efficacy, as well as contribute to a greater understanding of immunogenicity and the potential implementation of novel more sensitive antibody detection assays. As a first step the project group decided to revise the available template forms used by ATHN, CHESS, EUHASS and UKHCDO to collect data on safety and to ask regulatory agencies recommendations on what type of data should be collected to optimize the quality of the information, especially for long-acting products. This working group will have three/four conference calls during 2014/2015 and will meet during next ASH and/or ISTH meetings.    DISCUSSION Discussion regarding exposure day definition for longer acting product. Not so much information: we need to redefine better  both the exposure days and number of infusion. Regarding post-registration surveillance, Steven Pipe stated that post-infusion efficacy requires attention and dedicated studies. Moreover it is crucial to think about who is going to collect these data: it would be preferable to have an independent body.      SAFETY AND EFFICACY EVALUATION OF NOVEL DRUGS Session Chairpersons: Guy Young (USA) and Flora Peyvandi (Italy) A variety of new medications including factor replacement therapies with extended half-lives and non-replacement pro-hemostatic drugs are either already approved or in the pipeline. In the coming years, it is likely that clinicians will have a broad array of pro-hemostatic drugs to choose from to manage patients with hemophilia. There are two critical issues with respect to laboratory testing that have surfaced. First, the issue of potency labeling of the new products is crucial to ensure that what the amount of factor stated on the label is actually what is in the bottle, and second is the issue of therapeutic monitoring of the infused products in patients for PK assessments and surgery.  During this session, there were 3 sets of talks given. The first were by laboratory scientists. Dr. Hubbard presented the ISTH SSC recommendations for potency labeling of novel factor therapeutics. This was followed by a presentation by Elaine Gray from NIBSC demonstrating the high degree of variability of laboratory results from a field study evaluating various new factor products with the many different aPTT reagents for one-stage clotting assays as well as chromogenic assays. It was clear that depending on the assay used for specific products, the CV%s are often very high suggesting that results of such tests for specific products must be interpreted with caution. The final talk of this portion of the session was from Dr. Rosen from Rossix labs demonstrating a possible mechanism for why one specific novel agent, N9-GP, provides such poor results with the one-stage clotting assay. The next set of talks were given by representatives from 5 companies with novel products either recently approved or in the pipeline. The presentations were given by Dr. Turecek (Baxter), Dr. Muller (Bayer), Dr. Sommer (Biogen Idec), Dr. Bensen-Kennedy (CSL Behring) and Dr. Ezban (Novo Nordisk). Briefly, what is clear is that the various new drugs all present some problems with respect to therapeutic monitoring with standard one-stage clotting assays. While some of the products can be reliably assayed by some of the reagent/coagulometer combinations, some assays will overestimate and some will underestimate the amount of factor present. Strategies to overcome these issues include using a product-specific laboratory standard, recommending specific reagents/instruments to assay specific products, using chromogenic assays which overcomes the problems with one-stage clotting assays, or introducing a correction factor. What is clear is that laboratory monitoring of new products will be complex and many issues need to be addressed and resolved in the coming years. In the last part of the session, two talks from our regulatory colleagues, Annelise Hilger from EMA and Mark Weinstein from FDA provided the view from the perspective of the regulators.    Do we need to change the SSC recommendations for potency labeling with arrival of long acting products? Anthony Hubbard (UK) The recommendations on potency labelling were published last year and since then the interest from regulators and manufacturers has been very encouraging.  This was obvious during a workshop on the characterisation of new clotting factor concentrates held at the European Medicines Agency when almost every manufacturer of novel FVIII and FIX products reported their approach to potency labelling by reference to the published decision tree.  The central theme of the recommendations was to describe the necessary product characterisation on which the decision for potency labelling should be based.  This guidance remains relevant, however, it is now possible to comment further on some aspects: 1)  Decision on the method for potency labelling: It appears that all novel FVIII and FIX products, under development at present, can provide valid assays relative to the WHO IS Concentrates and manufacturers are intending to label using International Units (IU).  However, some products display potency discrepancies not only between the chromogenic and clotting methods but also within methods, for instance, when different APTT reagents are used in the clotting method.  In this situation, where several valid but different relative potencies are possible, it may be necessary to provide more guidance on the choice of method, including the reagent, for labelling.  An important consideration in this choice should be maintaining the equivalence of the IU between existing licensed products and the novel products which would ideally be administered using a similar IU/kg dosage albeit with different frequency for the "longer-lasting" therapeutics.  Large differences in the recommended IU/kg dosage between established products and the novel products could lead to confusion and undermine the credibility of the IU.  The choice of potency labelling method should therefore take into account potency comparisons with existing products in vitro, using the same methods and reagents, as well as information on comparative haemostatic efficacy from in vivo studies. 2) Importance of the manufacturer's in house product standard: Definition of the route for IU labelling of some novel products, relative to the WHO IS Concentrate, may need to specify not only the method used (e.g. clotting or chromogenic) but also the specific reagents (e.g. APTT reagent).  This may mean that the link of the product to the WHO IS Concentrate can be affected by the reliability and consistency of reagents beyond the control of the product manufacturer.  In these situations the consistency of product potency labelling will rely more heavily on the manufacturer's in house product standard and greater emphasis on its stability and replacement strategy will be necessary. 3)  Post-infusion testing: The recommendation that manufacturers should provide guidance to clinical laboratories on post-infusion testing remains valid.  The recovery of products where the measured potency is sensitive to the type of APTT reagent might not be suitably monitored when local clotting methods use different APTT reagents.  Although logistically challenging, it might be necessary for clinical laboratories to adopt specific laboratory test procedures for some products post-infusion, for instance, the use of recommended reagents or product-specific standards.     Report of the new generation FIX collaborative NIBSC study. Announcement of the replacement FIX standard study. Elaine Gray (UK) Seventeen laboratories participated in a study to investigate the comparability of recombinant and new generation factor IX products with the WHO 4th International Standard (IS) for FIX concentrate, the WHO 4th IS for FII, VII, IX, X, Plasma and the NIBSC recombinant FIX reference preparation.  Samples included in the study were a plasma-derived product, three recombinant FIX products and three long-acting products.  Assay methods included in the study were one-stage clotting assays using three common APTT reagents (APTT-SP, Actin FS and SynthAFax) two common chromogenic assay kits (ROX and Hyphen) and, in addition, laboratories’ own routine APTT reagent (n=12).  All samples can be assayed validly against either the 4th IS FIX concentrate, the 4th IS Factors II,VII,IX,X, Plasma or the NIBSC recombinant FIX reference preparation. Intra- and inter- laboratory assay variability was low for the plasma derived and the recombinant products for all methods against the three different reference standards. For the long-acting products, despite low intra-laboratory variation, inter-laboratory variability was high. Assay discrepancies were observed between the clotting assays using different APTT reagents and also between clotting and chromogenic assays for the recombinant and the long acting products when assayed against the concentrate or plasma IS. When the recombinant products were assayed against the recombinant reference preparation, assay discrepancies were reduced.  Based on the results of this study, it is proposed that an International Standard for Recombinant FIX be established to support potency labelling of recombinant FIX products and candidates will be evaluated in the forthcoming collaborative study for the replacement of the current International Standard for plasma-derived FIX Concentrate.    Activation kinetics of FIX by specific aPTT reagent explain discrepancies observed in one-stage assay for N9-GP. Rosén P, Bryngelhed P, Rosén S (Rossix AB) In order to investigate the cause of the large discrepancies observed in FIX potency determination of long-life rFIX preparations with one-stage (OS) methods depending on the aPTT reagent used, a study was performed with N9-GP, BeneFix and the 4th IS FIX Conc where subsampling was made in OS methods both during contact activation and after addition of calcium ions. Generated FXIa and FIXa was determined with sensitive chromogenic methods. Actin FS, APTT SP and SynthAFax were used as aPTT reagents. No difference in FXIa generation vs time was observed for the three FIX preparations within any given aPTT reagent. However, the actual level of generated FXIa with Actin FS was only about 2/3 vs APTT SP and SynthAFax. FIXa generation vs time was similar for the three FIX preparations with both SynthaFax and the chromogenic Rox Factor IX kit. The large discrepancy was instead caused by large differences in FIXa generation when using APTT SP and Actin FS on analysis of N9-GP. FIXa generation was impaired with Actin FS and, importantly, high levels of FIXa were generated with APTT SP already before addition of calcium ions. Our results identify the cause of the FIX potency discrepancy and demonstrate that different aPTT reagents may have a strong impact on FIXa generation.    Activity measurement of BAX 855, PEGylated recombinant full-length human FVIII Peter Turecek (Baxter) Baxter has developed BAX 855, a PEGylated recombinant Factor VIII product, which is currently in late stage clinical development. BAX 855 is based on the parent full length rFVIII molecule in ADVATE® produced from CHO cells. BAX 855 is chemically modified by conjugation with a proprietary stable PEG reagent from Nektar Therapeutics using comparable conjugation technology as had been successfully employed in other marketed and licensed PEGylated drug products. Baxter has decided to use the one-stage clotting assay for its BAX 855 product. The reason is that both clotting and chromogenic activity values for BAX 855 are better aligned than for BDD-rFVIII or other modified FVIII products which had been previously in clinical use or are in clinical development and where discrepancies between one-stage clotting and chromogenic values caused severe issues for patients [1-4]. The potency determination for BAX 855 uses Actin FSL which is the same activator reagent as also used for the one-stage clotting assay used for potency determination of ADVATE®. By using this specific set-up of the one-stage clotting assay good agreement with the chromogenic assay is achieved. The other assay parameters are also comparable to the one-stage clotting assay used for potency assignment for ADVATE® (automated coagulation analyzer BCS/XP, immunodepleted FVIII deficient plasma freeze dried, same incubation time and assay set-up, same in-house secondary standard for rFVIII). Using the one-stage clotting assay for potency assignment makes the determination of recovery in patients reliable as in clinical practice only the one-stage clotting assay is used. Preliminary results from the phase 1 study on BAX 855 showed that the mean incremental recovery (IU dL-1/IU kg -1) measured with the one-stage clotting assay was in the same range as for the study comparator FVIII product ADVATE®. According to EMA guideline CHMP/BPWP/144533/2009 on clinical investigation of recombinant and human plasma derived FVIII products "preferably, the same assay should be used for analysis of the product and the patient’s plasma”. At the same time the guideline recognizes that potency assignment for FVIII products has to be performed with the chromogenic assay as requested by European Pharmacopoeia monograph 1643, human coagulation factor VIII (rDNA). In contrast in clinical practice the one-stage clotting assay had remained for the last 50+ years as the preferred method to determine FVIII plasma levels. Therefore Baxter assumed that a new FVIII product should also be determined for potency by the same assay as used in clinical practice. References: Morfini et al, J Thromb Haemost. 2003 Nov;1(11):2283-9Gruppo et al., Haemophilia. 2004 Sep;10(5):449-51 Hubbard et al., Thromb Haemost. 2003 Dec;90(6):1088-93 Leong et al., Poster presented at the XXIII Congress of the ISTH, Kyoto, Japan, 2011  BAY 94‐9027; Evolution and Significance of Potency Assays used in the PROTECT VIII trial and recommendations for future clinical monitoring. Nikolaus Mueller+, Yvonne Katterle*, Lilley Leong+, Derek Sim+, Lisa Regan+, Prasad Mathew+, Georg Lemm* *Bayer Pharma AG Germany, +Bayer Healthcare Pharmaceuticals Inc. USA BAY 94‐9027 is B‐domain deleted rFVIII with site‐specific pegylation to reduce the clearance of FVIII. BAY 94‐9027 has a half‐life of approximately 19h in a phase I study, and is currently being investigated inadult and pediatric patients with Hemophilia A. The potency of BAY 94‐9027 in the final container is determined by chromogenic substrate assay (CSA) employing a product standard which is calibrated against the WHO 8th IS. BAY 94‐9027, sucrose formulated r‐FVIII, and plasma‐derived WHO 8th IS perform identically (superimposable calibration curves) in the CSA, indicating that this method is the most accurate for BAY 94‐9027 assay. Similar FVIII activity for BAY 949027 is attained using a One‐Stage aPTT assay with ellagic acid activators and with some silica activators, e.g. HemosIL SythaSil kit (IL), Pathromtin kit (Siemens). These results indicate that OS assays using ellagic acid activators and selected silica activators are acceptable alternatives to CSA. Both, CSA and OS have been validated to quantify BAY 94‐9027 in human plasma samples. The CSA was calibrated using recombinant BAY 94‐9027 release standard diluted in a FVIII deficient human plasma pool. A low (1.25 – 50 IU/dL) and a normal level (3 – 200 IU/dL) working range have been established. Accuracy and precision of the low level method ranged between 92.8 to 98.2% and 14.6 to 20.7 %, respectively, whereas for the normal level method, the values were 101 to 108% and 6.01 to 12.0%, respectively. An ellagic acid based OS method was validated in the range of 1.5 to 80 IU/dL using Siemens Behring Standard Human Plasma diluted with FVIII deficient human plasma pool as calibrator. Accuracy and precision ranged between 131 to 136% and 3.38 to 6.90%, respectively. Results from ongoing work with the OS method based on selected silica activators will be presented.Conclusion: Bay 94‐9027 can be best assayed by the chromogenic method. For the OS method, both ellagic acid and some silica‐based activators give comparable results to the chromogenic assay and qualify as well.     Assays for Therapeutic Monitoring of ELOCTATE™ and ALPROLIX™. Jurg Sommer (Biogen Idec) ELOCTATE, a recombinant B-domain deleted factor VIII Fc fusion product (rFVIIIFc) has comparable specific activity to native FVIII on a molar basis and no reagent or instrument dependent discrepancies were identified in the one-stage clotting assay.  A blinded field study conducted in 30 clinical hemostasis laboratories furthermore showed that ELOCTATE could be measured as accurately as Advate® in spiked plasma samples by a variety of one-stage clotting assays calibrated against common FVIII plasma standards.  The chromogenic FVIII activity of ELOCTATE measured in 11 of these laboratories using 5 different commercial kits was on average just 10 to 20% higher than the one-stage activity. The hemostatic activity of ELOCTATE was shown to be comparable to that of Advate in subjects that were evaluated by thrombin generation and whole blood clotting assays during the phase 3 clinical study.  Combined with the clinical efficacy results, these studies indicate that any of the existing one-stage or chromogenic assay methods may be used for monitoring rFVIIIFc activity in patients (without the need for a product specific standard) and, as is the case with currently marketed FVIII products, the activity determined by these methods is a suitable surrogate marker for in vivo efficacy of ELOCTATE.  In a phase 3 clinical trial, a prophylactic regimen of ALPROLIX™ designed to achieve trough levels of 1 to 3% FIX activity in individual subjects resulted in annualized bleeding rates (ABRs) that were consistent with the expected bleeding rates based on current FIX products. Global hemostasis assays performed during the clinical study also showed that ALPROLIX, while longer-acting, provided an ex vivo hemostatic activity per unit of plasma FIX activity that was comparable to the comparator rFIX product (BeneFIX®).  Meanwhile, a field study involving 30 clinical hemostasis laboratories demonstrated significant assay variability for both ALPROLIX and BeneFIX, though ALPROLIX had somewhat higher aPTT reagent dependent variability than BeneFIX. Despite the observed assay discrepancies, 80% of laboratories were able to measure a 0.8 IU/mL ALPROLIX sample within ± 30% accuracy. Laboratories using a kaolin based aPTT reagent were a notable exception, since they underestimated ALPROLIX activity by as much as 50%.  Overall, the majority of laboratories were able to measure ALPROLIX with reasonable accuracy using their existing one stage clotting assays and without using a product specific standard.  ALPROLIX activity by the one-stage clotting assay will thus be largely representative of its in vivo hemostatic activity. To address potential concerns about the accuracy of locally performed clotting assays, Biogen Idec has established a designated ALPROLIX and ELOCTATE Reference Laboratory and provides a rFIXFc Laboratory Sample Kit for research use.    rVIII-SingleChain and rIX-FP. Debra Bensen-Kennedy (CSL Behring) rVIII-SingleChain is a single-chain recombinant factor VIII molecule being developed by CSL Behring (CSLB) for prophylaxis, prevention, treatment of bleeding episodes and surgical prophylaxis in patients with hemophilia A.  Unlike other B domain deleted recombinant FVIII molecules, rVIII-SingleChain is a single polypeptide chain, B domain truncated recombinant factor VIII.  Since 1994, the chromogenic substrate (CS) assay has been the reference method of the European Pharmacopoeia for the assignment of factor VIII concentrate potency [Mikaelsson 2001].  CSLB uses the CS assay for potency assignment and regards the method to be of greatest value due to the evidence from other modified FVIII products that alterations of the B domain of FVIII can influence one-stage  (OS )assay results.  For rVIII-SingleChain, both the OS and the CS FVIII assays deliver parallel and linear results against the WHO International Reference Standard in terms of assay performance. As expected and in line with other recombinant and B domain deleted FVIII products, rVIII-SingleChain potency assignment results in a discrepancy between the two assay formats. In order to ensure the appropriateness of the method used to assign potency, a tiered approach was taken to determine which of the FVIII activity assays represent the true in vivo functionality of rVIII-SingleChain. CSLB’s approach was in line with recommendations from FVIII and FIX Subcommittee of the SSC of the ISTH [Hubbard 2013]. CSLB has demonstrated that results obtained in pre-clinical, PK and clinical studies utilizing the CS assay method are reflective of true potency. With respect to clinical monitoring, CSLB acknowledges that the OS assay remains the preferred method. For this reason, the development program was designed to test FVIII:C with both assay methods. Results obtained in clinical PK data were linear across the full range of values and consistent with what was observed in pre-clinical studies. The linear relationship between the results of CS and OS assays offers the ability to effectively guide clinicians utilizing OS methodology to interpret FVIII:C results and remain in alignment with CS testing results and potency assignment. This will lead to a simplified approach to calculate and target critical activity levels when treating patients. rIX-FP, a recombinant fusion protein linking coagulation factor IX with albumin, is being developed for prophylaxis, prevention, treatment of bleeding episodes and surgical prophylaxis in patients with hemophilia B. rIX-FP is a recombinant protein generated by the genetic fusion of the two cDNAs of human albumin to human coagulation factor IX to prolong the terminal half-life. Potency is assigned utilizing the OS clotting assay, in line with all other currently available FIX products. CSLB regards the OS clotting assay to be of greatest value for potency assignment and has demonstrated that results obtained in pre-clinical, PK and clinical studies support utilizing the OS method.  Clinical trial data utilizing paired patient samples measuring FIX:C in both local and central laboratories will also support recommendations for clinical monitoring.    Factor VIII and FIX long acting products: which assay should be used to evaluate their efficacy?  Mirella Ezban (NovoNordisk) The introductions of new longer half-life products for replacement therapy have the potential to improve the clinical outcome and reduce the burden of disease in hemophilia The one stage clot assay is primarily used for post-infusion monitoring. The assay is a biologic assay with inherent variation. Variation with existing factor products is considerable1. This existing variability and complexity is being increased with the new longer half-life products 2-4. N8-GP and N9-GP are GlycoPEGylated  recombinant FVIII and FIX molecules in late stage clinical development. The PEG moiety interferes with some aPTT reagents used in the one-stage clot assay.1,4 Careful assessment of the accuracy and precision of the assays for potency labelling and therapeutic monitoring is therefore necessary to support the evaluation of the efficacy and safety of these new long acting factor products. Potency assignment for both N8-GP and N9-GP follow the SSC recommendations 5 and the European Pharmacopeia (Ph.Eur) and the two molecules are calibrated against the respective WHO standards. The process for selecting a valid and accurate potency assay for each of the molecules will be described. N9-GP potency is assigned with the one stage clot assay using the aPTT reagent SynthAFax; the validity of which is supported by results obtained by other biological assays showing minimal PEG inference such as chromogenic assays and thrombin generation assays. A similar approach has been followed for N8-GP, however, a chromogenic assay has been used for potency assignment according to the Ph. Eur. The extent to which GlycoPEGylation interferes with post infusion monitoring has been analyzed in selected expert laboratories using spiked hemophilia plasma samples. For N8-GP acceptable values were obtained with chromogenic assays and with the most commonly used aPTT reagents. For N9-GP chromogenic assays gave accurate results but only a limited number of aPTT reagents could be used The clear question to be addressed is how to assure patient safety and to minimize complexity in laboratory work flow while providing patients the choice of treatment which may reduce the burden of their treatments and improve quality of life. . Expansion of the use of chromogenic assays may be the common choice for factor activity monitoring of new long-acting factor products. References:Viuff et al Haemophilia 2011; 17: 695-702 Barrowcliffe TW et al:Semin Thromb Hemost 2002; 28:247–56. Gu et al : Haemophilia 2014 [Epub ahead of print]doi: 10.1111/hae.12374 Sommer JM et al:ISTH, 2013, Amsterdam ; NL, Poster PB3.49-2 Holm PK, et al. ISTH 2013, Amsterdam, NL. Poster PB 3.49-1 Hubbard AR et al.,Journal of Thrombosis and Haemostasis, 11: 988–989 2013       Report on the 2013 EMA-EDQM Workshop on "Characterization of new clotting factor concentrates (FVIII, FIX) with respect to potency assays used for labelling and testing of post infusion samples”. Anneliese Hilger (EMA) The Workshop was held on 28-29 November 2013 at the European Medicines Agency (EMA) and organised jointly by EMA (with Biologics Working Party and Blood Products Working Party support) and the European Directorate for the Quality of Medicines and HealthCare (EDQM). The workshop discussed potency assays used for product labelling and testing of patient’s post-infusion samples for new clotting factor VIII and IX concentrates. A number of new recombinant products are in the late stages of development and it was felt that a more harmonised approach to assigning potency to these clotting factor concentrates was required. The objectives of the workshop were to provide a point of reference when making future decisions for licensing or changes to the relevant European Pharmacopoeia texts. Manufacturers of new products are undertaking a thorough characterization of the performance of new products in different assay systems as recommended by ISTH. For all products in development, valid assays versus the international concentrate standard were obtained and potency was expressed in IU. The decision tree defined in the ISTH recommendations is considered useful in many cases. However, for some products, valid assays could be obtained with both chromogenic and one-stage clotting assays but these different assays give very different potency values. Furthermore, for a number of the products, valid one-stage clotting assays give very different potency values depending on the APTT reagent used, especially for some long-acting products. Therefore, correlation with biological activity from in vitro, non-clinical and clinical efficacy studies is important in selecting the appropriate assay. Precise methods are needed for potency labelling of products (80-120%), whereas for clinical diagnosis sensitivity is important e.g. down to <1% and for clinical monitoring it is needed to cover dosing and trough levels. Clinicians wish to have one assay to be used to assign product potency and which correlates with patient factor levels. Different assays could lead to discrepant product plasma values measured post-infusion. A thorough characterization of Factor VIII and FIX products according to ISTH recommendation needs to be performed to develop the most appropriate assay for potency labelling. In addition, adequate measures to avoid any missdosing or potential problems regarding monitoring of patient plasma samples need to be proposed by the manufacturer.    FDA’s Current Considerations on Assays for Potency Determination of Long Acting Clotting Factor Concentrates. Mark Weinstein (FDA) There are multiple issues to consider when evaluating the potency of long acting clotting factor products. These include the fact that the one stage clotting assay (OC) along with a plasma reference standard are used by most laboratories worldwide to monitor FVIII/FIX activity,  whereas the labeled product potency may be determined using a chromogenic assay (CA ).  The validity of a particular method of potency assignment is based on its correlation with efficacy in clinical trials, and no one set of reagents or standards has been suitable to measure potency of all products. Recent studies have shown that activity results for a given product can vary greatly depending on the reagents and reference standards used, particularly for FIX products, and more so for the OC than for the CA. Manufacturers need reference standards and validated assays to ensure product potency, consistency of manufacturing and the stability of products over time.  Regulatory agencies have not reached consensus on the best approaches to potency determinations.  Given these considerations, US licensed manufacturers have followed a paradigm of assaying new FVIII and FIX products using both OC and CA tests (as appropriate for the product) to calibrate an internal reference standard against an International Concentrate Standard; assigning potency to a new product against the internal standard; determining levels of the product in plasma samples against the internal reference standard; and determining levels of the product in plasma samples against a plasma standard. However, for some products, an OC may simply not be possible, raising issues for product labeling.  With the diversity of new products and the potential of obtaining differing potency results depending on choice of assay and assay conditions, additional effort is needed to optimize potency labeling for end users. In situations where disparities are expected to exist between activity levels determined in clinical labs (generally using OC assays on plasma) and labeled product potencies (determined by a non-equivalent method), the user community needs to be educated how best to interpret the assay results.  Typically, such information would be conveyed by the manufacturer.         DISCUSSION In this session there was an important and fruitful discussion regarding which assay should be used for the longer acting product post infusion laboratory testing among scientists, physicians, manufacturers and regulatories (FDA and EMEA). All bodies declared to be knowledgeable regarding the assay issues for the longer acting products. It was clear from all presentations that chromogenic assay could be a more suitable assay for PEGylated products, particularly for B domain deleted ones. Both regulatories seem to be collaborative and "flexible” for any additional assays (including chromogenic assay if there would be evidence regarding its advantage to the one-stage clotting assay).       BUSINESS SESSION, SECOND PARTJune, 26th 2013     TOP-RATED ABSTRACT In vivo selection of genetically manipulated platelets corrects murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection for transduction. Qizhen Shi Objective: Our previous studies have demonstrated that lentivirus-mediated platelet-specific (2bF8) gene therapy can restore hemostasis in hemophilia A (HA) mice with or without inhibitors. In this study, we aimed to enhance platelet-FVIII (Plt-F8) expression while minimizing potential toxicities. Methods: A novel lentiviral vector (LV), which harbors dual genes, the 2bF8 gene and a drug-resistance gene, the MGMTP140K cassette, was constructed. Plt-F8 expression in HA mice was introduced by bone marrow (BM) transduction and syngeneic transplantation. After BM reconstitution, the recipients were treated with BG/BCNU monthly for 3 or 4 times. Animals were analyzed by PCR, qPCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and ROTEM analysis. Results: When an MOI (multiplicity of infection) of 1 was used for transduction, Plt-F8 expression in recipients was only 0.22±0.15 mU/108 platelets before the drug treatment, but remarkably increased to 4.33 & plusmn;5.48 mU/108 platelets (n=16) after BG/BCNU treatments, which is 2.89-fold higher than the data obtained from our regular 2bF8LV with an MOI of 10. 2bF8 proviral DNA was barely detectable (0.01±0.02 copies/cell) before chemoselection, but it increased to 0.42 & plusmn;0.15 copies/cell after BG/BCNU treatments. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. When an MOI of 10 was used, Plt-F8 expression was enhanced from 1.63±0.36 mU/108 platelets to 14.18±5.39 mU/108 platelets after 3 BG/BCNU treatments. Notably, no anti-FVIII antibodies were detected in the treated animals even after exogenous rhFVIII challenge. Conclusion: We have established a powerful in vivo selective system that allows us to enhance the therapeutic efficacy of 2F8 gene therapy and induce immune tolerance in hemophilia A mice.   DISCUSSION was on how this could help in clinical trials.      CLINICAL AND LABORATORY EVALUATION Session Chairpersons: Steven Pipe (USA) and Kathelijn Fischer (The Netherlands) In patients with hemophilia, the residual levels of clotting factor (FVIII or FIX) are considered as the most important prognostic factors. Based on that, hemophilia patients are classified into the severe, moderate and mild categories, despite several observations suggesting that this may be actually insufficient. For instance, about 10-20% of patients classified as having severe hemophilia A do not have major bleeding complications. On the other hand, the classification of patients with mild hemophilia is not entirely clear. Classification is based on laboratory testing, but 30% of patients with mild hemophilia exhibit discrepant FVIII:C levels depending on whether they are measured by the one- stage or chromogenic assay. In addition, patients with FVIII/IX levels >40% are not classified as having hemophilia, but they may bleed.    Report on the SSC project: Bleeding score in hemophilia: a prognostic tool for clinical outcome. Maria Elisa Mancuso (Italy) Despite the lack of knowledge, on the relation between bleeding symptoms and FVIII levels, treatment strategies are often implemented, sometimes pre-emptively, only on the basis of residual factor level (ie., prophylaxis vs. on demand therapies). Appreciation of the true prognostic value of residual factor level is therefore a major, clinically relevant, task to be undertaken by the SSC-ISTH Working Group on Clinical outcome evaluation. With this as background the Working Group (WG), together with 18 physicians expert in the field established, has initiated a 4 rounds Delphi method, to establish consensus-based clinical criteria to define the degree of severity of hemophilia independently from residual FVIII/FIX levels. The group came up with 4 major and 1 minor criteria and established that the presence of 2 major criteria was the minimum requisite to define a severe disease. A specific score was assigned to each criterion. The next step for the Working Group will be to propose a validation study of the predictive value of residual FVIII/FIX activity measured at diagnosis as compared with the definition of the severity of the disease done by using the aforementioned criteria.    Report on the SSC project: The definition of mild hemophilia A. Michael Makris (UK) The WG is examining the possibility of allowing the diagnosis with a higher FVIII:C level in the presence of a family history or pathogenic mutation. It is also investigating the possibility of raising the 0.40iu/ml for all, if there is sufficient published evidence for bleeding above this level. A further important issue is that the ISTH definition does not specify the type of FVIII:C assay to be used in the definition of hemophilia. The WG will propose that both 1-stage and chromogenic assays are performed on all mild hemophilic patients at diagnosis and that the ratios are reported as 1-stage over chromogenic. Ratios of <0.5 or >2.0 indicate significant discrepancy. The third area being addressed by the WG is the issue of inhibitors in patients with mild hemophilia A. The WG feels that all mild hemophilic patients should have their genetic defect identified as this can aid in the management if a high inhibitor risk mutation is present. All mild hemophilic patients exposed to FVIII concentrate should have a FVIII inhibitor level measured with the Bethesda assay six weeks after exposure.     DISCUSSION In the follow-up of the bleeding score project the Project group proposed to do a validation study to evaluate the score. The proposal is to include a minimum of 20 randomly selected patients born 1990-2010, irrespective of hemophilia type or severity. The outcome of the score would be correlated to FVIIII/IX levels. It was discussed that especially the moderate patients would be an interesting group, but this group may require central laboratory evaluation of the baseline FVIII/IX level. Several  participants discussed selection bias, as patients with a more severe phenotype would be started on prophylaxis sooner than those with a milder phenotype. Regarding the patients with FVIII/IX levels >= 40%, it was pointed out that we have no tools to distinguish bleeders from non-bleeders. The suggestion was to actively look for other laboratory abnormalities that may explain bleeding in these patients. The committee feels that genetic analysis is also important.       RECOMMENDATIONS ON HEMOPHILIA TREATMENT Session Chairpersons: Elena Santagostino (Italy) and Alok Srivastava (India) Dr Srivastava had apologized for not being present due to personal reasons.     Report on the SSC project: Consensus definitions and recommendations for immune tolerance induction (ITI) in hemophilia with inhibitors. Elena Santagostino (Italy) Consensus definitions of clinical and laboratory endpoints in the different subsets of patients undergoing ITI are required because these would allow more reliable data analyses and comparisons between studies, furthermore, prompting the initiation of international prospective surveys able to collect more homogeneous data during a prolonged follow-up period. The aim of this project is to provide definitions of ITI outcome standardizing the methodologies for the assessment of treatment endpoints. An additional aim is to establish harmonized strategies for ITI therapy in inhibitor patients belonging to different and well defined prognostic groups. Based on a literature review, several issues have been identified for discussion and deliberations within the working group. Critical issues include: ITI outcome defined on the basis of strict pharmacokinetic criteria not always reflect the clinical benefit obtained with ITI;the need for repeated pharmacokinetic assessment and inhibitor testing after a wash-out period makes these procedures very demanding;inhibitor testing by the modified Nijmegen-Bethesda assay is relatively insensitive to very low titers;FVIII pharmacokinetic measurements in the presence of low-titer inhibitors is a challenge with the available methodology.the definitions of good- and bad-risk patients based on historical and pre-ITI titers are more easily applied to young patients because this information may not be available in older patients.very heterogeneous patients are grouped together under the "umbrella” of bad-risk patients. The current presentation will briefly discuss these issues.    DISCUSSION There were many positive reactions to the proposals made by the Project Group. In addition, the group was asked to address the issue of when to discontinue ITI as well as consider the bleeding phenotype during ITI in some of the definitions (e.g. for partial response). Guy Young highlighted the importance of achieving an agreement on a certain ITI duration (many treatment courses have been prolonged for years without any clear benefit for the patient). Elena Santagostino commented that this is one of the issue to be defined, however, different treatment durations may be needed for different prognostic groups of patients. Victor Blanchette highlighted the need for standardized procedures for PK testing; his question was on the potential use of population-based PK models. Elena Santagostino answered that these models are not applicable to the heterogeneous group of patients with inhibitors. Massimo Morfini raised the issue of the lower limit of normal FVIII half-life, to date fixed at 6 hours: this is too short in his opinion. He also disagreed on having a measurable through level of FVIII as a criteria for tolerance; in his opinion other PK parameters such as FVIII clearance should be considered.  Alfonso Iorio highlighted the clinical relevance of bleeding during ITI; this should be included as a criteria to guide treatment choice.    Report on the SSC project: Prophylaxis in hemophilic patients without inhibitor. Victor Blanchette (Canada) on behalf of Peter Collins ,Kathelijn Fischer , Margareth Ozelo , Alok Srivastava  and Guy Young. The mandate of the Project Group is to prepare an evidence-based Report of long-term prophylaxis in persons with hemophilia A and B who are inhibitor negative . Topics to be considered are: 1) when and how to start prophylaxis; 2) strategies for adjusting prophylaxis regimens in different patient cohorts e.g. children , adolescents and adults; 3) outcome measures relevant to long-term prophylaxis aimed to prevent/delay progression of bleed-related arthropathy; 4) potential impact of prophylaxis on adverse outcomes other than joint damage e.g. intracranial hemorrhage , inhibitor formation; 5) adherence to prophylaxis and factors that influence adherence ; and 6) potential impact of long-acting factor VIII/IX concentrates on prophylaxis regimens in the future.  A formal literature search has been initiated that will start from the point of the Cochrane Review " Clotting factor concentrates given to prevent bleeding and bleeding related complications in people with hemophilia A and B ” [Iorio A et al. The Cochrane Library , Issue 9 , 2011]. An exact search strategy to that used in the Cochrane Review will be used to identify all additional relevant literature on prophylaxis for person with hemophilia from 2010 to the current time. Members of the Project Group will review all relevant material identified by the Cochrane search and the additional search to extract the information on the various aspects of prophylaxis. External experts, including health care providers, Agencies involved with decisions refunding of clotting factor concentrates and patient groups will be invited to provide commentary on the draft Report from the Project Group before it is finalized.     DISCUSSION As the mandate of the SSC was to provide clinical guidance on various aspects of prophylaxis, a formal search according to the rules of evidence based medicine generation may have to be reported separately. It was discussed that the Project Group should avoid duplication the work of the Cochrane collaboration. The question when and how to initiate prophylaxis in young boys with severe hemophilia was considered as the clinically most important question . Kathelijn  Fischer asked the audience to make proposals for research questions. Flora Peyvandi clarified that this is not the aim of SSC, she prompted the group to work on definitions as initially agreed upon.      BY-PASSING AGENTS Session Chairpersons: Midori Shima (Japan) and Yesim Dargaud (France)     FVIII / FIX SSC session on by-passing agents comprised five presentation on the two major topics i.e. development of new treatment strategies in patients with inhibitors and monitoring of by-passing agents. Several publications reported the potential interest of the global haemostasis assays including thrombin generation assay, thromboelastography and clot waveform analysis for the monitoring of by-passing therapy. Standardization of the assays is ongoing; recommendations were already published on the use of thromboelastography and clot wave form analysis.  A manuscript will be submitted to the J Thromb Haemost on standardization of thrombin generation measurement for hemophilia. Lyon Hemophilia Center plans to organize a multicenter clinical study in 2015, evaluating thrombin generation assay for individualizing by-passing therapy in patients needing elective surgeries. Potential investigator centers will be invited to participate.  A recent study organized at the Hemophilia Center of Milan reported that under the conditions used in this study, TGA was not able to predict either the hemostatic response to different by-passing agents used at different doses nor the risk of bleeding complications. With regard to clot wave form analysis, it has been recently shown that using a new reagent combining ellagic acid and tissue factor might be of interest for the monitoring of by-passing agents. Thrombin generation assay and clot wave form analyses were also suggested as potential monitoring methods for ACE910 which is a recombinant humanized anti-factor IXa  factor X bispecific monoclonal antibody mimicing a cofactor function of factor VIIIa. Differently from FVIII, ACE910 does not require the activation process. In addition, there is a substantial difference in the inactivation, in the binding affinity to FIX(a) and in the capability of binding to phospholipid. Therefore, global haemostasis assays that are not directly influenced by the difference of the activation process would be of interest for the monitoring of this new therapeutical approach. The use of RNA interference (RNAi) to target the natural anticoagulant antithrombin as a strategy to rebalance the hemostatic system and improve thrombin generation, and therefore hemostasis, in hemophilia was reported as a new therapeutical approach for hemophilia with inhibitors. ALN-AT3, a subcutaneously administered RNAi therapeutic targeting AT, is currently being developed for the treatment of hemophilia. Promising results were obtained in hemophilia mice models and non-human primates, with no significant adverse events reported. The potential for subcutaneous route of administration, infrequent dosing, and applicability to persons with hemophilia who have inhibitors, support the continued development of ALN-AT3.     Global assays for the evaluation of the efficacy of by-passing agents. Yesim Dargaud (France) Haemostasis and its abnormalities have been traditionally assessed by plasma clotting times. While factor assays based on these tests have defined the different coagulation disorders and are useful for diagnosis, they also have several limitations. Currently, there are tests that can quantitatively assess the overall haemostastic potential of blood. The process of thrombin generation and fibrin clot formation can be captured with greater sensitivity and completeness by assays that measure global haemostasis. These include i)the thromboelastography, ii)the clot waveform analysis and iii) thrombin generation assays using different instrument systems. There are several publications in the field of thromboelastography and hereditary or acquired bleeding disorders. Case reports and small case series have addressed the usefulness of thromboelastography in the management of bleeding or monitoring of treatment with bypassing agents. Recently, recommendations of the SSC working group on the standardization of  thromboelastography measurements in hemophilia were published. The utility of clot waveform analysis to define qualitative and quantitative differences at levels of FVIII:C less than 0.01 IU/mL has been previously reported, raising the possibility that the correlation observed, between the laboratory definition of severity and the clinical phenotype, could be improved by this approach. A recent publication suggested the interest of the assay for monitoring by-passing therapy. Recommendations of the SSC working party were recently published with regard to the standardization issues of the assay. Several groups reported a relatively good correlation between thrombin generation assays and the bleeding tendency of patients with hemophilia but has not been used to classify the severity of the disease so far. One of the greatest impact of thrombin generation assays in the field of hemophilia has been in the monitoring of by-passing agents. The ex vivo monitoring that would reflect achievement of haemostasis in vivo still needs further studies, though several attempts have already been initiated. In this respect, the thrombin generation assay might be used to predict the differential response to recombinant FVIIa and Feiba tested prior to in vivo administration, and might provide further insight into the optimal dose of therapy pre- and postoperatively. If one considers that there probably is a level of thrombin generated that predicts clinical efficacy, this assay could be used for monitoring of bypassing agents and for optimizing the infusion schedule by tailoring doses and frequency of injections individually for each single patient. However, correlation of the TGA parameters with in vivo clinical response needs to be further established if we believe that this assay may represent a surrogate marker for monitoring bypassing therapies. In order to establish the usefulness of this test in multicenter clinical studies, a standardized methodology needs to be developed. This is being carried out through a working group established by the SSC. The manuscript is currently in revision by the members of the working group and will be submitted for publication in July 2014. A multicenter clinical study evaluating TGA for the monitoring of by-passing therapy coordinated by Lyon Hemophilia Center will be organized in 2015.    Thrombin generation in Hemophilia treated-patients. Maria Elisa Mancuso (Italy) Background and aims: FVIII activity measurement is the mainstay of laboratory monitoring during surgery in patients with hemophilia A (HA) without inhibitors. No routine coagulation monitoring is available for hemophilic patients with inhibitors treated with by-passing agents. Global coagulation tests as thrombin generation assay have been considered has potential candidate to to predict hemostatic response in those with inhibitors treated with by-passing agents. The aim of this study was to evaluate if TGA is able to predict the hemostatic and clinical response to by-passing agents in patients with HA and inhibitors. Materials and Methods: TGA was assessed in platelet-rich (PRP) and platelet-poor (PPP) plasma with the addition of corn trypsin inhibitor (CTI) in 16 patients with severe HA and high-responding inhibitors aged 5-59 yrs (median: 39). Blood samples were collected in 2 different settings: in a non-bleeding state after a single infusion of one by-passing agent (either aPCC or rFVIIa) in order to assess a kind of PK after by-passing agent administration and during major orthopaedic surgery. Thirteen patients underwent the assessment in a non-bleeding state and 10 during surgery, being 7 those patients in whom TGA was measured in both conditions. PK assessment was performed after a standard 90-120 mcg/kg dose of rFVIIa, a high 270 mcg/kg dose of rFVIIa and/or a 80 IU/kg dose of aPCC. Blood samples were drawn at baseline, 30 minutes and 3 (if rFVIIa) or 6 (if aPCC) hours. During surgery TGA was assessed once daily prior and 30 minutes after by-passing agents injection starting from the pre-operative bolus and for at least 4 consecutive post-operative days. Haemostatic treatment to cover surgical procedures was established irrespective of TGA measurements. Results: In the non-bleeding state assessment TGA increased after each by-passing agents injection however it was not able to discriminate any difference between the two drugs and between the different doses of rFVIIa. Similarly, in the surgical setting TGA did not reveal different responses related to the type of drug, the dose used and/or the occurrence of bleeding complications (n=5). Moreover, a lack of response of the TGA curve was observed during the post-operative period irrespective of treatment regimen modifications in all patients. Conclusions: our results indicate that TGA is not a suitable tool to monitor hemostatic response during surgery in patients with hemophilia and inhibitors treated with by-passing agents. In fact, TGA was not able to predict either the hemostatic response to different by-passing agents used at different doses nor the risk of bleeding complications.     DISCUSSION In this session, there were two groups presenting opposite results on the use of thrombin generation assay. This clearly indicates that the use of thrombin generation assay requires standardization. To this regard Dr. Dargaud started discussion on the importance of pre-analytical conditions for global haemostasis assays. Questions on blood sampling, plasma preparation and the use of CTI were addressed. The educational DVD showing the key pre-analytical steps, generated by Lyon Hemophilia Center is available for clinical studies in the frame of the standardization of TGA.    Application of clot waveform analysis to hemostatic monitoring of bypassing therapy. Keiji Nogami (Japan) Background: Assays to determine the optimal hemostatic effects of bypassing therapy in hemophilia A (HA) patients with inhibitors are difficult to compare.  Clot waveform analysis (CWA), based on the continuous monitoring of routine coagulation parameters (PT/aPTT), offers a useful method for assessing global clotting function.  Objective: We investigated the technique of CWA for the hemostatic monitoring of bypassing therapy in HA patients with inhibitors.  Methods and Results: Ellagic acid (Elg), tissue factor (TF), or both (Elg/TF) were used as trigger reagents in CWA.  The standard parameters; clot time (CT), maximum coagulation velocity (|min1|), and acceleration (|min2|) were recorded.  An optimal monitoring was defined as (i) a significant difference in these parameters between plasmas from HA patients with inhibitors and normal plasmas, and (ii) a significant improvement in these indices in HA patients with inhibitors after bypassing therapy.  Experiments in vitro demonstrated that there were significant differences between plasmas from HA patients with inhibitors and normal plasmas with various triggers, in the order Elg > Elg/TF >> TF.  Addition of therapeutically achievable concentrations of bypassing agents, however, showed significant improvements in the different parameters only with Elg/TF, suggesting that this reagent provided the most appropriate assay.  A total of 20 plasmas from HA patients with inhibitors in which bypassing agents were infused were evaluated ex-vivo by Elg/TF-CWA.  The postinfusion parameters CT and |min2| reflected clinical effects, and were close to normal levels.  Furthermore, Elg/TF-CWA facilitated quantitative evaluation of perioperative hemostatic management of bypassing therapy in HA patients with inhibitors.  Conclusions: CWA is a promising method for the quantitative monitoring of bypassing therapy during routine automated clotting assays with a modified trigger reagent comprising a well-balanced mixture of Elg and TF.      Monitoring the hemostatic efficacy and safety of new therapeutic drugs (what assays should be used?) Restoring hemostatic balance in hemophilia by RNAi targeted silencing of antithrombin. Benny Sorensen (Alnylam) Introduction: Hemophilia A or B are congenital bleeding disorders caused by dysfunctional propagation of thrombin generation due to deficiency in factors VIII or IX in the presence of normal levels of anticoagulants resulting in an imbalance of the hemostatic system toward a bleeding phenotype. We are currently investigating the use of RNA interference (RNAi) to target the natural anticoagulant antithrombin (AT) as a strategy to rebalance the hemostatic system and improve thrombin generation, and therefore hemostasis, in hemophilia. ALN-AT3, a subcutaneously administered RNAi therapeutic targeting AT, is currently being developed for the treatment of hemophilia. Material and methods: Preclinical studies in hemophilia mouse models have investigated the ability of ALN-AT3 to silence AT and thereby correct thrombin generation as measured by Calibrated Automated Thrombin (CAT) generation assay, restore hemostatic plug formation in real-time laser injury clot formation visualization, and control traumatic bleeding in a saphenous vein bleeding model. Preclinical studies were also conducted in vehicle and ALN-AT3 treated non-human primates followed by infusion of high-dose anti-Factor VIII antibody to induce inhibitor hemophilia A and measurement of thrombin generation. The association between AT reduction and factor VIII and IX equivalence was assessed by in vitro thrombin generation studies using human hemophilia A and B plasma samples. Finally, a phase 1 clinical study in healthy volunteers and severe/moderate hemophilia A or B patients has been initiated. Part A in healthy volunteers has been completed and Part B in hemophilia patients is ongoing. Results: ALN-AT3 treatment targeting residual AT levels of 20-40% in hemophilia A and B mouse models increased thrombin generation, restored real-time localized hemostatic plug formation in the laser-injury model comparable to treatment with full-length recombinant factor VIII. ALN-AT3 controlled traumatic bleeding in the saphenous vein model with an increase in number of hemostatic events equivalent to that achieved with infusion of 25IU/kg full-length recombinant factor VIII. ALN-AT3 treatment targeting 20% residual AT levels normalized thrombin generation in non-human primates with induced high titer inhibitor hemophilia A. In vitro titration studies with factor VIII or IX in human hemophilia A or B plasma as well as decreasing AT showed that targeting residual AT levels of 40-60% is equivalent to factor VIII or IX trough levels ranging from 10-15%. In Part A of the Phase 1 study, healthy volunteer subjects received a single subcutaneous dose of ALN-AT3 and, per protocol, the maximum allowable level of AT knockdown was set at 40%. Initial results show that a single, low subcutaneous dose of ALN-AT3 at 0.03 mg/kg resulted in an up to 28-32% knockdown of AT at nadir that was statistically significant relative to placebo (p < 0.01 by ANOVA). This led to a statistically significant (p < 0.01) increase in peak thrombin generation, that was temporally associated and consistent with the degree of AT knockdown. ALN-AT3 was found to be well tolerated with no significant adverse events reported. Conclusion: Collectively, these data suggest that the use of a novel RNAi therapeutic targeting AT is a promising approach for restoring hemostatic balance in hemophilia, and potentially, other bleeding disorders. Further, the potential for subcutaneous route of administration, infrequent dosing, and applicability to persons with hemophilia who have inhibitors, support the continued development of ALN-AT3.    Monitoring the hemostatic efficacy and safety of new therapeutic drugs (what assays should be used?) ACE910, anti-FIXa/X bispecific antibody. Takehisa Kitazawa (Chugai Pharmaceutical Co, Japan) ACE910 is a recombinant humanized anti-factor IXa (FIXa)  factor X (FX) bispecific monoclonal antibody that places these two factors into spatially appropriate positions and mimics a cofactor function of factor VIIIa (FVIIIa). We previously demonstrated that ACE910 had an enough hemostatic activity even against on-going bleeds and completely prevented spontaneous joint bleeds in non-human primate models of acquired hemophilia A. In the phase I clinical study, ACE910 exhibited a long half-life of approximately 1 month and a good bioavailability with subcutaneous dosing in healthy volunteer subjects. The cofactor activity of ACE910 in plasma samples is principally measurable by the assays ever used for measuring factor VIII (FVIII) activity and/or effect, because ACE910 is a FVIIIa-mimetic. (Due to the high species-specificity of ACE910, the assays must include both human-origin factor IX (FIX)/FIXa and human-origin FX.) In terms of comparing ACE910 activity with FVIII activity, however, each assay demonstrates a different correlation between them. For example, ACE910 at 300 nM exhibited peak height of thrombin generation equivalent to 10 U/dL of FVIII did in vitro, while it shortened APTT beyond 100 U/dL of FVIII did. We consider that such inconsistency should attribute to the different properties between two molecules. Differently from FVIII, ACE910 does not require the activation process. In addition, there is a substantial difference in the inactivation, in the binding affinity to FIX(a) and in the capability of binding to phospholipid. We are now on the way to elucidating how such differences affect ACE910’s clinical hemostatic efficacy and thus on the way to identifying the most appropriate assay and/or assay condition for ACE910 in terms of the FVIII-relative hemostatic activity. Although I recognize that the data are not necessarily sufficient at the present, for starters I’d like to discuss here the possibility of the clot waveform analysis and thrombin generation assay that are not directly influenced by the difference of the activation process, referring to in vivo animal hemostatic data.    DISCUSSION The rationale for the use of a mixture of ellagic acid and tissue factor as the optimal reagent for the monitoring of by-passing therapy by CWA was discussed. For the monitoring of ACE910, FVIII activity assays or global haemostasis assays might be the more adapted laboratory  tools, investigations are currently ongoing in Dr Shima laboratory.       RARE COAGULATION DISORDERSSession Chairpersons: Danijela Mikovic (Serbia) and Michael Makris (UK)    Rare coagulation disorders resource room. Amy Shapiro (USA) Dr Shapiro had apologized for not being present due to personal reasons and the presentation was made by Dr. Flora Peyvandi. Introduction/background: Very rare bleeding and clotting disorders exist worldwide, yet knowledge of these conditions and their management is often suboptimal. The majority of healthcare professionals have little experience treating these disorders, with limited resources for their diagnosis and treatment. Moreover, as rare coagulation disorders represent a small potential commercial market, few, if any, specific therapies exist for these conditions. As a result, affected individuals often face delayed diagnosis, insufficient laboratory evaluation, and limited treatment options. The Rare Coagulation Disorders Resource Room (www.rarecoagulationdisorders.org) was created in 2013 to address these healthcare gaps. This dynamic, open-access website was developed through a collaboration of the international RBDD Registry (http://www.rbdd.org/), the Indiana Hemophilia & Thrombosis Center (www.ihtc.org), and the Rare Coagulation Disorders Working Group of the National Hemophilia Foundation (NHF), a group appointed by NHF’s Medical and Scientific Advisory Council. Aim The Rare Coagulation Disorders Resource Room aims to provide first-line educational resources for both healthcare providers and individuals with very rare coagulation disorders. The website represents an important step in a global initiative to enhance ongoing research and registry efforts and foster collaboration among a growing international network of care providers. The ultimate goal of the website is to improve the health and quality of lives of individuals with these rare disorders through improved awareness, diagnosis, and increased knowledge of the clinical manifestations and sequelae of these disorders. Methods: The website content was written by leading experts in coagulation disorders and represents revised updates of initial articles published in a 2008 supplement of Haemophilia. The content has been formatted to provide current and searchable information including the basic science, clinical management, available laboratory and genetic testing, clinical trials, and global research initiatives for very rare and heterogeneous coagulation disorders. Results: The website currently hosts content on 12 rare bleeding disorders* with an additional disorder (FXIII deficiency) to be added in 2014. Since its launch in October 2013, the website has garnered almost 12,000 page views in approximately 3,000 sessions, with new visitors accounting for 76% of sessions. Visitors viewed an average of 4 pages per session over an average 3.5 minutes, with 86% of sessions from a desktop computer. The top 5 countries for website visitors were the United States (57%), the United Kingdom (5%), France (3.7%), Canada (3%), and India (3%). Most of the visitors (34%) reached the website through an organic search, while 33% came directly to the site, 31% through referral, and 2.6% through social media. Visitors who came through referrals stayed longer on the website compared to visitors through an organic search (4 minutes, 6 seconds vs. 3 minutes, 8 seconds) and viewed more pages per session (4.80 vs. 3.45). The top websites for direct referrals were hemophilia.org (41.80%), rbdd.org (20.40%), and isth.org (9.42%). The 5 most commonly viewed disorders were platelet function defects (17% of pageviews), prothrombin deficiency (11.5%), fibrinogen deficiencies (11.2%), PAI-1 deficiency (11%), and plasminogen deficiency (10.2%). The website has been supported through a variety of organizations such as links posted on EUHANET (Haemophilia Central) and on the ISTH website in the resource section; an announcement by the National Hemophilia Foundation in their eNotes on 11/5/13 and on their website, and through their newsletter HemAware in 2014.  The Hemophilia and Thrombosis Research Society sent an e-blast to their membership and posted a link on their website; OrphaNews Europe provided an announcement in their newsletter, and the World Federation of Haemophilia posted a short blurb on their RBD webpage and in an e-blast. Summary/Future plans: The Rare Coagulation Disorders Resource Room serves as a platform for expanded global education and fosters data collection and international collaboration on clinical trial design. The Resource Room also serves as a repository for information on registries and provides valuable education on rare bleeding disorders. In the future, the website will include content on clotting disorders and patient education materials. ________________________________________ *Prothrombin (Factor II) Deficiency, Alpha-2 Antiplasmin Deficiency, Combined FV and FVIII Deficiency, Factor V Deficiency, Factor VII Deficiency, Factor X Deficiency, Factor XI Deficiency, Plasminogen deficiency, Congenital Deficiency of Vitamin K Dependent Clotting Factors, Congenital Platelet Function Disorders, Plasminogen Activator Inhibitor-1 (PAI-1) Deficiency, and Rare Congenital Fibrinogen Deficiencies.     EAHAD mutation databases on Factor VII and Factor XI. D. Hampshire, C. A. Ludlam, G. Kemball-Cook, A. Cairo, K. Gomez, A. Goodeve, J. McVey, S. Perkins, F. Peyvandi and P. Rallapalli on behalf of European Association for Haemophilia and Allied Disorders. Dr Ludlam had apologized for not being present due to personal reasons and only a summary has been presented. Readily accessible databases of sequence variation in coagulation factor genes have, over the past 20 years, been an important source of information for clinicians and researchers in haemostasis, e.g. HAMSTeRS for factor VIII and the VWF database. Recently an initiative by the European Association for Haemophilia and Allied Disorders (EAHAD) has led to the updating of databases for factors VIII and IX along with the addition of much additional protein structural information and sequence data for many animal species. This, along with the well-established VWF database, is available through the EAHAD database portal (www.eahad-db.org). A strength of these databases is that they also include phenotypic information in relation to each reported mutation, e.g. history of inhibitors and bleeding severity. This EAHAD database initiative is now in the process of making widely available equivalent genetic and phenotypic variation data for factors VII and XI. To achieve this the Steering Committee is collaborating with the Rare Bleeding Disorders Database and other databases for these two clotting factors to bring together and update these data to make them readily accessible. It is anticipated that this part of the project will be completed before the end of 2014. When the update has been completed these data will also be available through the EAHAD database portal. Correspondence and enquiries about the EAHAD Coagulation Factor Variant Database project should be addressed to Christopher Ludlam (CAL@Ludlam.org.uk), Geoffrey Kemball-Cook (Geoffrey@kemball-cook.co.uk) or Dan Hampshire (D.Hampshire@Sheffield.ac.uk).    Report on the SSC project: Factor V deficiency, clinical heterogeneity and treatment. Danijela Mikovic (Serbia),  Roberta Palla, Marzia Menegatti, Flora Peyvandi (Italy) Factor V (FV) deficiency represents 8-10% of all rare bleeding disorders (RBDs). Severity of symptoms is variable and correlates poorly with laboratory phenotype.  Although the most common symptoms are prolonged bleeding after trauma and mucosal bleeding, major spontaneous bleeding like haematoma, hemarthrosis, gastrointestinal, central nervous system and umbilical cord bleeding episodes occur in a significant number of patients who need replacement therapy. Replacement therapy of FV can be administered only through fresh frozen plasma, preferably virus-inactivated, since FV concentrates are not available and FV is not present in cryoprecipitate or prothrombin complex concentrates. The aim of the project is to achieve common definitions on different aspects of the FV deficiency using longitudinal harmonized data collection system. As the first step focused and concise questionnaire was filled up by each participating centre to collect basic data regarding number and structure of the group of patients with FV deficiency. Blood samples will be collected according to protocol to measure the levels of FV clotting factor in blood and to obtain DNA for mutation detection. Variables that will be prospectively followed include characteristics of bleeding episodes, type and intensity of treatment, treatment results and complications using existing web-application in "Prospective evaluation of the intensity of bleeding episodes in patients with coagulation factors deficiency” (PRO-RBDD) project. So far, 11 centers form 9 countries were enrolled. Data on 47 patients were entered. Among them there are 23 males and 24 females. Twenty-five patients are less than 18 years old while 22 are adult patients. There are 8 patients with FV activity <1IU/dl, 17 patient with FV activity between 1-10 IU/dl and 22 patients with FV activity >10 IU/dl. Data related to the availability of phenotype and genotype laboratory investigations as well as treatment possibilities in each center were entered. FV deficiency project will provide essential information on the course and optimal management of FV deficiency, allow design of new clinical trials and foster the development of specific treatment products.     Factor X concentrate. Registration of novel drugs for RBDs. Peter Feldman (Coagulation Factors Research & Development, Bio Products Laboratory – BPL) The development of a factor X concentrate for patients with factor X deficiency exemplifies the difficulties in registering such drugs for rare bleeding disorders. Orphan Drug Designations offer pre-registration regulatory assistance and subsequent market exclusivity. These benefits help to recover the development costs, which are the same as for any drug, but recoverable only from sales to a much smaller patient cohort. The clinical trial in this patient population required a single protocol which satisfied different regulatory agencies. Even with parallel US and European protocol assistance, different expectations required time-consuming iterations to achieve consensus. Other clinical challenges to the registration programme have been: identification and enrollment of eligible patients for the clinical study, with intrusive and lengthy sampling proving a disincentive; prospective establishment of investigator sites for a separate surgery study, because the need, location and timing for surgeries could not be predicted;  European paediatric clinical trial requirements delaying the registration and availability of product, even for the adult population. Licensing authorities must follow legally-binding regulations; if these regulations were amended, access to treatment could be facilitated. Adults and children in this patient group receive personalised care, individually modulated by dose recovery and efficacy. The requirement for lengthy clinical trials of dubious statistical power could be waived, in favour of marketing authorisation based on risk-benefit assessment. This is particularly appropriate for coagulation factor proteins which have a well-understood physiology.  Such an assessment could address: safety; indicative pharmacokinetics; broad indications in adults and children; and enhanced post-registration monitoring of clinical outcomes.    DISCUSSION There was a question about the frequency of updating the rare coagulation disorders resource room and duplication with other websites. This was anticipated by the authors who were due to meet to discuss the issue in Milwaukee but had to postpone their meeting due to Dr Shapiro’s absence. An update plan will be developed. After the EAHAD database presentation a question arose about whether the original FVIII and IX databases would remain live and the answer was no as these are now superseded by the new database. It was also pointed out that these are not the only FVIII and IX mutation databases. Note by Christopher Ludlam: People who attempt to access Hamsters at its usual address of http://hadb.org.uk/ will find a notice giving directions to the updated VIII database directly and via the www.EAHAD-DB.org  portal but also there is the option to continue to access Hamsters through the web site. The plan is to close Hamsters at the end of 2014. Mark Soucie from CDC reported that they also support databases for these two factors; it was suggested that the two groups work together to avoid duplication of effort. Moreover, there was a question whether the EAHAD group could be interested in genotyping patients affected with RBDs. Flora Peyvandi answered that at this stage the group works on already available data. There was a question about efficacy of rFVIIa in factor V deficiency. Although the factor V database collects data on this treatment this is simply about the fact that this treatment was used and no data on its efficacy is available. Dr Feldman presented on the challenges of bringing a new product for a rare bleeding disorder to market and was asked about the development costs. He indicated that so far BPL has spent 5-10 million UK pounds ($8-16million) on their new factor X concentrate program and they are hoping to get it licensed in the USA within months rather than years.
by F. Peyvandi
Wednesday, June 18, 2014
2013 Minutes Locked Topic 0 F. Peyvandi Factor VIII & Factor IX Chairman: Flora Peyvandi (Italy) Co-Chairs: Jan Astermark (Sweden), Kathelijn Fischer (Netherlands), Michael Makris (UK), Danijela Mikovic (Serbia), Steven W. Pipe (USA), Elena Santagostino (Italy), Midori Shima (Japan) ,Leonard A. Valentino USA) DRAFT MINUTES June 29, 2013 8.00 EDUCATIONAL SESSION Session Chairpersons: David Lillicrap (Canada) and Flora Peyvandi (Italy) Dr. Peyvandi and Dr. Lillicrap opened the session welcoming the audience. They apologised for the absence of Dr. Mikovic. She was not able to attend the meeting due to personal reasons, therefore more time will be dedicated to the other two presentations by Dr. Pipe and Dr. Collins. Inhibitor risk associated with switching products including PUPs and PTPs. Steven Pipe (USA) The prospect of a wave of new recombinant clotting factors advancing out of the development pipeline has highlighted concerns regarding how to assess the potential immunogenicity of these products. In addition, within the hemophilia community there remains a perceived increased risk of inhibitor formation among previously treated patients when switching between factor products, even in the absence of bioengineered modifications . This presentation reviewed the published experience with product switching, novel preclinical models for testing immunogenicity, as well as currently available inhibitor surveillance practices and based on data available in the literature it seems there is no increase of inhibitor rate after switching in PTO patients. Finally, the Delphi technique was introduced as potential approach for a more comprehensive approach to risk forecast/ assessment. DISCUSSION: The importance of post-registration surveillance was underlined. Since for the new longer acting modified rFVIII and rFIX products it is more difficult to predict the risk of inhibitor development due to their genetic modifications and potentially aggravating environmental factors it would be important to request long term post registration surveillance trials.It was also discussed to involve basic scientists (immunologists) to develop prospective cohort studies to verify a potential influence of the genetic modification and other environmental risk factors for inhibitor development. Acquired Hemophilia: Diagnosis and treatment. Peter Collins (UK) Management of acquired hemophilia A should be undertaken in close collaboration with a hemophilia center with expertise in the field. Treatment which is not required in 30% of patients involves controlling and preventing bleeds and immunosuppression to eradicate the auto-inhibitors. Prompt diagnosis is important to allow early haemostatic treatment and prevent unessential invasive procedures. First line hemostatic treatment should be with a bypassing agent and both recombinant activated factor VII and the activated prothrombin complex concentrate anti-inhibitor complex concentrate (FEIBA) are equally efficacious. Both rFVIIa and AICC are associated with thrombotic events when used in acquired hemophilia. The pivotal trial with porcine rFVIII awaits results and could be a treatment alternative, with the promise of fewer thromboembolic events and well measurable FVIII levels. Immunosuppression should be started as soon as a diagnosis has been confirmed. The combination of steroids and cyclophosphamide may induce remission in more patients than steroids alone. Current data do not suggest that rituximab results in better outcomes, rather slightly inferior. Relapse is common (10-20%) in the first 6 months after immunosuppression is stopped and patients need to be followed up regularly to allow early diagnosis and treatment of relapse.. is currently under development DISCUSSION Questions and comments from the audience: Effect of the delay of immunosuppression on eradication. Data should be thoroughly analysed, however, no association with mortality has been shown so far.Should immunosuppression be stopped if there is no clinical response? Numbers too small to reach a final conclusion, but 3-5 weeks should be OK. Dr. Lillicrap: could VWF be involved in unexplained bleeding after treatment? There are no convincing data supporting this. Dr Mikovic had apologized for not being present due to personal reasons. Summary of her abstract Laboratory and clinical phenotype comparison in rare bleeding disorders. Danijela Mikovic (Serbia) According to clinical experience there is a heterogeneous association between coagulation factor activity level and clinical bleeding severity in patients with rare bleeding disorders (RBDs). The aim of the report is: To give an overview of data on the association between the residual FV levels and clinical bleeding symptoms in patients with different RBDs. To identify factor levels necessary to reduce major spontaneous bleeding To point the inadequacy of the current available assays to define a minimum residual level of all coagulation factors and suggest the further research in potential role of global coagulation assays in accurate prediction of the hemorrhagic risk To indicate that a more detailed evaluation on each single factor deficiency is required for future planning of optimal diagnosis and management. 9.00 REPORT ON SSC FVIII&FIX ACTIVITY 2012 – 2013. Flora Peyvandi (Italy) SSC reorganization SOA FVII&FIX SSC (report on on-going and closing projects) Dr Peyvandi opened the SSC business session by introducing the new SSC rules and strategy plan. She thanked all co-chairs of this subcommittee for the work done in the last year and she welcomed Dr. Michael Makris, Dr. Danijela Mikovic and Dr. Elena Santagostino as new co-chairs wishing them a fruitful and successful collaboration. She announced that the SSC executive committee approved the new name of this SSC: Factor VIII, Factor IX and Rare Coagulation Disorders. She reported about the state of the art of the SSC: 1. Recommendations published as Official SSC Communications on JTH Consensus definitions in rare bleeding disorders (Peyvandi et al, JTH 2012:10) Pharmacokinetics (Bjorkman and Collins, JTH 2013:11) Potency labelling of clotting factor concentrates (Hubbard et al, JTH 2013. doi: 10.1111/jth.12167)Standardization of methods for performing the clot wave form analysis (Shima et al, JTH 2013. doi: 10.1111/jth.12287)Reports posted on the ISTH website for further comments and discussion 2. Recommendation submitted as Official SSC Communications to JTH Consensus definitions in haemophilia (Chair: V. Blanchette) Standardization of methods for performing the thromboelastogram (Chair: G. Young and M. Chitlur) 3. Recommendation in preparation based on available data by SSC on Control of Anticoagulation (JTH 2011;9:1859–61) Standardization of methods for performing the thrombin generation test 4. Closing project: Clinical trial design for haemophilia (Chair: D. DiMichele). This project has been completed and the manuscript is in preparation. Dr. Peyvandi presented new projects that started in late 2012 or early 2013: Evaluation of hemostatic efficacy of novel FVIII / FIX concentrates and FVIII-inhibitor by-passing agents (Chairs: A. Lawrie and A. Tripodi) – 2012/2014Bleeding score in hemophilia: a prognostic tool for clinical outcome (Chairs: E.M. Mancuso and A. Tosetto) – 2012/2014The definition of mild Haemophilia A (Chair: M. Makris) – 2012/2014Factor V deficiency, clinical heterogeneity and treatment(Chair: D. Mikovic) – 2013/2015Standardization and quality management of genetic assays for diagnosis of hemophilia (Chair: V. Jenkins) – 2013/2015Prophylaxis in patients with and without inhibitors (Chairs: C. Escuriola and V. Blanchette) – 2013/2015Consensus definitions and recommendations for immune tolerance induction (ITI) in hemophilia with inhibitors (Chair: E. Santagostino) – 2013/2015Standardization of anti-FVIII inhibitor assays (Chair: K. Moertens) – 2013/2015 Finally Dr. Peyvandi highlighted the importance to start a new project on post-registration surveillance in patients using novel modified rFVIII and rFIX drugs. Dr. Lillicrap underlines the importance of SSC now and in future. Dr. Peyvandi reported the importance of collaboration between different SSC sub-committees in order to answer overlapping issues (e.g. bleeding in women affected with hemophilia, bleeding score ( BS) which could involve either FVIII, FIX and women Subcommittee or the issue of FDA- EMA harmonization on new drug registration. EMA requires the full clinical data base in children as part of the submission dossier for registration while FDA requires only data in adults (>12 years of age) for initial registration . The FVIII, FIX and Rare Coagulation Disorders SSC and the Pediatric SSC subcommittees should discuss and release a common recommendation. 9.20 CLINICAL OUTCOME EVALUATION - METHODS Session Chairpersons: Michael Makris (UK) and Kathelijn Fischer (The Netherlands), Dr. Fisher introduced the session. The 2001 SSC communication defining the different levels of severity of haemophilia was a landmark publication that had a major impact in the field both at clinical and research levels. Two other SSC projects will attempt to refine the definitions above by examining the impact of FVIII:C and other parameters which might affect them. Another project on mild haemophilia will examine the issues of borders, discrepancy and inhibitor development. SSC project: Bleeding score in hemophilia: a prognostic tool for clinical outcome. Alberto Tosetto (Italy) In patients with haemophilia, the residual levels of clotting factor (FVIII or FIX) are considered as the most important prognostic factors. Based on that, haemophilia patients are classified into the severe, moderate and mild categories, despite several observation suggesting that this may be actually insufficient. For instance, about 10-20% of patients classified as having severe haemophilia A do not have major bleeding complications. Furthermore, there are very few data supporting a strong relationship between factor VIII level and bleeding symptoms in patients with moderate or mild haemophilia A. Despite this lack of knowledge, treatment strategies are often implemented, sometimes pre-emptively, only on the basis of residual factor level (ie., prophylaxis vs. on demand therapies). Appreciation of the true prognostic value of residual factor level is therefore a major, clinically relevant task to be undertaken by the SSC-ISTH Working Group on Clinical outcome evaluation. With this as background the Working Group will establish consensus-based clinical criteria to define the degree of severity of hemophilia independent from residual FVIII/FIX levels and will validate the predictive ability of such residual levels in a pre-defined cohort of haemophilia patients. Such consensus-based criteria on the degree of clinical severity of hemophilia will be established by means of the Delphi method. If FVIII/FIX predictive ability is not entirely satisfactory, the Working Group will define a protocol for eligible measurements to be tested for prognostic improvement over residual factor in patients with hemophilia. SSC project: The definition of mild haemophilia A. Michael Makris (UK) The current ISTH definition for severity of haemophilia defines mild haemophilia as a condition with FVIII:C levels between >0.05-0.40u/ml. This definition does not comment on how patients with FVIII:C between 0.40u/ml and the lower end of the normal range should be classified. This working group (WG) will examine the evidence to decide on whether the 0.40u/ml cut-off is justified or needs to be altered. A further important issue is that the ISTH definition does not specify the type of FVIII:C assay to be used in the definition of haemophilia. It is now recognised that approximately 30% of patients with mild haemophilia exhibit discrepant FVIII:C levels depending on whether they are measured by the one- stage or chromogenic assay. The WG will define discrepancy when applied to FVIII assays so that different studies can be compared and will investigate the most appropriate assay for use in the definition of mild haemophilia A. The third area to be addressed by the WG will be the issue of inhibitors in patients with mild haemophilia A to try and capture evidence as to which mutations are associated with inhibitor development, whether it is possible to confidently state which mutations are not associated with inhibitors and provide guidance on genetic testing of mild haemophilic individuals. DISCUSSION Questions and comments from the audience: SSC project on BS prediction: suggestion on using the software based on Delphi approach. Moreover it was suggested that it would be important to collect data also on patients not receiving any or very few treatments. Which assay should be used to define mild haemophilia or unexplained mild bleeding disorder? One-stage assay or Chromogenic assay? 10.20 INHIBITOR DEVELOPMENT – METHODS AND RESULTS Session Chairpersons: Steven Pipe (USA) and Marijke van den Berg (The Netherlands) Dr. van den Berg introduced the session. SSC project: Inhibitor assays standardization. Koen Mertens (The Netherlands) The development of a FVIII inhibitor standard has been initiated at the 2003 Subcommittee Meeting, and subsequently endorsed by WHO and EMEA. The initial study, which included 22 laboratories and 5 candidate materials, has been performed by Dr. Raut from NIBSC. In 2006 he presented an interim analysis at the SSC meeting in Oslo and at the WHO-ECBS meeting in Geneva. Unfortunately, none of the candidate materials was considered suitable for establishment as the 1st International Standard for FVIII Inhibitors. This was due to a variety of concerns, including the origin of some of the materials (rabbit versus human), and the overall poor quality of the data generated. The rabbit Mab was deemed most suitable, though not accepted by the competent authorities. Both inter- and intra-laboratory variability were undesirable high (CVs up to 36%), and recalculating the results relative to any of the 5 candidate preparations did only marginally improve the inter-laboratory CVs. Moreover, information was lacking regarding the statistical validity of the individual inhibitor assays performed. In 2007, a Working Party was established (Chair: K. Mertens) with the aim to perform a ‘post-hoc’ statistical analysis on the raw data in order to assess the validity of the participants’ inhibitor titre estimates. Remarkably, this revealed that most participants performed inhibitor assays in a format that does not allow any statistical evaluation at all. As proposed at the SSC meetings in 2008 and 2010, the only option to proceed would be a new, more controlled collaborative study. This would require an assay format that complies with statistical requirements, and additional candidate materials, including some of human origin. As for new materials, Dr. Mertens’ laboratory has produced new recombinant antibodies which are cloned from the immune repertoire from inhibitor patients. Whether these monoclonals are suitable for "like versus like” testing of polyclonal patient samples remains to be investigated. At NIBSC, Dr. Raut has performed studies on a new assay format as an alternative to the regular Bethesda/Nijmegen assay. These recent developments may provide a starting point for bringing this project to some completion. The project group will focus on: (1) recommendations for the assessment of inhibitor titres, and (2) characterization of reference reagents (not necessarily standards) to facilitate inhibitor testing. In absence of these tools, the establishment of an International Standard for FVIII Inhibitors may remain an unrealistic goal. Any comments or suggestions from the Subcommittee are welcome at k.mertens@sanquin.nl for discussion in the FVIII inhibitor standard project group. Membrane composition can alter results of factor VIII inhibitor assays. Gary Gilbert (USA) The C2 domain of factor VIII binds to phospholipid membranes and is a dominant epitope for inhibitory antibodies. These antibodies alter the interaction with phospholipid membranes but, in many cases, do not entirely block membrane binding. Thus there is a need for basic insights that explain the mechanism through which anti-C2 antibodies inhibit activity of factor VIII. There are qualitative differences in the interaction of factor VIII with membranes of low phosphatidylserine content (<12%) where binding results from stereoselective interaction with phosphatidyl-L-serine vs. membranes with high phosphatidylserine content, where negative net charge is critical. Thus, we have hypothesized that the poor predictive value of factor VIII assays may be related to measurement on membranes of non-physiologic composition. mAb ESH4, directed against the factor VIII C2 domain, interferes with membrane binding and is a prototypic factor VIII inhibitor. ESH4 is a type II inhibitor, with residual factor VIII activity in the presence of saturating antibody concentrations. ESH4 decreased the apparent affinity 4-fold for membranes of 4% PS (KD = 4.8 ± 0.4 M without and 21 ± 4 M with ESH4) but only 2-fold on 15% PS vesicles (KD = 1.3 ± 0.2 M without and 2.5 ± 0.5 M with ESH4). The apparent affinity for factor X in the presence of saturating phospholipid and ESH4 was higher on 15% PS vesicles (KM = 129 ± 18 nM) than on 4% PS vesicles (KM = 284 ± 30 nM). To determine the mechanism through which ESH4 inhibits membrane-bound factor VIII activity, we measured the fluorescence anisotropy of factor IXa-Fl-EGRck. ESH4 decreased anisotropy of the factor VIIIa-factor IXa-factor X complex from 0.280 ± 0.002 to 0.272 ± 0.002 indicating that ESH4 alters the Vmax of the membrane-bound factor VIIIa-factor IXa complex through a change near the factor IXa active site. ESH4 inhibited 28% and 36% of factor VIII activity in aPTT-based and 2-stage commercial factor VIII assays, respectively. In contrast, factor VIII inhibited 78 ± 12% of factor VIII activity supported by thrombin-stimulated platelets. The results indicate that anti-C2 domain antibodies can inhibit factor VIII activity through modulation of the membrane-bound factor VIIIa-factor IXa complex. The 2-fold difference between inhibition in commercial factor VIII assays vs. an assay supported by thrombin-stimulated platelets suggests a strategy for improving the predictive value of factor VIII assays in patients with inhibitory antibodies. DISCUSSION Questions and comments from the audience: The difference among all new products coming on the market is a big source of variability: one standardized assay for all of them or specific assays for each one? The community decided to go step by step and first focus on one standard assay, which is sensitive and can be used for all new replacement therapy products . SIPPET study: power and weakness of the study. Pier Mannuccio Mannucci (Italy) Clinical data stemming from a systematic review that summarized a few retrospective studies suggest that the source of factor VIII used for replacement therapy (plasma or recombinant DNA technology) might have some influence on the cumulative incidence of inhibitors [1]. A more recent systematic review and meta-analysis found a two-fold higher inhibitor incidence with the use of recombinant factors, but the difference in favor of plasma-derived factor VIII was no longer statistically significant when variables such as study design, study period and frequency of inhibitor testing were included as confounders [2]. To tackle this state of current uncertainty on this issue, the randomized, prospective independent SIPPET trial [3], ongoing now in 19 countries from 5 4 continents, is planned to enroll 300 previously untreated or minimally treated patients with severe hemophilia A at risk of developing factor VIII inhibitors, in order to establish whether or not plasma-derived factor VIII products are less immunogenic than recombinant products. At this time, SIPPET has already enrolled 241 patients with 208 patients having received at least one dose and an interim analysis of the available data is planned within the current year when 50% of the planned patients have achieved at least 20 exposure days. So far 40 inhibitor patients have been identified. References Paisley S et al. Haemophilia 2003;9:405-17. Iorio A et al. J Thromb Haemost 2010;8:1256-65.Mannucci PM et al. Haemophilia 2007;13 (Suppl 5):65-8. RODIN study: power and weakness of the study. Marijke van den Berg (The Netherlands) The PedNet registry was initiated in 2004, included patients born from beginning of 2000 and apart from that aimed to collect prospective data of all children diagnosed with severe hemophilia born in one of the participating Haemophilia treatment centers (HTC’s). The RODIN study was the first satellite study and it was set up to investigate both genetic and treatment related factors for inhibitor development. As of May 2011 the 516 PUPs reached the study end point with major center variability in incidences of inhibitor patients. From September 2012 also the N=8 ex-RODIN centers became part of the PedNet study group. The aim for the 30 centers is also to include newborns from 2010 onwards of both hemophilia A and B with factor VIII/IX levels <25%. The strength of multi center observational studies in rare diseases is the possibility to collect large numbers of patients. This makes it possible to study correlation between different variables that have an impact on disease outcome. Selection bias is an important confounder and could largely be prevented in the PedNet study group. Inhibitor development in PTP: available registries and harmonization. Alfonso Iorio (Canada) The development of inhibitors in patients previously exposed to factor concentrates is a rare event, with estimates be in the order of 2 per 1000 patient year (95% CI 1-4)(1). Both the UK and Canadian registry have shown an increase of this rate with the age of the patient (2,3). Historically, the interest in inhibitors in PTPs has been mainly related to the use of this population as the preferred one to assess the immunogenicity of new factor concentrates before their approval for clinical use (4). More recently, the analysis of the development of inhibitors in PTPs has been proposed for the comparison of the immunogenicity of different products and for the assessment of the risk of developing inhibitors as a consequence of switching factor concentrates (1,5). Furthermore, recent publications and ongoing trials have employed or suggested the use of PUPs to answer similar or overlapping questions (6,7). Several challenges make this issue interesting for the ISTH SSC on factor VIII. To the purpose of the assessment of inhibitor rates, the rarity of the event requires large scale data collection to gather relatively stable estimates; diagnostic modalities (upon clinical suspicion versus routine testing, laboratory assay used, frequency and modality of testing) should be recorded if standardization and central testing is unfeasible; size of the exposed population under surveillance has to be reliably assessed to approximate as much as possible an inception cohort; the clinical details of the events occurring before inhibitor development and the clinical course of the inhibitors should be collected in a simple but standardized way. To the purpose of performing surveillance in patients undergoing a concentrate switch, surveillance should be performed in a consistent and identical way in switcher and not switcher over a reasonable time frame. Recent surveys of the information currently collected by existing registries and surveillance schemes outline the need for a harmonization process. For most of these aspects, no evidence exists to guide practice, so that empirical protocols would be better developed after gathering advice from immunologist and epidemiologist, and endorsed by the SSC. References Iorio A et al. Blood, 2012;120:720-7. Hay CRM et al. Blood, 2011;117:6367–70. Webert KE et al. Haemophilia, 2012;18; e254-e259. White GC et al. Thromb Haemost, 2001;85:560. Aledort LM et al. J Thromb Haemost, 2011;9:2180–92. Gouw SC et al. New Eng J Med , 2013;368:231–9.Iorio A et al. J Thromb Haemost, 2010;8:1256–65. DISCUSSION Questions and comments from the audience: Dr. Rosendaal states that probably we should not talk anymore of retrospective and prospective studies creating a paradox: data are ALWAYS collected after the event. 14.00 NEW HEMOSTATIC DRUGS: CLASSIFICATION, DOSE STANDARDISATION AND MONITORING Session Chairpersons: Flora Peyvandi (Italy) and David Lillicrap (Canada) Dr. Lillicrap introduced the session. SSC project: Clinical trial design for hemophilia. Donna Di Michele (USA) The Project Group on Clinical Trials for New Products in Hemophilia (CTPG) was created in February 2011 by the FVIII/IX Subcommittee of the International Society for Thrombosis and Hemostasis (ISTH) to examine current rationale and propose alternative strategies for the design of pre- and post-authorization (licensure) clinical trials and studies for new therapeutics in hemophilia. Its mandate arose from pragmatic concerns about the feasibility of populating and conducting multiple simultaneous new clotting factor trials. These concerns led to the question of whether innovative and evidence-based approaches to trial simplification might increase feasibility without compromising assessment of product safety and efficacy. Moreover, the project group was envisioned as a forum for the participatory discussion of the EMA’s and FDA’s respective regulatory goals for new product clinical trials, and the potential for inter-agency harmonization of the most critical regulatory requirements. Based on this rationale, the CTPG was tasked with exploring alternative statistical models for the pre- and post- licensure study of so called ‘me-too’ and novel biologics for hemophilia A and B (with /without inhibitors), while giving due consideration to: a) safety and efficacy data required by regulators for marketing authorization; b) number of potential subjects available world-wide for multiple simultaneously- conducted trials; and c) innovative clinical trial design suitable for rare diseases such as hemophilia. The group was constituted according to member expertise in clinical care and investigation, immunology, clinical trial design and statistics methodology, and included regulatory science representatives FDA and the EMA. The group met monthly between February 2011 and June 2013 to 1) review the known clinical data on factor VIII and IX product immunogenicity (and any emerging data for novel biologics) in previously treated (PTPs) and previously untreated (PUPs) patients; 2) explore the potential impact of alternative statistical modelling and innovative trial design on the pre-authorization regulatory requirements for product safety and efficacy determination; 3) examine the current scientific concepts of immunogenicity and neo-antigenicity and their potential influence on clinical trial design and novel approaches to antibody surveillance; 4) develop consensus safety and efficacy clinical endpoint definitions (building on the work of the Definitions Project Group); and 5) formulate recommendations. Progress reports were provided and input from critical stakeholders in the hemophilia community was solicited at international meetings that included the Scientific and Standardization Committee of the ISTH (2011, 2012); World Federation of Hemophilia (WFH) meeting (2012); and the WFH Global Research Forum (2011; 2013). The hemophilia biologics industry was also surveyed in 2012 for its input with respect to perceived regulatory barriers to efficient and timely product registration; alternative paradigms for clinical safety and efficacy monitoring in pre- and post- licensure studies; and potential benefits to be derived from strategic harmonization of key FDA and EMA regulatory requirements. Finally, comments were solicited from selected experts in clinical trial design methodology as well as the FVIII/IX Subcommittee membership prior to publication. The CTPG ultimately narrowed the project scope to the following recommendations for an alternative approach to statistical modelling and the clinical trial design of pre-authorization trials for both ‘me-too’ and novel factor VIII (FVIII) biologics in PTPs. This approach was rooted in the combined agency regulatory goals for pre-licensure studies, and incorporates both current immunological theories of neo-antigenicity and consensus clinical efficacy endpoint definitions. The group considered several innovative approaches to the clinical design of new product safety (immunogenicity) trials. All proposed strategies were based on the known epidemiology and immunology of FVIII inhibitor development in congenital haemophilia A. The CTPG explored how epidemiology might best inform the traditional design of the single arm pre-licensure new product study with respect to subject number and duration, and considered alternative clinical designs that incorporate either Bayesian modelling or adaptive design. The group also recognized that the optimal design of pre-and post-authorization clinical trials remains hampered by poor understanding of the precise nature of the interactions between the therapeutic FVIII products (both ‘me too’ and novel biologics), and the recipient’s immune system. This is in part due to the incomplete characterization of the PTP subject’s immune status relative to the therapeutic product(s) received prior to recruitment into a new product trial such that immunological ignorance is not routinely distinguished from active tolerance. Critical information about genetics, cross-reactive material (CRM)-status and treatment intensity history is also frequently lacking. The CTPG therefore advocated for the systematic and harmonized collection of clinical and biological data from subjects entering pre- and post authorization new product studies. It envisioned complementary but integrated data sets may be required to satisfactorily address regulatory, scientific and national/ international surveillance priorities. The ultimate intent of this harmonized data collection would be a more evidence-based rational design of clinical trials for new therapeutics in which a smaller but well characterized and homogenous subject cohort would more precisely define the risk for inhibitor development in specific populations. The CTPG also recommended that the FVIII/IX Subcommittee establish a new project group dedicated to the feasible implementation of international harmonized post-authorization studies using existing national and international database infrastructure and consensus standardized minimum datasets. Consideration should be given to the implementation of consensus clinical outcomes definitions that, while tailored to the specific period of observation, are themselves harmonized with pre-authorization clinical endpoints in order to maximize the potential for continuous longitudinal data collection on well characterized populations. The CTPG suggests that such a coordinated post- marketing effort could collect strategic data that contribute to a greater understanding of immunogenicity and the potential implementation of novel more sensitive antibody detection assays. Finally, the group advocated for greater EMA/FDA harmonization in critical regulatory areas such as evolving landscape of inhibitor assays and the approach to product authorization in children. A manuscript summarizing this PG’s activities and recommendations is nearing final draft form and will be sent shortly to the FVIII/IX Subcommittee membership for final comment prior to submission for publication. Immunogenicity of novel products and its evaluation. Sebastien Lacroix-Desmasez (France) The Project Group on Clinical Trials for New Products in Hemophilia (CTPG) was created in February 2011 by the FVIII/IX Subcommittee of the International Society for Thrombosis and Hemostasis to examine current rationale and propose alternative strategies for the design of pre- and post-authorization (licensure) clinical trials and studies for new therapeutics in hemophilia, with a focus on FVIII biologics. The CTPG has recognized that the optimal design of pre-and post-authorization clinical trials for FVIII biologics in previously treated patients (PTPs) with hemophilia A is limited by the poor understanding of the precise nature of the interactions between the therapeutic FVIII products and the recipient’s immune system, and by the imprecisely characterized rate of allo-immunization to FVIII therapeutics. Optimization of clinical trials therefore requires the systematic collection of information that is potentially available but not routinely registered. The latter information includes i) the immune status of the PTPs relative to the therapeutic FVIII products received prior to recruitment in a new product trial, which should distinguish between immunological ignorance and active tolerance, ii) the cross-reactive material (CRM)-status of the patients, and iii) the genetics and the treatment intensity history. Further, the true rate of inhibitor development in PTPs should be determined including parameters such as age, treatment intensity/bleeding pattern, product use and product switch. Development of novel clinical trials would then proceed in two phases. Firstly, a mandatory international registry should be established for the systematic and harmonized collection of clinical and biological data from subjects entering pre- and post-authorization product studies. A more rationale design of clinical trials could then be based on registry-derived evidence, and might be expected to include a smaller but well characterized and homogenous subject cohort with a more precisely defined risk for inhibitor development. Efficient model of safety evaluation of novel products. Frits Rosendaal (The Netherland) New opportunities in drug design and modification pose a paradoxical challenge of luxury: after a decade in which clotting factor concentrates have remained largely unchanged, we are currently observing a series of innovative drugs in various stages of development that offer a view of major advances in, for instance, extended half-life and ease of administration. In addition, biosimilar compounds will be developed that offer the benefits of currently available products at a far reduced price, which may close the treatment gap between the rich and the poor. New drugs need to be tested for efficacy and safety, and here is where the paradox comes in, i.e., that haemophilia is such a rare disease that stringent criteria for licensing studies may lead to a shortage of patients to be entered into clinical trials. Several solutions are conceivable: firstly, in revisiting the criteria. Secondly, in making informed choices and only allow products in the phase of clinical testing that indeed have the potential of a major advance. Thirdly, to use the most efficient study design. For the latter, we will discuss the approach to establishing safety with regard to neo-antigenicity (inhibitor development), which is currently the sample-size-limiting aspect of required studies. Several approaches can be applied that all share some characteristics. The first is two apply prior experiences with product-related neo-antigenicity, and two develop study designs that incorporate these patterns. A second is to use an adaptive trial design, in which predefined stages of subsequent trials are set, not unlike predefined interim analysis. The third is to use Bayesian approaches, which is the method that relies most strongly on predefined expectations, and which may be appropriate for biosimilars. Timelines of approval of new products: patients’ view. Brian O'Mahony (Ireland) The Haemophilia patient community are positively anticipating the licencing and availability of a new generation of longer acting factor concentrates which will provide an additional therapeutic option and the possibility to transform the current treatment paradigm. The availability of these new products, if introduced on a cost sustainable basis, could also lead to a change in the economics and availability of the current recombinant and plasma derived factor concentrates. There are real possibilities of improving access to treatment for countries at all levels of current treatment availability. We are greatly concerned that these positive developments may be negated by delays in licencing of these products in Europe and the impractical application of orphan drug market exclusivity to these products. The requirement to submit paediatric PTP data before initial market approval may delay access to these products for two to three years in the EU compared to the USA and Canada. Apart from depriving adult patients from more attractive product options, this, may lead to unacceptably high launch prices in Europe for these products based on historically higher prices in the USA and Canada. This could create difficulty in reimbursement of these products in Europe. Many of the new products under development have been granted Orphan Drug designation. However, it is the view of the patient community in Europe that market exclusivity should not be granted to the first of the longer acting FVIII or FIX products to be licenced. The products under development use demonstrably different protein modification or enhancement methodology and in our view should not be classed as similar products for this purpose. If market exclusivity is granted, this will lead to a lack of therapeutic options, a monopoly with consequent higher costs and a very significant reduction in the potential benefit to the community. Global assays standardization: what is needed? Guy Young (USA) Over the past decade, through the work of a number of investigators worldwide, there has been substantial progress made regarding the understanding of the use of global hemostatic assays in the field of hemophilia. Important studies have evaluated different methodologies, demonstrated the ability of these assays to distinguish different phenotypes of hemophilia, and have also demonstrated in small pilot studies the potential clinical applications for monitoring of factor therapy including bypassing agent treatment in inhibitor patients. In order to move the field forward and importantly to develop these assays as a clinical tool, the first step is to have a unified, standardized approach to conducting the testing. Thus far, among a number of global assays that have been studied, two specific assays have emerged as the leading candidates for clinical application: thrombin generation test and thromboelastography. With respect to thrombin generation testing, the calibrated automated thombogram has become the main device used in hemophilia studies and a standardized protocol has been developed by one center (1) although this protocol is yet to be accepted as an international standard (2). The situation for thromboelastography is similar although with 2 key differences. First, there are two devices, the TEG®5000 Analzyer from Haemonetics and the ROTEM® delta from Tem International which are available. While the devices differ slightly in their specifications, their output in terms of quantifiable parameters and graphical representations are identical. The second difference is that the Working Party on Thromboelastography Standardization has developed a standardized protocol which will be published shortly as an SSC Communication in the Journal of Thrombosis and Hemostasis (3) and will allow investigators in this area move forward with a unified approach. For a detailed discussion of the state-of-the-art with respect to these assays, the following reference is recommended (4). References Dargaud Y et al. Thromb Res 2012; 130:929-934.Loeffen R et al. J Thromb Hemost 2012; 10:2544-54.Chitlur M et al. Recommendations for the methodology to perform thromboelastography/thromboelastometry in patients with hemophilia: A SSC communication from the project on standardization of methods for performing thromboelastography. Submitted to J Thromb Hemost.Young G et al. Blood 2013; 121:1944-1950. SSC project: Evaluation of hemostatic efficacy of novel FVIII / FIX concentrates and FVIII-inhibitor by-passing agents. Andrew Lawrie (UK) INTRODUCTION: Assessment of new therapeutic agents that are not directly comparable to a WHO (plasma derived) concentrate standard is a topic that concerns. As Pfizer have already identified that this is an issue and produced a ReFacto AF Laboratory Standard, it was felt that this product specific reference preparation could be used to investigate possible alternative analytical methods such as thrombin generation or thromboelastography that could be used to evaluate the potency of new products for treatment in haemophilia. METHODS: Initial studies were performed using one-stage clotting and chromogenic factor VIII (FVIII) assay techniques, employing two analytical systems (Reagent / Analyser combinations). The analysers were calibrated using the 8th International Standard Factor for VIII Concentrate (07/350) pre-diluted to ~1 IU/mL in system specific FVIII deficient plasma. The 13th British Standard Factor VIII Concentrate (10/188) and ReFacto AF Laboratory Standard (RAFLS Lot 10/246) were pre-diluting as for 07/350. Thrombin generation was performed using the Calibrated Automated Thrombogram (CAT) with Thrombinoscope PPP reagent low. Rotational thromboelastometry (ROTEM) was performed using Sekisui Diagnostics dilute prothrombin time reagent (reaction concentration ~3 pmol/L Tissue Factor. For both the CAT and ROTEM 0.1mL of concentrate was added to 0.9 mL factor VIII deficient plasma from: Precision BioLogic Inc, Siemens and IL RESULTS / DISCUSSION: Both the one-stage FVIII clotting assays and chromogenic FVIII assay the BS FVIII (10/188) and ReFacto AF (10/246) gave linear dose response curves that were parallel to the IS FVIII (07/350) calibration curve. Mean relative potency estimates for the BS FVIII were similar by each of the assay techniques; however the mean relative potencies for ReFacto AF (10/246) were clearly influenced by the assay system. Thrombin generation showed that the three sources of factor deficient plasma had different TGT characteristics in terms of lag time, time to peak, peak and endogenous thrombin potential (ETP). These characteristics shown by the deficient plasmas were mirrored in the thrombin generation curved produced when they were spiked with FVIII concentrates. For each of the substrate plasmas of BS FVIII (10/188) and ReFacto AF (10/246) ETP and Peak thrombin were normalised relative to the values from IS FVIII (07/350), however this failed to produce similar results for each of the deficient plasmas systems. Rotational thromboelastometry performed on factor deficient plasmas and those deficient plasmas spiked with the concentrate exhibited only minor changes in the ROTEM trace, those changes relating only to the alpha angle i.e. the rate at which clot firmness increased. CONCLUSIONS: Under the study conditions results from the CAT and ROTEM were dependent on the source of factor FVIII deficient plasma used to dilute the FVIII concentrate. Therefore these alternative techniques do not appear to offer any advantages over the conventional assay systems. DISCUSSION Questions and comments from the audience: On the opportunity to use Bayesian approach or two stage approach as statistical analysis: Prof. Rosendaal answers that this is an issue the scientific community have to decide on.Is it possible to approach the standardization of pharmacokinetics studies in paediatric patients in the same way Dr. DiMichele approached in the clinical trial design recommendation for the safety and efficacy evaluation? This will help to avoid delay due to regulatory requests?: Dr. DiMichele answers this could be done, but it is necessary to set the method first.What will be the timeline and do we need to obtain harmonization between two regulatory bodies of FDA and EMA for performing clinical studies with novel drugs?: Dr. O’Mahony answers he is afraid it won’t be possible to reach this goal, while Dr. Srivastava invites to continue to look for such an important agreement. He further re-iterated that the community has not strongly enough stood together to allow the EU guideline regulating the ped data base being a pre-requisite for submission of dossier for product approval in EU. 15.30 ASSAYS STANDARDIZATION Session Chairpersons: Jan Astermark (Sweden) and Midori Shima (Japan) Dr. Astermark introduced the session. Precision in estimation of the FVIII and FIX levels is a prerequisite for accurate correlation between activity levels and clinical symptoms. It is also crucial for true characterization and prediction of phenotypes. Assay discrepancies are a well-known occurrence in the case of FVIII measurement, but less so for FIX. In addition, genetic analyses are now more readily available and used for both characterization of the disease as well as for carrier and prenatal testing. These assays, however, require standardization and quality controls. This session will highlight two important projects in the area of laboratory testing Relationship between results with different FIX assays in post concentrate infusion samples. Steve Kitchen (UK NEQAS Blood Coagulation) Approximately 60 centres participated in a collaborative study assessing FIX assays. Samples were collected from a haemophilia B carrier and 2 subjects with severe haemophilia B after infusion of FIX concentrates with written informed consent. Two subjects were infused with recombinant FIX (BeneFIX, Pfizer) and one with high purity plasma derived concentrate (Replenine, BPL ). The details were as follows Sample Baseline FIX (IU/dl) Concentrate sample collection details 01 37 IU/dl BeneFIX 20 mins after 3000 units 02 <1 IU/dl Replenine 20 mins after 3000 units 03 <1 IU/dl BeneFIX 20 mins after 6000 units All samples were lyophilized protocols in 0.5 ml aliquots using established UK NEQAS BC and dispatched to participating centres through the post. Centres were requested to assay these as thought they were routine samples for monitoring patients during treatment with FIX concentrates. Median results for centres calibrating assays using plasma standards are shown in the table below Assay n sample 01 sample 02 sample 03 median IU/dl CV median IU/dl CV median IU/dl CV Chromogenic (Rossix) 3 71 - 37 - 61.5 - Chromogenic (Hyphen) 2 77 - 37 - 74.5 - One stage IL reagents 27 89 10.1% 39 7.3% 92 11.1% One stage Siemens reagents 16 87.2 6.3% 32.5 9.6% 82.5 7.5% All chromogenic 5 71 - 37 - 63.5 - All one stage 61 87.5 10.3% 37..7 12.7% 87 13.3% One stage results with the reagent set from IL (Synthasil APTT reagent, IL deficient plasma IL reference plasma) were significantly greater than those obtained with the Siemens reagent set (AFS APTT reagent, Siemens deficient plasma, Siemens reference plasma) for samples 02 ( p<0.0001) and 03 (p<0.02). When results obtained by different methods were combined chromogenic assay results were significantly lower than one stage results for samples 01 ( p<0.002) and 03 ( p<0.01). Our data indicate that FIX results vary according to the assay methods used in some samples from patients treated with recombinant or plasma derived concentrates. SSC project: Standardization of genetic assays for diagnosis of hemophilia. Vincent Jenkins (Ireland) Mutation analysis of haemophilia A and haemophilia B is now routinely available in many countries and increasingly available internationally. Carrier and prenatal diagnosis by molecular analysis are now standard techniques. Despite the growth in molecular testing there are relatively few guidelines and external quality assurance schemes for the molecular analysis of the F8 and F9 genes. This project will survey current practices in diagnostic laboratories by means of a questionnaire. The aim is to harmonise diagnostic practice especially the reporting of genetic results, and encourage the development of appropriate quality assurance. DISCUSSION Questions and comments from the audience: Percentage of non-participating centres to quality exercise is high, does it mean there are lot of inadequate labs?: no, only that they decide not to take part.It seems there is different specific type of intron 22 inversion mutation .Is there any different method to identify these type of variants for intron 22 inversion mutation? how to solve this problem during an exercise? Dr. Jenkins explains that the test we are using now is very basic one and at the beginning of this exercise we just need to understand the basic information and later one we could include more specific questions. 16.25 ORPHAN DRUGS IN RARE BLEEDING DISORDERS (RBDS) Session Chairpersons: Elena Santagostino (Italy) and Danijela Mikovic Dr Santagostino introduced the session, explaining that Dr Mikovic had apologized for not being present due to personal reasons and the SSC Project will be presented by Dr. Roberta Palla. SSC project: Factor V deficiency, clinical heterogeneity and treatment. Roberta Palla (Italy) Inherited factor V (FV) deficiency is an autosomal recessive rare bleeding disorder with estimated prevalence of one per million in the general population. The phenotypic expression is variable. Homozygotes and compound heterozygotes show mild to severe bleeding symptoms while heterozygotes are usually asymptomatic or experience mild symptoms. Main clinical manifestations are mucosal and postoperative haemorrhage but haemarthroses and intracranial haemorrhages can also occur. The reasons for clinical heterogeneity are largely unknown. No clear correlation between genotypes and the clinical phenotypes has been identified. Clinical severity shows weak association with laboratory phenotype. There is no ready explanation for the differences in bleeding phenotype between patients with equally low or undetectable FV levels. Several observations indicate the crucial role of platelets in FV deficiency. Although most FV is present in plasma, approximately 20% of the circulating FV is found within platelet alpha-granules. Residual platelet FV might be responsible for the vast differences in bleeding phenotype observed among patients with equally undetectable plasma FV levels. Moreover, recent findings show that inter-individual differences in tissue factor pathway inhibitor (TFPI) plasma levels might be contributing factors. Replacement therapy with fresh frozen plasma (FFP), preferably virus-inactivated, remains the mainstay of treatment in the absence of FV concentrate. In most patients FFP is administered on demand, rarely prophylactically in severely affected patients. Potential risks of treatment with FFP are allergic reaction, infection and volume overload. In refractory cases or in patients with inhibitors platelet transfusions and recombinant activated FVIIa were used, with variable efficacy. The SSC project will enable collection of prospective information related to FV deficiency patients using web-application PRO-RBDD database. Aims of the project are to determine potential genetic, laboratory and clinical predictors of bleeding severity, to evaluate efficacy and safety of treatment options in order to improve care due to increased use of standardized therapeutic approach. Collection of sufficient data should allow design of a clinical trial of newly produced FV concentrate. Novel products for treatment of RBDs: clinical trial design and methods for efficacy evaluation (antithrombin RNAi) Amy Simon (Alnylam Pharmaceuticals , USA) Rare Bleeding Disorders (RBD) impact over 14,000 people worldwide, according to the World Federation of Hemophilia 2010 Global Survey. Recently, there has been increased understanding about the epidemiology, clinical course and treatment of these different RBDs due to the establishment of national and international networks and registries. While treatment options are available for RBD patients, such as fresh frozen plasma, activated prothrombin complex concentrates, or factor concentrates in some cases (e.g. FVII, FXIII), there is still an unmet need for better therapeutic options in RBD patients. In addition, there are a subset of RBD patients with a severe clinical phenotype that could benefit from a more feasible prophylaxis option. To this end, we have initiated a therapeutic program targeting the natural anticoagulant antithrombin (AT) through RNA interference (RNAi) as a mechanism to enhance thrombin generation and clot formation in patients with bleeding disorders. Supportive data for this therapeutic approach can be found in the literature from animal models as well as humans with hemophilia who have coinheritance of thrombophilic mutations, such as AT deficiency. We have developed a small interfering RNA (siRNA), ALN-AT3SC conjugated to a hepatocyte targeted ligand Gal-NAc, that results in the potent AT reduction as seen by liver mRNA as well plasma AT levels (by antigen and activity assays) in multiple species, with an ED50 of 1 mg/kg after a single subcutaneously administered dose. Chronic weekly dosing with 6 or more doses at 0.125, 0.25 or, 0.50 mg/kg was well tolerated in wild type non-human primate (NHP) and resulted in approximately 50%, 65% and 85% AT suppression, respectively. Importantly, , data from both in vitro studies using hemophilia patient plasma and in vivo studies from NHP have demonstrated that 50% or greater reduction in plasma AT resulted in increased thrombin generation as seen by a 2 to 4-fold increase in the ETP. Furthermore, reduction of AT by 50% or more resulted in improved hemostasis after laser injury in hemophilia A mice. These combined preclinical studies provide validation for AT as a target for enhancing thrombin generation and improving hemostasis. Current studies are ongoing in large animal models of hemophilia, and clinical studies in man will commence in 2013. If ALN-AT3SC is found to improve hemostasis in clinical trials, it may offer a novel, attractive prophylaxis option for RBD patients, given its subcutaneous route of administration and long duration of action. Novel Factor V concentrate: orphan drug and clinical trial. Claudia Nardini (Kedrion, Italy) Patients affected by coagulation factor V deficiencies, classified as rare diseases, cannot lean on a suitable clinical therapy, since so far no factor V concentrate is available on the market. FFP infusion remains indeed the mainstay of treatment, implying several and established side effects, such as volume overload, risk of allergic reactions and pathogens transmission. The aim of Kedrion’s project is to bridge the gap between patients’ needs and existing therapies, developing a plasma derived factor V concentrate targeted to on demand/prophylaxis replacement treatment, in order to overcome all the drawbacks deriving from the administration of huge volumes of undefined pathogen safety plasma. The development of such a product implies the planning and execution of several steps in order to make it available for patients’ treatment. First a purification strategy has been defined, including virus reduction steps and product characterization. Then a pilot production plant has been set up with the aim of manufacturing GMP clinical batches of orphan drugs. On the regulatory side an application for Orphan Drug Designation is planned to be submitted to the regulatory authorities with the aim to obtain the designation within 2013 and preliminary in vitro preclinical studies are being started. The purification process set up has been developed, bearing to a stable factor V concentrate, suitable for parenteral infusion in deficient patients. The GMP certification application for the pilot plant has already been submitted with the goal of receiving authorization first and to start with the production of cGMP batches within 2013. In conclusion a factor V concentrate has been developed for clinical use in deficient patients and first steps have already been taken from the regulatory point of view, in order to make it available on the market as soon as possible. 17.15 OPEN SESSION ON NEW PROPOSALS Session Chairpersons: Alok Asrivastava (India) and Leonard Valentino (USA) Dr. Srivastava introduced the session. Assessment of CFC- The end points towards long term outcomes. Alok Srivastava (India) Dr. Srivastava introduced this session describing the current situation of the evaluation of clotting factor concentrates (CFC). He mentioned that in the lifecycle of the a new CFC, there is a reasonably well defined process of assessment with the pivotal clinical study that evaluated short term safety particularly with regard to incidence of inhibitors in PUPs and hemostastic efficacy in prevention and treatment of bleeds as well as surgical hemostasis. The work of this subcommittee through several project groups has helped in this process by providing definitions of clinical events and treatment responses that should help standardize some of the short term endpoints. However, there is currently no well defined and implemented process for the systematic assessment of long term safety and efficacy of CFCs. This can lead to delays or non detection of potential adverse events in the post marketing phase. There are examples of this happening in the past. The question therefore is whether such processes should be put in place and if so, what should they be and finally what should the additional responsibilities therefore of the manufacturers, regulators, treating physicians and very significantly the patients who need to participate in many of these additional assessments. New European pharmacovigilance legislation: first experiences. Anneliese Hilger (EMEA) New pharmacovigilance legislation (Regulation EU 1235/2012 and Directive 2010/84 was adopted by the European Parliament and European Council in December 2010 and amended by Regulation 520/2012, 1027/2012 and Directive 2012/26. The legislation was the biggest change to the regulation of human medicines in the European Union since 1995. It has significant implications for applicants and holders of EU marketing authorizations including products for haemophilia treatment. Impacts of the new legislation comprise adverse drug reactions reporting only into EudraVigilance data base. Safety monitoring as laid down in periodic safety update reports has been simplified e.g. electronic reporting, single assessment for same active substance instead of individual routine PSUR reporting. There is a strengthened legal basis for the requirement of post-authorization safety and efficacy studies. In view of the limited availability of patients suffering from haemophilia, data from pre-authorisation studies only are considered to provide basic knowledge at the time of marketing authorisation, especially with respect to immunogenicity. Therefore, to collect additional clinical data and to ensure consistency in the long-term between the outcome from pre-authorisation clinical studies and from routine use, post-marketing investigation should be performedfor Factor VIII and IX products. This post-authorisation investigation concept is included in the Risk-Management-Plan and thus, will be approved and evaluated by the recently established Pharmacovigilance Risk Assessment Committee (PRAC). New FDA regulation for post-marketing surveillance. Nisha Jain (FDA) In 2007, the provisions made under the FDA Amendments act gave the FDA authority to mandate post-marketting surveillance. This could include studies or clinical trials if there were credible information to suggest that there were potential issues with safety or efficacy of the licensed products. For approval of a product, if a serious risk is identified in the clinical trial or there is potential for serious mandate a post-marketting surveillance. For an approved product, if the FDA becomes aware of of a new safety information, then a post-marketting study / clinical trial can be also be mandated. Prior to mandating a post-marketting study, the FDA has to establish that routine pharmacovigilance will not be adequate to identify these risks. Some examples of such post-marketting studies include pharamaco epidemiologic studies and clinical trials with specific safety end-points. DISCUSSION Questions and comments from the audience: Have been post-marketing surveillance methods for new products already set? Too premature.Dr. Mannucci asked why do not make easier rules for the pre-marketing registration and then applying demanding rules for the post-marketing surveillance? Dr. Hilger reported that both regulatories are evaluating this point but strongly believe that the requested number of patients to be enrolled is an essential requisite to licence treatment products to the market. NEW SSC PROJECTS Consensus definitions and recommendations for immune tolerance induction (ITI) in hemophilia with inhibitors. Elena Santagostino (Italy) In recent years some randomized clinical trials have provided high levels of evidence in the field of hemophilia including immune tolerance induction (ITI) treatment. In fact, the International ITI Study (Hay and DiMichele, Blood 2012) provided milestone results on treatment of children with good prognosis and pointed out the safety issue of the bleeding risk during ITI. Nonetheless, heterogeneous dosing regimens are used in routine practice and additional efforts are needed to optimize and standardize ITI therapy. Solid evidences concerning ITI treatment in other patient groups (i.e., patients with bad prognostic factors at first ITI course, adults with long-standing inhibitors or patients candidate to a rescue ITI) are scant and the premature closure of the randomized Resist study (Gringeri A, Haemophilia 2007) betrays the challenge of conducting rigorous studies in this setting. At this stage, consensus definitions of clinical and laboratory endpoints in the different subsets of patients undergoing ITI are required because these would allow more reliable data analyses and comparisons between studies, furthermore, prompting the initiation of international prospective surveys able to collect more homogeneous data during a prolonged follow-up period. Based on this background, the aim of this project proposal is to provide consensus definitions of ITI outcome standardizing the methodologies for the assessment of treatment endpoints. An additional aim will be to establish harmonized strategies for ITI therapy in inhibitor patients belonging to different and well defined prognostic groups. The rationale, the methodology and the workflow of the project was presented and is posted on the ISTH SSC website. Prophylaxis in hemophilic patients with inhibitor. Carmen Escuriola (Germany) Increasing more long term prophylaxis is applied in inhibitor patients using Novo 7 or FEIBA alike. Novo 7 is only licensed for prophylaxis very few countries. Both treatment options lead to a reduction of ABR by approximately 60% independent of the status of the inhibitor patients, i.e. early secondary, secondary/tertiary. The down-side, though is that there is an unpredictable response, the long term outcomes as to protection from joint degeneration, not measurable factor levels and the incidence of thromboembolic events. It is strongly requested to optimize prophylaxis regimen/dose levels and establish a clear analysis of cost/ benefit ratio. The details of the composition and mandate of this project group is posted on the ISTH SSC website. Prophylaxis in hemophilic patients without inhibitor. Victor Blanchette (Canada) Dr. Blanchette described the current status of prophylaxis in patients with hemophilia. He presented data to drive home the point that despite decades of observational data and more recent data from prospective clinical trials, randomized or otherwise, there is no clear directions on how prophyalxis should be initiated. To address this situation a project group has been created. This group will look at the following issues that are relevant to long-term prophylaxis in persons with hemophilia and will focus on the following key areas: definitions of prophylaxis; when and how to start prophylaxis; dose regimens for use in prophylaxis; strategies for adjusting prophylaxis regimens in different patient cohorts e.g. children, adolescents, adults; outcome measures relevant to long-term prophylaxis aimed to prevent/delay progression of bleed related arthropathy; potential impact of prophylaxis on adverse outcomes other than joint damage e.g. intracranial hemorrhage, inhibitor formation; and adherence to prophylaxis regimens and factors that influence adherence. The potential impact of long-acting factor VIII/IX concentrates on prophylaxis in the future will also be reviewed and discussed. The details of the composition and mandate of this project group is posted on the ISTH SSC website.
by F. Peyvandi
Wednesday, June 19, 2013
2012 Annual Minutes Locked Topic 0 F. Peyvandi Factor VIII and IXSubcommittee Minutes Friday, 29th June, 2012 Chairman: Flora Peyvandi (Italy) Co-Chairmen: Jan Astermark (Sweden), Kathelijn Fischer (The Netherlands), Claude Negrier (France), Steven Pipe (USA), Midori Shima (Japan), Leonard Valentino (USA) 09.30 REPORT ON SSC-FVIII&FIX ACTIVITY 2011-2012 – Flora Peyvandi (Italy) Dr Peyvandi opened the SSC session by thanking Dr. Alok Srivastava for the outstanding work done in the last 4 years as chair and co-chair of this subcommittee, and Drs. Charles Hay, Johannes Oldenburg and Prof. Edward G Tuddenham for their valuable contribute as co-chairs; and she welcomed Dr. Steven Pipe as new co-chair wishing him a fruitful collaboration. She reported about the state of the art of the FVIII&FIX SSC: Recommendation submitted as Official SSC Communications to JTH: Project on Consensus definitions in rare bleeding disorders (Chair: F. Peyvandi) International Standards under submission as Official SSC Communications to JTH: 2nd WHO International Standard for Factor VII concentrate4th international standard for Factors II & X, concentrate Reports posted on the ISTH website for further comments and discussion Project on Consensus definitions in haemophilia (Chair: V. Blanchette)Project on Pharmacokinetics (Chair: P. Collins)Project on Potency labelling of clotting factor concentrates (Chair: A. Hubbard)Project on Standardization of methods for performing the clot wave form analysis (Chair: M. Shima)Project on Standardization of methods for performing the thromboelastogram (Chair: G. Young and M. Chitlur) Report soon available on the ISTH website: Project on Standardization of methods for performing the thrombin generation test (Chair: C. Negrier) On-going project: Clinical trial design for haemophilia (Chair: D. DiMichele) Dr Peyvandi presented her proposals shared with the co-chairs and invited participant to propose others. Dr Peyvandi presented two new projects: Standardisation of laboratory tests for evaluation of FVIII / FIX inhibitor by-passing agents (using global assays) and novel drugs (using one stage/chromogenic and global assays), chaired by Drs Andrew Lawrie (UK) and Armando Tripodi (Italy)Bleeding score in haemophilia: a prognostic tool for clinical outcome, chaired by Drs Elisa Maria Mancuso (Italy) and Alberto Tosetto (Italy). 09:40- 10.20 PROJECTS REPORTS I Session Chairpersons: Guy Young (USA) and Alok Srivastava (India) Dr Young introduced the session. Three years ago, the SSC formed 3 Working Parties (now called Project Groups hereafter PG) aimed at providing standardized methodologies for global hemostasis assays. The SSC chose to focus on 3 assays: clot waveform analysis, thromboelastography, and thrombin generation assays. The PG chairs were selected by the SSC FVIII/FIX subcommittee and the chairs formed their respective groups. Each group was tasked with reviewing the literature on their specific assay and to recommend a standardized approach for the methods of each of these assays as it applies to hemophilia and rare bleeding disorders. The groups have recently completed their work and presented their final recommendations. 09:40 Standardization of whole blood viscoelastic measurement of clot formation and clot stability; Meera Chitlur (USA) Thromboelastography is based on the assessment of the viscoelastic properties of whole blood during the dynamic process of clotting. There are two devices that are currently available, the TEG®5000 Analyzer and ROTEM®Gamma. The devices rely on the same principles and while there are some mechanistic differences, the clot parameters that are produced are essentially the same. The following are the basic recommendations for using the devices that will be discussed. Blood collection: Blood should be collected atraumatically with no or minimal tourniquet application into either standard sodium citrate (kaolin or INTEM) or into citrated tubes with pre-loaded corn trypsin inhibitor(CTI) 0.1 mg/mL for EXTEM or tissue factor triggered assays. The sample should be run within 2 hours of collection. Recommendations for the Kaolin/INTEM Method: Intrinsic system activation is simple and has been the standard method for thromboelastogrpahic analysis in the operating room setting for many years. For the (TEG®5000), 1 mL of whole blood should be transferred into the kaolin vial and 340?L then transferred into the TEG cup into which 20?L of calcium chloride had been placed. For the ROTEM®Gamma, the blood is transferred directly into the device for automated pipetting. Since both reagents are supplied by the manufacturers, their potency is standardized ensuring reliable results. Recommendations for the Tissue Factor method: Tissue factor has been used as an activator since it is deemed to be a more physiologic representation of the in vivo coagulation process and has been adopted for use in hemophilia. Utilizing the commercially available TF Innovin®, 2 dilutions have been studied, a high concentration of approximately 0.35pM (1:17,000 dilution) and a low concentration of approximately 0.15pM (1:42,000 dilution). Which concentration is best remains unresolved. We suggest performing preliminary studies with both dilutions to determine the most appropriate one for the specific study one is performing. Once prepared, 20µL of the dilute TF is placed in the specimen cup, followed by 20 µL of calcium chloride for recalcification of the citrated blood sample. Since the maximum volume in the specimen cup cannot exceed 340 µL, 300 µL of blood is added instead of 340 µL as with Kaolin in the TEG®5000. The same procedure may be applied to both the TEG®5000 and the ROTEM®Gamma with manual pipetting. Otherwise, for the ROTEM®Gamma, the EXTEM reagent may be used as the extrinsic pathway activator, but it is important to be aware that the results may differ from that obtained using Innovin® described above as the activators are not identical. Comparison of the TF and Kaolin methods: This remains a much debated and controversial question since both activators have strengths and weaknesses. The advantage of kaolin or INTEM is that the method is very simple and the reagents are standardized. The main disadvantage is the non-physiologic nature of contact system activation. The advantage of TF is the physiologic activation of clotting while the disadvantages include the lack of standardization of the potency of the TF and the requirement with the TEG®5000 of making a "home-made” TF reagent using commercial TF intended for other uses. In a study comparing kaolin to TF, kaolin performed as well as a low concentration of TF and better than a higher concentration. Conclusion: This WP has concluded that thromboelastography can be performed with either of the available instruments and that both the contact activation and tissue factor activation methods continue to be used in clinical trials. Further research to determine a correlation between the laboratory results and clinical outcomes are required before these assays can be recommended for clinical use. 09:50 Standardization of methods for performing the thrombin generation test; Claude Negrier (France) Abstract will be soon available 10:00 Standardization of methods for performing the clot wave form analysis; Midori Shima (Japan) Clot waveform analysis (CWA) is based on the continuous monitoring of light transmittance or absorbance during routine aPTT. The clot waveform can be illustrated using the various aPTT reagents, 0.025mM CaCl2, and reference plasma. However, the reagent validated for FVIII or FIX measurement is recommended. Furthermore, it is possible to perform tissue factor triggered CWA. The complete clotting process recorded in the CWA is categorized into the three parts; the pre-coagulation phase, the coagulation phase and the post-coagulation phase. Pre-coagulation is described as the first segment of the trace, from the beginning of the signal to the onset of coagulation. Usually, this phase is horizontal, except in waveforms in patients with DIC induced by septicemia. After the onset of coagulation, light transmittance is decreased in association with the formation of fibrin and is defined by a slope in the waveform. At the end of coagulation, light transmittance tends to stabilize and is characterized again by a linear segment. The advantages of utilizing CWA are provided by the quantitative assessment of various parameters derived by mathematically processing the waveform data. Slope 1 is initial slope in the pre-coagulation phase, before the onset of coagulation. tmin2 is the time at the onset of coagulation, and min2 is the minimum value of the second derivative of the transmittance. The absolute value of min2 (|min2|) reflects maximum coagulation acceleration. tmin1 is the time at the mid-point of the coagulation phase. min1 is the minimum value of the first derivative of the transmittance. The absolute value of min1 (|min1|) reflects the maximum coagulation velocity. tmax2 is the time at the end of the coagulation phase, and is recorded at the maximum deceleration rate of coagulation. Max2, is derived from the second derivative. Delta is amplitude of signal change, and this value reflects fibrinogen concentration. Several currently available coagulometers can be utilized for CWA if the raw data of light transmittance or absorbance can be extracted. Relative transmittance is utilized to derive the waveform, and this is readily calculated using standard statistical software. The first derivative of the relative transmittance is then used to describe coagulation velocity, and similarly, further calculation of the second derivative gives a measure of coagulation acceleration. These values can be calculated easily using straightforward statistical algorithms provided in computer programs such as Microsoft Excel DISCUSSION Questions and comments from the audience: It should be appropriate to standardize the global assays in other haemorrhagic disorders than haemophilias. 10:20-11.00 PROJECT REPORTS II Session Chairpersons: Flora Peyvandi (Italy) and Michael Makris (UK) Dr. Makris introduced the session. The Project reports II Session presented the deliberations around the clinical trial design in haemophilia. The small number of patients, especially of children with haemophilia B, poses particular problems to manufacturers, the haemophilia community and regulators. The first two presentations addressed the new developments in clinical haemophilia trial regulations in the USA and Europe. The final presentation was an update of the discussions by the project group on the optimal design of haemophilia trials. FDA and EMEA regulatories: clinical trials requirements: 10:20 New regulation of FDA for rare disorders; Nisha Jain (USA) Under the Federal Food, Drug and Cosmetic Act a rare disease or condition means any disease or condition which affects less than 200,000 persons in the United States, or affects more than 200,000 in the United States and for which there is no reasonable expectation that the cost of developing and making available in the United States a drug for such disease or condition will recovered from sales in the United States of such drug. Since the implementation of the Orphan Drug Regulations FDA has reviewed over 3,350 requests for orphan-drug designation of drugs for rare diseases and conditions. FDA now proposes revisions to this regulation. These revisions include (1) demonstration of an appropriate ‘‘orphan subset’’ of persons with a particular disease or condition that otherwise affects 200,000 or more persons in the United States, for the purpose of designating a drug for use in that subset; (2) eligibility for orphan-drug designation of a drug that is otherwise the same drug for the same orphan indication as a previously approved drug; (3) eligibility for multiple orphan-drug exclusive approvals when a designated orphan drug is separately approved for use in different subsets of the rare disease or condition; (4) requirement for demonstrating clinical superiority for the purpose of orphan-drug exclusive approval; (5) requirement for submitting the name of the drug in an orphan-drug designation request; (6) required drug description and scientific rationale in a designation request and other minor revisions. These revisions are expected to clarify the existing regulations. 10:30 New European guidelines for clinical trials in rare disorders; Anneliese Hilger (Germany) The EU-requirements on clinical development for Factor VIII and Factor IX products are laid down in guidelines and core Summary of Product Characteristics. The guidelines cover clinical investigations to be conducted pre- and post-marketing authorisation. Guidance is also provided for authorised products where a significant change in the manufacturing process has been made. Clinical trials, addressing efficacy and safety are required in patients of all age groups for an application for a marketing authorisation. In addition, depending on the type of factor product (e.g. novel protein modifications) studies in previously untreated patients should be performed to investigate efficacy and safety in this specific patient population. In view of the limited availability of patients suffering from haemophilia, data from pre-authorisation studies only are considered insufficient to estimate all aspects of therapy with factor FVIII/IX products, especially with respect to immunogenicity. Therefore, to collect additional clinical data and to ensure consistency in the long-term between the outcome from pre-authorisation clinical studies and from routine use, a post-marketing investigation should be performed. The clinical development for factor FVIII/IX products should follow a stepwise approach in order to have some experience in adults and older children before investigating younger children. The clinical investigation in children needs to be supported by an approved paediatric investigation plan. The guidelines exist since more than 10 years and have been recently revised to fulfil progressing legal, scientific and regulatory requirements. The new guidelines became into operation in February 2012. 10:40 Clinical trial design for haemophilia; Donna DiMichele (USA) Dr. Peyvandi informed the audience that unfortunately Dr. DiMichele was not able to reach the session to present her results. Dr. Srivastava made a brief summary of the on-going activity of this project. The Clinical Trial Design for Hemophilia is a project group (PG) of the Factor VIII/IX Subcommittee of the SSC. The PG began its deliberations in February 2011. The aim of this PG is to develop a set of recommendations for the optimal design of pre- and post-authorization clinical studies and trials for new clotting factor concentrates (CFCs) for hemophilia A and B. Clinical trial design recommendations will be based on four priority considerations: 1) the harmonized safety and efficacy data required by regulators for product registration; 2) the post-licensure information on product safety and efficacy required by all stakeholders; 3) the realistic number of eligible and available study subjects for pre- and post-registration studies in hemophilia A and B; and 4) the availability of innovative clinical trial design strategies and models that may be suitable for rare diseases such as hemophilia. In an effort to ensure that its recommendations are relevant and based on scientific rationale and evidence, the PG is seeking guidance from all stakeholders. Its deliberations within the PG itself are being informed by clinical investigators, immunologists, clinical trial methodologists, and representatives of the FDA and EMA. The PG is also soliciting input through consultation from hemophilia physicians, patients and the biologics industry. Small clinical trials, such as those conducted in the rare bleeding disorders, require specific approaches to clinical trial design and statistical evaluation. Therefore the initial approach to this task has been to consider the basis for existing requirements while exploring alternative clinical trial methodology (e.g. Bayesian and Adaptive Design) and statistical modeling for the design of pre-licensure trials for new unmodified and novel FVIII, FIX and FVIII/FIX bypassing therapeutics in PTPs. PUP studies will subsequently be considered. With the goal of study optimization, the PG is examining the impact of these alternative strategies on the type and number of subjects, as well as the CFC exposure days required to achieve the current safety and efficacy endpoints for product authorization. As part of this exercise, the group will also evaluate the statistical targets for the pre-licensure determination of product safety (defined by neoantigenicity) for both novel and unmodified FVIII and FIX CFCs. Additionally, the PG is considering the feasibility of using post-licensure studies to validate current immunological definitions of neoantigenicity and to study emerging immunological biomarkers of treatment-related antibody development for future incorporation into exploratory clinical trial design models. The PG is examining the current tenets of clinical efficacy determination in a similar way. In collaboration with the Definitions PG, this PG is pursuing the potential implementation of more precise definitions for subject inclusion criteria and clinical outcome endpoints as a way to maximize data generation on clinical efficacy in pre-registration studies. Furthermore, the group will consider the possible role of surrogate markers (e.g., pharmacokinetics) in ascertaining clinical efficacy in pre-registration trials when complimented by mandatory rigorous data collection on clinical effectiveness through prospectively designed post-licensure studies. All activities are ongoing and the PG’s final report will be presented at the SSC meeting in 2013. DISCUSSION Questions and comments from the audience centred on the following areas: Number of requested patients affected with haemophilia B and other rare disorders should be differentiated from haemophilia A, since these disorders are less prevalent compared to haemophilia A.It is necessary to gain different request from regulatory bodies on the number of patients to involve.The regulators indicated they will consider a staged process of trials prior to registration.Regarding the safety and immunogenicity of products in PUPs study, there was some discussion whether it is reasonable to treat PUPs for 50 exposure days or it is better to make it shorter and follow them with post-marketing trial.The definition of an exposure day when dealing with products with prolonged half-life was questioned; no answer to this comment.It was pointed out that major problems with inhibitors, such as those seen in the Netherlands in the early 1990s, will be easy to detect in phase 2/3 trials. However to detect subtle changes will need very large numbers and the trial requirements are far too small to detect this. The number of minimum exposure days has been discussed since there is no evidence for this choice. 11.10 – 12.00 PROJECTS REPORTS III Session Chairpersons: Steve Kitchen (UK) and Yesim Dargaud (France) Dr. Dargaud introduced the session. 11:10 Using pharmacokinetics to individualize treatment: update; Peter Collins (UK) Prophylaxis in severe haemophilia A is usually prescribed on the basis of weight and this strategy has been shown to result in good short and long term outcomes.[1,2] However, because the pharmacokinetics (PK) of factor VIII (FVIII) varies between patients, weight-based dosing results in markedly different FVIII trough levels between individuals.[3,4] Furthermore, because half-life increases with age5 the trough levels achieved by standard weight-based prophylaxis are, on average, higher in adults than children.[3] Trough FVIII is related to breakthrough bleeding during prophylaxis, although the level that is appropriate for an individual is likely to vary dependent on numerous patient-related and environmental factors. However, if tailoring prophylaxis to an appropriate trough level is desirable then knowledge of the patient’s PK will be useful. Furthermore, tailoring prophylaxis using individual PK, in addition to observed bleeding pattern and activity, is likely to result in more cost effective treatment and potentially expand access to prophylaxis in countries where health care budgets are constrained.[6] The standard way to measure FVIII PK requires a washout and [8-10] FVIII measurements over a 48 hour period.[7] Calculation of the half-life and assessing the implications of this result for dosing prophylaxis is a complex undertaking beyond the ability of most haemophilia centres. Population PK and Bayesian analysis can be used to measure FVIII PK without a washout using a sparse sampling schedule. Measuring FVIII levels 3 times in a 48 hour period gives almost as much information as the full [8-10] point schedule.[8] This information can be used to estimate the effect of prophylactic regimens on trough FVIII levels and tailor the regimen to a target level appropriate for the individual. These techniques have been used routinely for many years to monitor and adjust the dose of drugs such as aminoglycosides.[9] Recently a population PK model of rFVIII (Advate) has been published.[5] This model covers both adult and paediatric patients and can therefore be used to measure PK with sparse sampling in all age groups. The model is suitable for all standard rFVIII preparations but cannot be used to measure PK in people with low titre FVIII inhibitors or for assessing longer half-life molecules. The introduction of this population PK model into routine practice is limited because, at present, user friendly software that can be used by haemophilia centres in not available. A potentially useful programme called TCIwork® has been developed at Otago University and initial studies suggest that it may be applicable to haemophilia.[10] However, because the programme does not use age as a covariate in the analysis, it is not suitable for all patients with haemophilia. The programme is currently being updated to allow age to be included and this programme is being investigated in the context of haemophilia. A manuscript describing the measurement of PK with sparse sampling using population PK and Bayesian analysis has been prepared and submitted for review. An appendix will be made available giving detailed instructions on how to download, install and use TCIworks once the updated version becomes available. Conclusions: FVIII PK can be measured by Bayesian analysis and population PK models using about 3 samples taken at appropriate times after an infusion. No washout is needed and the times of the samples can be flexible. This information can be used to help dose prophylaxis in haemophilia A and potentially make dosing much more cost effective. Simple to use software to calculate PK and prophylaxis dosing is potentially available but is being updated so that it is applicable to haemophilia. This software will need to be validated in routine practice. Studies on the use of PK tailored prophylaxis are required to establish the safety and efficacy of this approach. A document describing population PK as it applies to haemophilia has been posted on the ISTH website for comments and this will be revised after the meeting on the basis of any feedback. References Nilsson IM, Berntorp E, Löfqvist T, Pettersson H. Twenty-five years‘ experience of prophylactic treatment in severe haemophilia A and B. J Int Med. 1992;232:25-32.Manco-Johnson MJ, Abshire TC, Shapiro AD et al. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe haemophilia. N Engl J Med. 2007;357:535-544.Collins PW, Björkman S, Fischer K et al. Factor VIII requirement to maintain a target plasma level in the prophylactic treatment of severe haemophilia A: influences of variance in pharmacokinetics and treatment regimens. J Thromb Haemost. 2010;8:269-275.Collins P, Faradji A, Morfini M, Enriquez MM, Schwartz L. Efficacy and safety of secondary prophylactic versus on-demand sucrose-formulated recombinant factor VIII treatment in adults with severe hemophilia a: results from a 13-month crossover study. J Thromb Haemost. 2010;8:83-89.Björkman S, Oh M, Spotts G, Schroth P et al. Population pharmacokinetics of recombinant factor VIII: the relationships of pharmacokinetics to age and body weight. Blood. 2012;119:612-618.Collins PW, Fischer K, Morfini M, Blanchette VS, Björkman S. On behalf of International Prophylaxis Study Group (IPSG) Pharmacokinetics Expert Working Group. Implications of coagulation factor VIII and IX pharmacokinetics in the prophylactic treatment of haemophilia. Haemophilia. 2011;17:2-10.Lee M, Morfini M, Schulman S, Ingerslev J and the Factor VIII/Factor IX Scientific and Standardization Committee of the International Society for Thrombosis and Haemostasis. The design and analysis of pharmacokinetic studies of coagulation factors. http://www.med.unc.edu/isth/ssc/communications/factor8and9/fviiipharmaco.pdfBjörkman S, Folkesson A, Jönsson S. Pharmacokinetics and dose requirements of factor VIII over the age range of 3-74 years: A population analysis based on 50 patients with long-term prophylactic treatment for haemophilia A. Eur J Clin Pharmacol. 2009;65:989-998.Ruffo S, Messori A, Grasela TH et al. A calculator program for clinical application of the Bayesian method of predicting plasma drug levels. Comput Progr Biomed. 1985;19: 167-177.Björkman S. Evaluation of the TCIWorks Bayesian computer program for estimation of individual pharmacokinetics of FVIII. Haemophilia 2010 (e-published 22 August) 11:20 Potency labelling of clotting factor concentrates: update; Anthony Hubbard (UK) Since development of the World Health Organisation International Standards (WHO IS) for Factor VIII (FVIII) and Factor IX (FIX) Concentrates all plasma-derived and recombinant therapeutic concentrates have been labelled in International Units (IU) defined by in vitro biological activity. The development of new products, with novel properties introduced through structural or chemical modifications (e.g. truncation, pegylation, fusion), may challenge this traditional approach and present different routes of potency labelling with the risk of discordance between licensing authorities and subsequent confusion to users. In order to address this issue the project group has considered the options for the potency labelling of new FVIII and FIX concentrates and have drafted recommendations for manufacturers and regulators. The project has also attempted to reconcile the methods used for potency labelling with the local practices for post-infusion testing by clinical laboratories. The draft recommendations are presented in four sections and are summarised below: Manufacturer’s characterisation of new product potency. This section recommends a thorough characterisation of the new products by clotting and chromogenic methods, relative to the WHO IS, in order to determine if statistically valid estimates are possible and to identify methods discrepancies and/or the influence of different reagents. Valid assays of modified products relative to the WHO IS, in terms of parallel and linear dose/response relationships, supports potency labelling in IU. Where there are methods-based potency discrepancies it will be necessary for licensing authorities to agree on the approach to potency labelling. Where assays relative to the WHO IS are invalid it may be necessary to label in product-specific units defined by a product reference.Calibration of manufacturer’s product reference. Depending on the validity of assays relative to the WHO IS, the product reference should be calibrated in IU relative to the WHO IS or in product-specific units.Manufacturer’s pharmacokinetic studies. It is recommended that pharmacokinetic studies should include both clotting and chromogenic methods with potency estimation relative to both the product reference and a plasma reference. The pharmacokinetic study should provide information on the relationship between the infused dose, based on the labelled potency, and the expected measured recovery in the patient when different methods and references are used on post-infusion samples. This information should be made available to clinicians and may allow the use of local methods and reference materials.Post-infusion testing in clinical laboratories. Although the optimal approach to quantification of post-infusion samples requires testing relative to a product reference it is recognised that this may be difficult to implement in a routine clinical laboratory. Local assay systems could be used subject to manufacturer’s guidance on the interpretation of assay results. 11:30 Consensus definitions in haemophilia; Alok Srivastava (India) This project group charged with the mandate of developing definitions of clinical events and endpoints in clinical studies has completed its work as described last year. It has carried out an extensive review of the existing literature combined with discussions with a range of stake holders including expert physicians around the world, patients, industry representatives and regulators. Apart from endorsing the existing definitions of the severity of the condition and high and low titre inhibitors, new definitions are being provided for significant inhibitor titres as well as transient and persistent inhibitors. Factor replacement protocols including different types of prophylaxis are also being defined. In addition joint bleeds, rebleeds, target joints and muscle bleeds are being defined. Responses to treatment including that of joint bleeding and adequacy of surgical hemostasis are being covered. 11:40 Consensus definitions in rare bleeding disorders; Flora Peyvandi (Italy) Over the past five years, the "Consensus definitions in Rare Bleeding Disorders” Project focused on the need for comprehensive data collection regarding the association between laboratory phenotype and bleeding severity in RBDs. Different networks and registries, established in Europe (European network of Rare Bleeding Disorders [EN-RBD]), United Kingdom (United Kingdom Haemophilia Centre Doctors' Organisation registry [UKHCDO]), USA (The North American Rare Bleeding Disorders Registry [NARBDR] and the American Thrombosis Hemostasis Network [ATHN] Rare Coagulation Disorder database) and India started to collaborate with an initial task of understanding what data are currently available through each system. We report the results of a review of available data that explored the association between residual plasma factor activity and the clinical bleeding profile for each of the RBDs. The results are based on three different sets of data: A detailed review of the available English language literature from 1990 to March 2012 (search engine: Medline PubMed), this included all reports evaluating the laboratory phenotype and clinical characteristics and having a sample size ?5 patients: a total of 51 relevant original publications were retrieved;Overview of data from the UKHCDO, NARBDR and Indian registries on a total of 3745 patients affected with RBDs. The laboratory phenotype severity was categorized differently by the three registries. Clinicians reporting to the UKHCDO registry are not asked to classify RBDs by severity and do not report bleeding events. For this analysis data were analysed using the criteria defined in the Table. A minority of reported patients had severe deficiency (level not always reported).Overview of data from the EN-RBD, which evaluated the association between residual plasma factor activity as a continuous metric and clinical bleeding severity in 489 patients with different RBDs. Based on the interpretation of published data and the experiences from the networks and registries, it was evident that: There is a heterogeneous association between coagulation factor activity level and clinical bleeding severity in different RBDs. The strongest association was observed for fibrinogen, FX and FXIII deficiencies; although different thresholds are needed to ensure patients remain asymptomatic or to prevent major spontaneous bleeding.These data indicate that a more detailed evaluation on each single factor deficiency is required for future planning of optimal diagnosis and management; and that it is not appropriate to use a single criterion of classification for all types of RBDs. The results of this project were submitted as Official SSC Communications to the Journal of Thrombosis and Haemostasis with a manuscript entitled "Classification of rare bleeding disorders (RBDs) based on the association between coagulant factor activity and clinical bleeding severity”. Laboratory phenotype classifications according to the three registries, compared to the laboratory classification of haemophilia. DISCUSSION The audience has been asked to comment on the manuscripts reporting results of these projects available on the ISTH website. All manuscripts should be hopefully published within 2012. Saturday, 30th June, 2012 9.00 – 10.10CLINICAL ISSUES Session Chairpersons: Marilyn Manco-Johnson (USA), Carmen Escuriola Ettinghausen (Germany) Dr Escuriola introduced the session. The Clinical Issues Session addressed the most recent insights into inhibitor incidence, its risk factors, the potentially protective effect of prophylactic treatment, treatment of patients with inhibitors, and provided information on on-going studies on the same and related topics. 09:00 The incidence of inhibitors in long terms experience in prophylaxis; Erik Berntorp (Sweden) Eric Berntorp focusses on the incidence of inhibitors in long terms experience in prophylaxis. Inhibitors develop in about 30% of patients with severe hemophilia A and up to 5% of those with severe hemophilia B (1). Various issues might affect inhibitor development, including type of hemophilia, factor VIII/IX mutation type, race, immune response genes and environmental factors. These factors include type of replacement therapy i.e. plasma derived vs. recombinant, purity of products, treatment regimen and age at start of treatment. Recently the risk of inhibitor development in hemophilia A in relation to long-term prophylaxis has gained great interest and some studies have indicated a protected effect of prophylaxis (2,3) although certainly more studies are needed to be convincing (4). The so called danger theory proposed by Matzinger (5,6) has shead new light on the idea that early, long-term prophylaxis may prevent inhibitor development. If the inhibitor response leading to inhibitor development is an effect of immunological danger to the organism, rather than self-versus non-self, it is appealing to believe that less risk of bleeding during prophylaxis compared to on demand treatment gives less inflammation and less danger to the immune system. Striking results along this line have been published from Germany where institution of early prophylaxis in low dose and avoidance of obvious danger signals have dramatically reduced the inhibitor incidence (7,8). This preliminary experience has prompted a larger study Early Prophylaxis Immunologic Challenge (EPIC) Study (ClinicalTrials.gov NCT01376700) where patients (FVIII?2%) up to the age of one year will start on weekly prophylaxis 25±5 FVIII IU/kg and minimization of immunological danger signals very similar to what has been reported by Auerswald and colleagues. It is hard to believe that such a regimen will abolish the risk of inhibitor development, especially in patients with high risk FVIII mutations, but even a reduction by a few % will mean a lot for patients, their families and for society. References Astermark J. Overview of inhibitors. Seminars in hematology. 2006;43(2 Suppl 4):S3-7.Gouw SC, van der Bom JG, Marijke van den Berg H. Treatment-related risk factors of inhibitor development in previously untreated patients with hemophilia A: the CANAL cohort study. Blood. 2007;109:4648-4654.Santagostino E, Mancuso ME, Rocino A et al. Environmental risk factors for inhibitor development in children with haemophilia A: a case-control study. Br J Haematol. 2005;130:422-427.Calvez T, Laurian Y. Protective effect of prophylaxis on inhibitor development in children with haemophilia A: more convincing studies are required. Br J Haematol. 2006;132:798-800.Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol. 1994;12:991-1045.Matzinger P. The danger model: a renewed sense of self. Science. 2002;296:301-305.Auerswald G, Bidlingmaier C, Kurnik K. Early prophylaxis/FVIII tolerization regimen that avoids immunological danger signals is still effective in minimizing FVIII inhibitor developments in previously untreated patients--long-term follow-up and continuing experience. Haemophilia. 2012;18:e18-20.Kreuz E, Ehrenforth S, Funk M et al. Immune tolerance therapy in paediatric haemophiliacs with factor VIII inhibitors: 14 years follow-up. Haemophilia. 1995;1:24-32. 09:10 Research of determinants of inhibitor development among previously untreated patients with haemophilia (RODIN study); Marijke van den Berg (The Netherland) Marijke van den Berg presents on behalf of the RODIN Study group (www.rodinstudy.eu) the first findings from the RODIN Study. In the RODIN study, prospective data were collected from 29 haemophilia centres. The first analysis of May 2011 concerned 606 PUPS born between 2000 and 2010 with severe haemophilia A. In total 179 patients (32.0%) developed inhibitors, of whom 118 (22.2%) with high titre inhibitors. The rather high total inhibitor incidence can be explained by the fact that in this study all eligible patients were included, which means also patients with major bleedings in the neonatal period. Prophylaxis in severe haemophilia A has been associated with a decreased risk of inhibitory antibodies. In 412 patients prophylaxis was started during the first 75 exposure days, but only a small decrease of inhibitor incidence was demonstrated. In the early phase of factor VIII treatment, inhibitor incidence was not associated with prophylaxis; after about 20 exposure days, however, prophylaxis was associated with a slightly decreased inhibitor incidence. The relative risk of prophylaxis was 1.01 after 1 to 10 exposure days, 0.95 after 11 to 20 days, 0.22 after 21 to 30 days, 0.27 after 31 to 40 days and 0.32 after 41 to 75 exposure days. An effect of prophylaxis on inhibitor incidence was only observed for patients with low risk gene mutations. Furthermore, peak treatment moments of at least 5 days were associated with a 50% increased inhibitor incidence, while dose and frequency of dosing were not associated with an increase in incidence. 09:20 Extending prophylaxis around the world – What doses can we start with?; Alok Srivastava (India) Dr Srivastava apologises for declining to make this presentation. 09:30 Immunotolerance induction using plasma derived products; Carmen Escuriola Ettinghausen (Germany) Carmen Escuriola Ettinghausen presented the newest insights on immune tolerance induction using plasma derived products. Immune tolerance induction (ITI) is a powerful approach to eliminate inhibitors. Success rates of ITI may vary depending on patient variables (e.g. inhibitor titre at start of ITI, maximum inhibitor titre, F VIII genotype etc) and on factors related to the therapeutic regimen (e.g. age at start of ITI, Interval between dx and start of ITI, dosage and frequency of F VIII etc). The product chosen for ITI particularly the presence of von Willebrand Factor (VWF) may play a supportive role in inducing immune tolerance to FVIII. Reports from Germany showed stable success rates around 90% using VWF-containing concentrates in the frame of the Bonn protocol (1) But in the early 1990s a substantial decline of ITI success rates to 29% after the introduction of F VIII concentrates lacking of VWF with an unchanged use of the Bonn protocol was observed (2,3). After a switch to VWF/FVIII complex concentrates the overall success rates of ITI of around 90% have been achieved again (2). Since then VWF/FVIII complex concentrates have been used particularly in high responders with poor prognosis or as a salvage therapy after earlier ITI failure: complete or partial success could be achieved in 50 to 100% of these patients (4-9). In vitro data showed a lower inhibitory activity against FVIII complexed with VWF compared to pure F VIII inhibitor plasmas with anti-C2-specifity (10-14). A higher recovery was observed when the infused F VIII concentrate contained VWF during the treatment of patients with haemophilia A and inhibitors against the light chain of F VIII (10,15). An association between the clinical outcome of ITI with the inhibitor epitope profile and the type of concentrate used has been described in a small patient cohort: in inhibitor patients with an anti-C2 specificity treated with a VWF-containing F VIII concentrate the inhibitor could be successfully eliminated whereas those who had an anti-A2 specifity failed ITI significantly more often. These findings suggest the possibility to tailor ITI to the individual demands of the inhibitor patient by testing the inhibitor epitope specificity or the inhibitory activity against different types of F VIII concentrates. More results from the RESIST- as well as the ObsITI- study are warranted to evaluate success of ITI and to determine patient- and therapy-related variables influencing ITI outcome particularly such as the type of F VIII product. References Brackmann HH, Oldenburg J, Schwaab R. Immune tolerance for the treatment of factor VIII inhibitors – twenty years `bonn protocol`. Vox Sang. 1996;70(Suppl 1): 30-35.Kreuz W, Escuriola Ettingshausen C et al: Immune tolerance induction (ITI) in haemophilia A with inhibitors: the choice of concentrate affecting success. Haematologica. 2001; 86(Suppl 4):16-20.Auerswald G, Spranger T, Brackmann HH. The role of plasma-derived factor VIII/von Willebrand concentrates in the treatment of haemophilia A patients. Haematologica 2003;88(Suppl 9):21-25.Orsini F, Rothschild C, Beurrier P et al. Immune tolerance induction with highly purified plasma-derived factor VIII containing von Willebrand factor in haemophilia A patients with high-responding inhibitors. Haematologica. 2005;90:1288-1290.Gringeri A, Musso R, Mazzucconi MG et al. Immune tolerance induction with a high purity von Willebrand factor/VIII complex concentrate in haemophilia A patients with inhibitors at high risk of a poor response. Haemophilia. 2007;13:373-379.Kurth MA, DiMichele D, Sexauer C et al. Immune tolerance therapy utilizing factor VIII/von Willebrand factor concentrate in haemophilia A patients with high-titre factor VIII inhibitors. Haemophilia. 2008;14:50-55.Greninger DA, Saint-Remy JM, Jacquemin M et al. The use of factor VIII/von Willebrand factor concentrate for immune tolerance induction in haemophilia A patients with high-titre inhibitors: association of clinical outcome with inhibitor epitope profile. Haemophilia 2008;14:295-302.Kurth M, Puetz J, Kouides P et al. The use of a single von-Willebrand factor-containing, plasma derived F VIII product in hemophilia A immune tolerance induction: the US experience. J Thromb Haemost. 2011;9:2229-2234.Bidlingmaier C, Kurnik K, Escuriola Ettingshausen C et al. Immune tolerance induction with a factor VIII concentrate containing von Willebrand factor (Haemoctin SDH®) in 14 patients with severe haemophilia A. Haemophilia 2011;17: e831-e848.Berntorp E, Ekman M, Gunnarsson M et al. Variation in factor VIII inhibitor reactivity with different commercial factor VIII preparations. Haemophilia. 1996;2:95-96.Suzuki T, Arai M, Amano K et al. FVIII inhibitor antibodies with C2 domain specificity are less inhibitory to FVIII complexed with VWF. Thromb Haemost. 1996;76:749-754.Gensana M, Altisent C, Aznar JA et al. Influence of VWF on the reactivity of human FVIII inhibitors with FVIII. Haemophilia. 2001;7:369-374.Kallas A, Talsep T. Von Willebrand factor in factor VIII concentrates protects against neutralization by factor VIII antibodies of haemophilia A patients. Haemophilia. 2001;7:375-80.Astermark J, Voorberg J, Lenk H et al. Impact of inhibitor epitope profile on the neutralizing effect against plasma-derived and recombinat factor VIII concentrates in vitro. Haemophilia. 2003;9:567-572.Inoue T, Shima M, Takeyama M et al. Higher recovery of factor VIII (FVIII) with intermediate F VIII/von Willebrand factor concentrate than with recombinant FVIII in a haemophilia A patient with an inhibitor. Haemophilia. 2006;12:110-113. 09:40 International study project; Charles Hay (UK) He presented an administrative report and plan of future data analysis and reports. Abstract will be soon available DISCUSSION Questions and comments from the audience centred on the following areas: Dr Berntorp showed that the incidence of inhibitors increased during the period 2000-2007 compared to 1980-1999. It was hypothesized that this could be due to 1) higher immunogenicity of new rFVIII products or 2) general increment of the autoimmune disease in childrenDetails on the study methodology (quality of life, countries of origin, time interval, etc) were asked to the presenters.It was asked if analysis based on brand and types of products had been carried out. Speakers reported that huge sample size would be necessary and meta-analysis could be the appropriate study design. 10:10-10.50 STANDARDISATION ISSSUES Session Chairpersons: Raimondo De Cristofaro (Italy) and Anthony Hubbard (UK) Dr Hubbard introduced the session, explaining that Dr De Cristofaro had apologized for not being present due to personal reasons. Biological standards remain the foundation for the quantification of coagulation factors in therapeutic products. The complexity of the clotting activity of factors II, VII and X cannot be measured using physicochemical methods and their estimation relies on the relative comparison of products with the relevant WHO International Standard (WHO IS) which has an agreed assigned value in International Units (IU). The IU convention has promoted global harmonisation in the content and labelling of therapeutic concentrates for over 3 decades and the need for the replacement of dwindling stocks of WHO IS is a measure of the success of this approach. The projects presented by Drs Gray and Thelwell describe the value assignment of the replacement WHO IS for the estimation of factors II, X and VII in purified products. The estimation of inhibitory antibodies to FVIII remains a critical aspect of haemophilia care and initiatives to improve on current methodologies through facilitation of methodology and reduction of variability are required. Dr Raut presented a modification to the current Bethesda/Nijmegen method which involves the replacement of FVIII-deficient plasma by buffered normal plasma. This approach promises reduced cost of testing and the potential of reduced variability. Initial studies have indicated equivalent results to the conventional method. 10:10 4th International Standard for FII and X, Concentrate; Elaine Gray (UK) Twenty-eight laboratories from 14 different countries participated in a collaborative study to value assign the proposed 4th International Standard for Blood Coagulation Factors II and X, Concentrate (11/126) by assay relative to the WHO 3rd International Standard for Blood Coagulation Factors II and X, Concentrate (98/590). Overall intra-laboratory variability was low with over 70% of the laboratories having geometric coefficients of variation (GCV) of less than 5% indicating that the participants performed assays for factors II and X reproducibly and with high precision. Inter-laboratory variability was also low for estimates of both factors with GCV below 5%. There was a small but significant difference observed for the candidate preparation (11/126) between clotting and chromogenic methods for both factors II and X. For Factor II, the overall potency from all methods was 9.44 IU/ampoule, with inter-laboratory GCV of 3%. The estimate from the Prothrombin Time based assays (9.53) was 1% higher, and the chromogenic potency (9.24) was 2% lower. For Factor X, the overall potency was 8.13 IU/ampoule, with the Prothrombin Time and chromogenic potencies being 8.29 and 7.81 IU/ampoule (2% higher and 4% lower), respectively, and inter-laboratory GCV being 5%. Taking into consideration the low inter-laboratory variation and the relatively small discrepancies between the potencies by the different methods, it was proposed to assign the overall geometric mean potency of 9.4 IU/ampoule for FII and 8.1 IU/ampoule for FX to the proposed 4th International Standard for Blood Coagulation Factors II and X, Concentrate (11/126). 10:20 A report on the collaborative study to calibrate the WHO 2nd International Standard for Factor VII concentrate; Craig Thelwell (UK) An international collaborative study was organized to calibrate a replacement for the WHO 1st International Standard for Factor VII concentrate (97/592). The study involved 24 laboratories from 11 different countries representing manufacturers, clinical and regulatory groups. Laboratories were asked to measure the FVII content of two freeze-dried candidate materials: sample A (10/250) and B (10/252), using clotting and/or chromogenic methods. For each laboratory, potencies were calculated for A and B relative to the WHO 1st IS, and the geometric mean potency was calculated for each of the candidates independently for clotting and chromogenic methods. Statistical analysis revealed significant differences in potencies determined by clotting and chromogenic methods for both candidates. In addition there was a significant assay method bias within the clotting results for candidate A, with laboratories using recombinant thromboplastin reagent producing lower potencies compared to those using a natural purified thromboplastin. For candidate B there was no method bias observed, however the potency determined by clotting methods was significantly higher than the chromogenic potency. Clotting methods for FVII potency are sensitive to the amount of activated FVII (FVIIa) present whereas chromogenic methods are not. Candidate B was found to contain higher levels of FVIIa compared to candidate A and this may explain the relatively higher potency for candidate B determined by clotting methods. Since the difference between the potency estimates by the different methods was too large to reconcile by a mean value it was proposed that candidate B (10/252) should be established as the WHO 2nd IS for factor FVII concentrate with a potency of 9.8 IU for chromogenic methods and 10.6 IU for clotting methods. 10:30 FVIII Inhibitors Assay (SMIA): A new approach in measurement; Sanj Raut (UK) Previous studies have shown high variability between laboratories when measuring FVIII inhibitors in patient samples, with CVs ranging from 40-200%. The Nijmegen Modification is currently the gold standard inhibitor assay, in which patient’s inhibitor titres are measured relative to a "Control” mixture consisting of equal volumes of buffered normal pooled plasma and FVIII-deficient plasma (FDP). This project questioned the need for FDP, which was introduced into the assay as a "like for like” diluent for the Reference "Control” and which may actually introduce variability due to the many different FDPs now commercially available. As the inhibitor titre is based on % of FVIII in the Reference ("Control”), it was hypothesised that it may be possible to substitute the FDP with a more "like for like” diluent such as buffered normal plasma. The unknown inhibitor titre in this modified assay (South Mimms Inhibitor Assay - SMIA) is expressed relative to 200% FVIII in the Reference "Control” (rather than 100% FVIII previously). This approach may remove the variability of FDP and significantly reduce the cost of an inhibitor assay. Results showed that, for high titre samples (5 - 40 BU/ml), equivalent inhibitor titres could be obtained using the Nijmegen method and the modified method. For low titre (1.0 - 0.15 BU/ml) samples, the Nijmegen method was able to detect inhibitor titres down to ~0.6 BU/ml, whilst SMIA was able to detect inhibitor titres down to ~0.2 BU/ml. DISCUSSION Dr Sanj Raut was asked to propose a new project within the FVIII&FIX SSC on the use of the SMIA assay for the evaluation of clinical outcomes. 11:00-12:45 CRITICAL ISSUES ON EVALUATION OF EFFICACY OF TREATMENT IN HEMOPHILIA Session Chairpersons: Donna DiMichele (USA) and Steven Pipe (USA) Dr Pipe introduced the session, communicating that Dr DiMichele had not been able to attend the meeting. Global tests of blood coagulation were superseded by more specific tests as the mechanism of blood coagulation unfolded in the latter part of the 20th century. Specific assays used in clinical trials demand the use of national and international standards. Each assay result depends on the precise nature of the substance to be measured, the standard, assay system and reagents used and even the equipment employed. In particular, recombinant factors have special assay characteristics which can be difficult to manage, especially when results from clinical trials are extrapolated to the various assay platforms and standards used in clinical practice. Potency assignment and biological activity of FVIII and FIX products are expected to correlate with in vivo efficacy and plasma activity measured in clinical assays. Within the constraints of current regulatory guidelines, manufacturers have limited means of assigning potency to a novel product, either by a one-stage clotting assay in the case of FIX, or, additionally, by a chromogenic substrate assay for FVIII, both of which must be calibrated against the respective WHO standard. FVIII:C quantitation with chromogenic and the classical one-stage assay may differ up to 40% and dose adjustments may be necessary depending on the region. With the recent advent of novel, modified FVIII or FVIIa products with increased clotting efficiency and/or prolonged half-life, clinical trials are changing from replacement therapy trials to pharmacological trials with quantifiable clinical endpoints. Classical clotting or chromogenic assays to quantify the modified products may not be applicable any longer or may need assay modification (e.g. different activators). Nevertheless regulatory bodies still require objective and laboratory based evidence to support claims of efficacy and safety in clinical trials relating to the expansion of use of existing products and the development of new products. One needs to ensure that current clinical assays can accurately determine the activity of these novel products in plasma samples. This is best demonstrated through comprehensive field studies that include reagents, instruments and calibrators commonly used in clinical hemostasis laboratories. Another solution being explored is the use of global hemostasis assays such as thrombin generation assay or thromboelastography that do not measure the infused compound directly but rather quantify and qualify the response of the hemostatic system to the therapy. This approach has the potential to not only exclude product related specific assay artifacts but to also personalize therapy by individualization of dosing while maintaining effective factor levels in the patients to prevent bleeding. Global clotting assays may attain greater relevance in the description of the pharmacodynamic actions of the novel products and their ability to serve as surrogate markers for efficacy. However, standardization and pre-analytical challenges still need to be resolved. An alternative approach would be to design assays that specifically measure the procoagulant activity of modified coagulation factors. In one example, an assay was developed that allows selective determination of CHO cell derived recombinant human proteins in the presence of their human plasma-derived equivalent. These examples of new assay strategies – global hemostasis assays and highly product specific assays – have the potential to substantially improve the array of laboratory endpoints used in clinical trials investigating novel molecules to treat hemophilia and related disorders. In contrast to replacement therapy, the common clinical laboratory coagulation assays of clotting factor activity do not reflect the clinically relevant hemostatic activity of bypassing agents. Furthermore, no validated assay is available to measure the in vivo efficacy of these agents or predict individual patient responses to treatment. Recently published data from one prospective trial in haemophilia patients with inhibitors undergoing surgery have shown a correlation between in vivo clinical response to the bypassing agents and thrombin generating capacity in a TGT assay. Novel recombinant factor VIIa molecules also introduce unique assay challenges. The design of one molecule, by example, reduces the affinity of anti-FVII commercial antibodies leading to miscalculation of antigen level as compared to the native FVII. One company has developed a product specific ELISA utilizing a product specific antibody enabling a more accurate approach for its quantitation. The session discusses all of these approaches in turn. 11:00 Measuring clinical efficacy and laboratory parameters in patients treated with porcine factor VIII"; Martin Lee (Inspiration Biopharmaceuticals) The advent of recombinant porcine factor VIII has meant the renewed ability to treat patients with auto- and allo-antibodies to human factor VIII with a biological agent that allows for the direct measurement of the circulating levels of the infused material. However, this brings with it the challenge of assaying porcine factor VIII in human plasma (which is additionally compounded by the fact that this product is B-domain deleted), as well as the determination of anti-porcine antibodies (which, of course, requires a directed factor VIII assay). We have considered the effect of assay methodology (one-stage clotting assay versus chromogenic) as well as the standard used (plasma versus plasma-based concentrate versus recombinant-based concentrate). These comparisons shed light on the differences and similarities with the usual issues raised with assaying human recombinant factor VIII and inhibitors. Clearly, resolution of these factors is key to the appropriate and titrated use of porcine factor VIII in patients with either acquired hemophilia or congenital hemophilia A with inhibitors. We report on an inter-laboratory study of the assay and standard differences recorded and provide recommendations for clinical and laboratory use. In addition, we will comment on a scale that is being used to evaluate clinical efficacy when recombinant porcine FVIII is used in surgery or a serious bleeding episode in these patients. 11:10 Development of new rFVIII and rFIX molecules: clinical and laboratory evaluation of safety and efficacy; Stephanie Seremetis (Novo Nordisk) The author asked to change the tile of the presentation to: "Vatreptacog alfa: Assays in the development program” Vatreptacog is a rFVIIa analogue with three point mutations in the protease domain at amino acid positions 158, 296 and 298. Vatreptacog alfa has a conformation similar to that of tissue factor (TF) -bound FVIIa, even in the absence of TF. On the surface of activated platelets this translates to greater activity than rFVIIa, resulting in a fast and large thrombin burst and the rapid formation of a tight and stable clot. On the other hand, in the presence of TF similar activity is measured for vatreptacog alfa and rFVIIa. Bypassing agents do not restore the normal pathways of hemostasis in hemophilia, but rather boost thrombin generation in spite of a lack of platelet-surface FVIIIa/FIXa ("tenase”) activity. In contrast to replacement therapy, the common clinical laboratory coagulation assays of clotting factor activity do not reflect the clinically relevant hemostatic activity of bypassing agents. Furthermore, no validated assay is available to measure the in vivo efficacy of these agents or predict individual patient responses to treatment. Recently published data from one prospective trial in haemophilia patients with inhibitors undergoing surgery have shown a correlation between in vivo clinical response to the bypassing agents and thrombin generating capacity in a TGT assay. In our phase 2 clinical trial for vatreptacog alfa we have focussed on securing robust and reliable safety assays including measures of pharmacokinetics and potential antibody formation. For the PK analysis we used the standard FVIIa clot assay. The development of antibodies is followed closely in all clinical trial by a stepwise tiered approach using validated antibody assays. Samples are collected on a regular basis and screened for presence of antibodies binding to vatreptacog alfa using a radio-immuno assay. If any antibodies are detected these will be further characterised with respect to cross-reactivity to FVIIa and neutralising capacity. The neutralising effect of the antibodies will be investigated using two functional clot assays. One assay detects antibodies specifically neutralising vatreptacog alfa, and one assay detecting cross-reacting antibodies neutralising endogenous FVIIa. 11:20 Evolution of clinical trials in hemophilia: From simple replacement to pharmacological evidence; Rupert Sandbrink (Bayer) Dr Prasad Mathew made this presentation on behalf of Dr Sandbrink Until recently, clinical development in hemophilia was predominantly replacement therapy with wild-type factor VIII or IX concentrates or recombinant versions thereof. Pharmacokinetic demonstration of the pertaining factor activity in the plasma at expected levels and duration was usually sufficient to reassure pharmacodynamic action. With the emergence of the two-stage chromogenic substrate assay (CSA) to measure FVIII:C in hemophilia A and its adoption for release of FVIII products in the EU, issues arose for some FVIII products. FVIII:C quantitation with CSA and the classical aPTT based one-stage clotting assay may differ up to 40% and dose adjustments may be necessary depending on the region. The clinical impact of this assay issue is currently investigated in the clinical development of the full-length FVIII product BAY 81-8973. With the recent advent of novel, modified FVIII or FVIIa products with increased clotting efficiency and/or prolonged half-life (e.g. BAY 94-9027; BAY 86-6150), clinical trials are changing from replacement therapy trials to pharmacological trials with quantifiable clinical endpoints. Classical clotting or chromogenic assays to quantify the modified products may not be applicable any longer or may need assay modification (e.g. different activators). Global clotting assays such as RoTEM and TGA may attain greater relevance to describe the pharmacodynamic actions of the novel products. However, standardization and pre-analytical challenges still need to be resolved. 11:30 Correlation of Potency Assignment and Clinical Performance of rFVIII-Fc and rFIX-Fc Fusion Proteins through Field Studies, Ex Vivo Coagulation and Global Hemostasis Assays; Jurg Sommer (Biogen Idec) Potency assignment and biological activity of FVIII and FIX products are expected to correlate with in vivo efficacy and plasma activity measured in clinical assays. Within the constraints of current regulatory guidelines, manufactures have limited means of assigning potency to a novel product, either by a one-stage (OS) clotting assay in the case of FIX, or by a chromogenic substrate (CS) assay for FVIII, both of which must be calibrated against the respective WHO standard. In the case of the recombinant FVIIII-Fc fusion protein (rFVIIIFc), both the OS and CS assays provide comparable results with a specific activity equivalent to that of native FVIII. Plasma FVIII level by the OS assay may thus be an appropriate surrogate marker for monitoring the activity and pharmacokinetics of this product during the clinical trials. The in vitro specific activity of rFIXFc is approximately 50% lower than that of native FIX by both the OS and the CS assays and this activity correlates with in vitro thrombin generation activity. To further verify that the potency assignments for rFVIIIFc and rFIXFc reflect their in vivo activity, global hemostasis assays (TGA and ROTEM) are being performed as part of the pharmacokinetic analysis in the clinical efficacy (Phase III) studies. These exploratory tests are expected to demonstrate comparable ex vivo thrombin generation and hemostatic activity for rFVIIIFc and Advate® (or rFIXFc and BeneFIX®) at equivalent factor FVIII/FIX levels. Ex vivo hemostatic activity of these products should also be consistent with their respective PK profiles. Lastly, in order to successfully monitor rFVIIIFc and rFIXFc therapy in patients, one needs to ensure that current clinical assays can accurately determine the activity of these novel products in plasma samples. This is best demonstrated through comprehensive field studies that include reagents, instruments and calibrators commonly used in clinical hemostasis laboratories. 11:40 Clinical Research Challenges in Developing Novel Compounds to Treat Hemophilia: The rIX-FP and rVIII-SingleChain Experience; Debbie Bensen-Kennedy (CSL Behring) Abstract will be soon available 11:50 Standardised specific assays or general tests of haemostatic potential? Pfizer’s view on precise science or clinical relevance; Brian Colvin (Pfizer) Global tests of blood coagulation were superseded by more specific tests as the mechanism of blood coagulation unfolded in the latter part of the 20th century. Specific assays used in clinical trials demand the use of national and international standards. Each assay result depends on the precise nature of the substance to be measured, the standard, assay system and reagents used and even the equipment employed. In particular, recombinant factors have special assay characteristics which can be difficult to manage, especially when results from clinical trials are extrapolated to the various assay platforms and standards used in clinical practice. To address the latter challenge Pfizer has utilized two approaches for management of clinical FVIII coagulation factor assays. One BDD rFVIII product (Xyntha) has a potency assignment aligned to the one stage clotting assay that is commonly used in clinical practice. For the other BDD rFVIII product (ReFacto AF) a product specific assay standard has been introduced. Perhaps more challenging is the utility of factor VIII and IX assays and pharmacodynamics as surrogate markers for efficacy. Pfizer is currently considering the importance and relevance of the factor IX assay in relation to the clinical efficacy and optimal use of BeneFIX in weekly prophylaxis. Preliminary results suggest that a clinical effect may extend further than the results of laboratory analysis might imply. In the management of inhibitors, no bypassing agent so far developed has provided the level of reliable therapeutic response associated with the use of factor VIII or IX concentrates in treatment of the inhibitor-free patient. Dosage estimation and frequency of administration for aPCCs and rVIIa have never been based on specific assays and the monitoring of treatment for inhibitor patients continues to be guided by clinical criteria. Use of pharmacodynamic markers to measure therapeutic effect, while part of the pre-clinical characterization for Pfizer’s investigational rFVIIa variant, remains experimental and, to date, has not been validated for guiding treatment of the inhibitor patient. Nevertheless regulatory bodies still require objective and laboratory based evidence to support claims of efficacy and safety in clinical trials relating to the expansion of use of existing products and the development of new products. Pfizer will discuss these issues in the context of current clinical studies of BeneFIX and preclinical trials of a modified rVIIa. 12:00 Novel assays for novel hemophilia therapies – Practice of clinical testing during clinical studies 2012 and beyond; Peter Turecek (Baxter) Accurate dosing is key for treatment success with any medication. Due to the broad therapeutic range of coagulation factors in hemophilic patients safety issues in hemophilia therapies related to misdosing are mainly related to under-dosing and result in lack of efficacy. Also hemophilia therapies are costly and therefore - at least in certain geographies and with specific treatment regimens - only minimally required amount of factor is used for treatment of patients. Under these circumstances it represents a serious issue when potency assignments do not match the factor activity recovered and the one measurable in patients - as had been an issue for B-domain deleted rFVIII for more than a decade [1]. Recent recommendations therefore request post-infusion testing against a product reference composed of the same material as that which is infused. At the same time manufacturers should establish a relationship between the dose based on the labeled potency and the expected factor recovery in the patient. This can be particularly challenging as demonstrated by the example of a cysteine modified B-domain deleted FVIII that showed a severe discrepancy between the one-stage clotting and the chromogenic assay [2]. One way out of this situation would be to use global hemostasis assays such as thrombin generation assay or thromboelastography that do not measure the infused compound directly but the response of the hemostatic system to the therapy. This would not only exclude product related specific assay artifacts but would also personalize therapy by individualization of dosing while maintaining effective factor levels in the patients to prevent bleeding. An alternative approach would be to design assays that specifically measure the pro-coagulant activity of modified coagulation factors. We recently developed and validated a novel modification dependent activity assay (MDAA) to selectively measure PEGylated human recombinant factor VIII in human plasma without interference from non-PEGylated FVIII. The MDAA combines the use of an anti-PEG antibody with a FVIII activity assay in an ELISA like assay system. A completely different challenge comes from the fact that current assays for coagulation factors do not differentiate recombinant from endogenous plasmatic proteins. This is an issue for treatment monitoring of mild forms of factor deficiency where endogenous levels of protein are existing as background. To allow measurement of a recombinant protein in the presence of the plasmatic equivalent we established a platform that allows selective determination of CHO cell derived recombinant human proteins in the presence of their human plasma-derived equivalent. This was first developed for determination of recombinant von Willebrand factor (rVWF) and was used during the preclinical drug development phase in animal studies. The assay principle is a lectin based immune assay with specific detection of VWF. Validation showed that the assays was highly specific for the CHO derived protein and could quantitatively measure rVWF derived from CHO cells in the presence of plasma-derived VWF. The use of this assay could be expanded to determination of rVWF in mild forms of human VWD with circulatory levels of endogenous VWF. These examples of new assay strategies described here – global hemostasis assay and highly product specific assays – have the potential to substantially improve the array of laboratory endpoints used in clinical trials investigating novel molecules to treat hemophilia and related disorders. References Barrowcliffe TW, Raut S, Sands D, Hubbard AR. Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations. Semin Thromb Hemost. 2002;28:247-256.Leong L, Evans V, Ramsey P, et al. Evaluation of methods for potency testing of pegylated FVIII (Peg-Fviii, Bay 94-9027) J Thromb Haemost 2011;9,Suppl. 2 12:10 A clinically validated technology for elongating the half life of coagulation factors, enabling a prolonged haemostatic activity in hemophilic animal models; Gili Hart (Prolor Biotech) PROLOR Biotech Inc. (NYSE: PBTH) is a clinical stage public company developing biobetter long acting versions of therapeutic drugs utilizing CTP technology. CTP is a naturally occurring peptide evolved to provide long durability of hCG and has the potential to dramatically reduce injection frequency, drug load and side-effects for most therapeutic proteins. CTP is already clinically validated - Merck’s long acting FSH-CTP named Elonva® received EU marketing approval in 2010. PROLOR’s current pipeline includes long acting human growth hormone, long acting FVIIa and long acting anti obesity/type II diabetes drug. PROLOR recently announced successful completion of a Phase II trial in GH deficient adults, initiation of phase II study in GH deficient children and expects to initiate its Phase III in 2012. PROLOR has developed a long acting Factor VIIa-CTP, demonstrating a markedly enhanced pharmacokinetics, increased exposure, increased recovery and a prolonged haemostatic effect in hemophilic animals model with a comparable biological activity. PROLOR is in the initial stage of preparations for Phase I-IIa study in hemophilic patients, and due to the nature and properties of the modified, long acting FVIIa, the company is facing the following challenges in the in-vitro and in-vivo characterization of the product. FVIIa-CTP quantitation: Assessment of FVIIa-CTP Ag level in -vitro and in-vivo is performed using two methods, FVII ELISA and A280. Following the attachment of CTP the FVII content is reduced, requiring an analytical adjustment for A280 calculations. The presence of the highly O-glycosylations on the CTP portion of the molecule reduces the affinity of anti FVII commercial Abs leading to miscalculation of Ag level as compared the native FVII is. The company has developed a FVII-CTP specific ELISA, utilizing a product specific Ab enabling a more accurate approach for its quantitation.Evaluation of FVIIa-CTP clotting activity: One of the major challenges in the preparation for the non-clinical and clinical studies is assessing FVII-CTP and FVIIa-CTP in vitro and in vivo activity, utilizing the well established methods. The fusion of CTP reduces the relative content of FVIIa, therefore the specific activity is lower. A conversion factor, considering the molar ratio between FVII and CTP was established and might lead to further adjustment of the injected doses in clinical setting.CTP Immunogenicity paradigm: The immunogenicity of FVIIa-CTP is an important parameter in the ongoing non-clinical and planned clinical trials of the product. Fusion of CTP to FVIIa can affect the structure and properties of the molecule. Moreover, the enhanced longevity and exposure are additional unknown factors. Generation of anti-FVIIa Abs in hemophilic patients with inhibitors is a major concern, therefore a detailed immunogenicity paradigm for the detection and characterization of anti FVIIa-CTP antibodies was developed according to FDA and EMA recommendations. DISCUSSION Questions and comments from the audience centred on the assay methods used to detect inhibitor and test the potency labelling of new products and the utility of the global assays. Manufacturers set up their own methods and criteria to evaluate new products. Therefore it was suggested that FVIII&FIX ISTH-SSC constitutes a committee to establish, in collaboration with manufacturers, common criteria to study new products. The FVIII/FIX SSC agreed on the idea that industry can have a mature relationship with the medical and scientific community and with bodies such as ISTH.
by F. Peyvandi
Friday, September 14, 2012
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