de Haas, M (Masja)

Huiskes, E

Porcelijn, L

Schipperus, M

van der Holt, Ronnie

Zwaginga, JJ

English

EUR Research Repository

To the editor:Lack of detectable platelet autoantibodies is correlated with nonresponsiveness to rituximabtreatment in ITP patientsLeendert Porcelijn,1 Elly Huiskes,1 Martin Schipperus,2 Bronno van der Holt,3 Masja de Haas,1,4-6 and Jaap Jan Zwaginga,5,6for the Dutch HOVON 64 Study Group1Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, The Netherlands; 2Department of Internal Medicine, HagaZiekenhuis, DenHaag, The Netherlands; 3Haemato Oncology Foundation for Adults in The Netherlands (HOVON) Data Center, Erasmus MC Cancer Institute–ClinicalTrial Centre, Rotterdam, The Netherlands; 4Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam,Amsterdam, The Netherlands; 5Center for Clinical Transfusion Research, Sanquin Research, Leiden, The Netherlands; and 6Department ofImmuno-hematology and Blood Transfusion, Leiden University Medical Center, Leiden, The NetherlandsRituximab, a chimeric CD20 monoclonal antibody that causesdepletion of B cells, shows short-term treatment efficacy in 40% to60% of immune thrombocytopenia (ITP) patients.1,2 Our recentlypublished multicenter randomized open-label phase 2 trial comparing3 rituximab dosing schemes showed 50% responses in 138 ITP pa-tients.3 Unfortunately, the treatment efficacy of rituximab so far cannotbe predicted. Here, however, we show absent levels of platelet-boundantibodies to be associated with refractoriness to rituximab. Assayson platelet-bound antibodies may hence lead to a more personalizedtreatment approach for these patients.ITP was diagnosed in accordance with the recommendation of theAmericanSocietyofHematology (ASH),4 bypatient and familyhistory,physical examination, and laboratory investigations (eg, normal whiteblood cell count and differentiation, normal red blood cell count, redblood cell indices andmeanplatelet volume, absenceofHIV,hepatitisBvirus, hepatitis C virus, Helicobacter pylori, and antinuclear factor,antinuclear antibody, antiphospholipid antibodies). Furthermore, low tonormal thrombopoietin levels supported thrombocytopenia to be due toincreased platelet destruction.5 Eligible patients were 18 years of age orolder, with an ITP relapse or refractoriness (minimally 2 platelet counts,30 3 109/L), at least 3 weeks after high-dose ($1 mg/kg) cor-ticosteroids, who were randomized between 3 rituximab dosingschemes, that is, 2 or 4 once-weekly standard 375 mg/m2 doses and atwice-weekly 750mg/m2 regimen.3 Complete response (CR), good/partial response (PR), and moderate response (MR) were definedas platelet counts of 1503 109/L or more, 503 109/L or more on 2consecutive occasions, and a platelet count over 30 3 109/L with atleast twice the baseline count, respectively. The different dosages ofrituximab did not lead to changes in response rate.3Prior to starting the first rituximab dose and subsequently for10 weeks, weekly EDTA-anticoagulated blood sampling wasrequested to assess the presence of platelet-associated immuno-globulin G (IgG) and IgM autoantibodies by the direct plateletimmunofluorescence test (PIFT) and the eluate-(indirect) PIFT, asdescribed previously.6 In the current report, a positive “PIFT”refers to the combination of a reactive (5 11 to 31) direct as wellas eluate PIFT. One or both tests with negative reactions weredefined as a negative “PIFT.” Additionally, in samples withsufficient platelet numbers, both the PIFT and a direct monoclonalantibody immobilization of platelet antigens (MAIPA) assay wereperformed; the latter todetect IgG-class autoantibodies.7Allmonoclonalantibodies, CLBthromb/1 (CD41, anti-glycoprotein IIb [GPIIb]),MB45(CD42a, anti-GPIX), and SW16 (CD42d, anti-GPV) were supplied bySanquin Reagents (Amsterdam, The Netherlands).For statistical analysis, correlation between continuous variableswas calculated by the Spearman rank test, association betweencategorical valuables was calculated by the 2-tailed Fisher exact prob-ability test, and 95% confidence intervals (CIs) were calculated usingStata version 14.0 (StataCorp LP).Prerituximab treatment samples from 112 of 138 patients andfollow-up samples during and after rituximab treatment from 80patients were sent to our laboratory (supplemental Figure 1, availableon the BloodWeb site).In 99 of 112 patient samples (88%), the platelet count inprerituximab treatment samples was sufficient to enableplatelet-associated autoantibody detection by PIFT. Of these, arepresentative 47 (47%) responded to rituximab (CR, n5 17; PR,n 5 22; and MR, n 5 8, respectively) whereas 52 (53%) werenonresponders (NRs). For all tested patients (n 5 99), directPIFT results corresponded with the eluate-PIFT results. Antibodieswere present in 79 patients, of whom 43 responded (54%) with 16complete (21%). In contrast, the absence of antibodies in 20 patientswas associated with 4 responses (20%) of which only 1 (5%) wascomplete (Table 1; P 5 .006). In summary, undetectable plateletautoantibodies in the direct PIFT strongly predict absence of or lessthan complete response to rituximab (Table 1; negative predictivevalue, 95.0%; CI, 73.2%-99.25%, positive predictive value, 20.3%;CI, 17.7%-23.1%).Vice versa, 16 patients (94%) with a complete response torituximab showed positive to very-strong-positive direct PIFTreactions (mean level, 21) whereas the percentage of non-responsive patients with a positive direct PIFT was much lower(69%; P 5 .051). In patients with partial or moderate plateletresponses, positive PIFT occurred with similar frequency in 19(86%) and 8 (100%), respectively.Serial autoantibody testing was performed for 80 of thesampled patients (71%). Of these, the presence of plateletautoantibodies and corresponding platelet counts were evaluated“per response group” at baseline and at the time of the highestplatelet counts that were reached within 10 weeks after start ofrituximab treatment (Figure 1; supplemental Figure 2). Of 30patients predominantly responding (10 CR, 10 PR, 3 MR), theplatelet count also enabled the direct MAIPA. Both PIFT andMAIPA results in these patients appeared in full concordance, thatis, 7 patients (4 NR and 3 PR) were negative with both PIFT anddirectMAIPA and 23 (3 NR, 3MR, 7 PR, and 10 CR) positive withboth PIFT and direct MAIPA.CR in the Haemato Oncology Foundation for Adults in TheNetherlands (HOVON) 64Studywas defined as a rise in platelet countsto 150 3 109/L or more. Nowadays, ITP is defined as plateletcounts ,100 3 109/L.8 Recategorization of the response groups,using 100 3 109/L did not result in different response numbers.BLOOD, 22 JUNE 2017 x VOLUME 129, NUMBER 25 LETTERS TO BLOOD 3389For personal use only.on August 9, 2018. by guest  www.bloodjournal.orgFrom Anti-CD20 monoclonal antibodies deplete B cells for periodsvarying frommonths to.1 year. In this respect, B cells are essential topresent antigens to CD41 T cells and moreover secrete cytokines-activating macrophages, dendritic cells, and immune-regulatory cellsand thus an ongoing autoimmune response.9-11Additionally, rituximabis reported to normalize both the abnormalities and dysfunction ofthe T-cell compartment in ITP patients.9,12-14 However, depletion ofpre–plasma cell B cells, andwith it decreased autoantibody production,is regarded as the most likely mechanism of action. But, so far not oneof these explanations could be correlated to rituximab’s variabletherapeutic effects.Our results are in agreement with the latter mechanism, whereasall serially tested CR patients (n 5 16) showed decreasing antibodyresults and32of 39nonresponsive patients havenondecreasing autoanti-bodies after or undetectable autoantibodies before rituximab treatment(Figure 1).Apossible explanation for nondecreasing antibodies could beinsufficient eradication of antibody-producing plasma cells. Indeed,long-lived plasma cells were detected in the spleen of ITP patientsnonresponsive to rituximab treatment and it has been suggested thatB-cell depletion promotes the differentiation and settlement of theselong-lived plasma cells in the spleen.15Although nonresponsive patientswithout detectable platelet-associated autoantibodies fit the ASHcriteria for ITP, these patients might represent a subgroup of ITPpatientswith possibly T-cell–mediated platelet destruction. Indeed,Audia et al in this respect showed the importance of activatedsplenic CD81 T cells in ITP patients unresponsive to rituximab.16Furthermore, CD81 T cells were shown to contribute to the murinesplenocyte’s ability to induce thrombocytopenia; recently, Arthur et alshowed CD81 T-cell–mediated antibody-independent platelet clear-ance in a murine model for refractoriness to platelet transfusions.17,18Immune-suppressive treatment in combination with rituximab, asrecently published, may improve the limited response for rituximab assingle treatment.19 In this respect, itwill be interesting to study the effectof combination therapy in ITP patients with and without detectableplatelet autoantibodies.In conclusion, our results show absence of platelet-boundantibodies to be associated with a low response to rituximab. Ad-ditionally, response to rituximab appeared strongly associated witha decline in platelet-bound antibodies. Our findings importantlyimplicate that both antibody negativity vs strong antibody presencemight enable a more individualized therapeutic approach in thisgroup of ITP patients.The online version of this article contains a data supplement.Contribution: L.P. was principal investigator, carried out the initial analysis,reviewed and revised the manuscript, and approved the final manuscript assubmitted; E.H. designed the data collection instrument, coordinated andsupervised data collection, critically reviewed the manuscript, and approved0123Direct PIFT results0Pre post Pre post Pre post PreNR n=40MR n=8PR n=16CR n=16post200100300400Platelet count (x 10E9/L)ABFigure 1. Direct PIFT results and platelet counts prior toinitiation with rituximab and at the time of the highestplatelet counts reached within 10 weeks after start ofrituximab treatment of 80 serially tested ITP patients. X-axisfor panel B also applies for panel A. (A) CR, n5 16; PR, n5 16;MR, n 5 8; NR, n 5 40. Direct PIFT scores before (pre) and atthe time of the highest platelet count within 10 weeks after(post) treatment with rituximab: 0, negative; 1, positive; 2, strongpositive; and 3, very strong positive. Changes in antibodydetection results are indicated by means of connecting lines. (B)Platelet counts before and the highest platelet count within 10weeks after treatment with rituximab.Table 1. Platelet-associated antibody detection prior to initiationwith rituximabP = .006In the NR group, we found significantly (P 5 .006) more patients withoutdetectable autoantibodies than in the response group (CR 1 PR 1 MR).3390 LETTERS TO BLOOD BLOOD, 22 JUNE 2017 x VOLUME 129, NUMBER 25For personal use only.on August 9, 2018. by guest  www.bloodjournal.orgFrom the final manuscript as submitted; M.S. submitted samples and helped analyzethe initial rituximab data; B.v.d.H. coordinated and supervised data collection;L.P., J.J.Z., and M.d.H. conceptualized and designed the study, drafted theinitial manuscript, and approved the final manuscript as submitted; and theHOVON 64 Study Group included patients and executed the trial.Conflict-of-interest disclosure: The authors declare no competing financialinterests.A complete list of themembers of the Dutch HOVON 64Study Group appears in“Appendix.”Correspondence: Leendert Porcelijn, Immunohematology Diagnostics, Ples-manlaan 125 (Q2), 1066CX Amsterdam, The Netherlands; e-mail: l.porcelijn@sanquin.nl.Appendix: study group membersThe members of the Dutch HOVON 64 Study Group are: Jaap J.Zwaginga (Department of Immunohematology and Blood Trans-fusion, LeidenUniversityMedical Centre and the Centre for ClinicalTransfusion Research, Sanquin-Leiden University Medical Centre,Leiden, The Netherlands), Bronno van der Holt (HOVON DataCenter, Erasmus MC Cancer Institute–Clinical Trial Centre,Rotterdam, The Netherlands), Peter A. te Boekhorst (Departmentof Hematology, Erasmus MC, Rotterdam, The Netherlands), Bart J.Biemond (Department of Hematology, Academic Medical CentreAmsterdam, Amsterdam, The Netherlands), Mark-David Levin(Department of Internal Medicine, Albert Schweitzer Hospital,Dordrecht, The Netherlands), René van der Griend (Department ofInternal Medicine, Diakonessenhuis, Utrecht, The Netherlands),Anneke Brand (Department of Immunohematology and BloodTransfusion, Leiden University Medical Centre and the Centre forClinical Transfusion Research, Sanquin-Leiden University MedicalCentre, Leiden, The Netherlands), Sonja Zweegman (Departmentof Hematology, VU University Medical Centre, Amsterdam, TheNetherlands), Hans F. M. Pruijt (Department of Internal Medicine,Jeroen Bosch Hospital, Den Bosch, The Netherlands), Vera M. J.Novotny (Department of Hematology, Radboud University MedicalCentre, Nijmegen, The Netherlands), Art Vreugdenhil (Departmentof Internal Medicine, Máxima Medical Centre, Veldhoven, TheNetherlands), Marco R. de Groot (Department of Internal Medicine,Medisch Spectrum Twente, Enschede, The Netherlands), Okke deWeerdt (Department of Internal Medicine, St. Antonius Hospital,Nieuwegein, The Netherlands), Elisabeth C. M. van Pampus(Department of Laboratory Medicine, Radboud University MedicalCentre, Nijmegen, The Netherlands), Tanja M. vanMaanen-Lamme(Department of Internal Medicine, Westfriesgasthuis, Hoorn, TheNetherlands), Shulamiet Wittebol (Department of Internal Medi-cine, Meander Hospital, Amersfoort, The Netherlands), MartinR. Schipperus (Department of Internal Medicine, HagaZiekenhuis,Den Haag, The Netherlands), Matthijs H. Silbermann (Departmentof Internal Medicine, Tergooiziekenhuizen, Blaricum, The Nether-lands), Peter C. Huijgens (Department of Hematology, VU Univer-sity Medical Centre, Amsterdam, The Netherlands), MarleenLuten (HOVONDataCenter, ErasmusMCCancer Institute–ClinicalTrial Centre, Rotterdam, The Netherlands), Rene Hollestein(HOVON Data Center, Erasmus MC Cancer Institute–ClinicalTrial Centre, Rotterdam, The Netherlands), Jan A. C. Brakenhoff(Department of Internal Medicine, Waterland Hospital, Purmerend,The Netherlands), Jolanda G. Schrama (Department of InternalMedicine, Spaarne Hospital, Hoofddorp, The Netherlands),FransjeA.A.Valster (Department of InternalMedicine, LievensbergHospital, Bergen op Zoom, The Netherlands), Gerjo A. Velders(Department of Internal Medicine, Gelderse Vallei Hospital, Ede,The Netherlands), and Harry R. Koene (Department of InternalMedicine, St. Antonius Hospital, Nieuwegein, The Netherlands).References1. Auger S, Duny Y, Rossi JF, Quittet P. Rituximab before splenectomy in adultswith primary idiopathic thrombocytopenic purpura: a meta-analysis. Br JHaematol. 2012;158(3):386-398.2. Patel VL, Mahévas M, Lee SY, et al. Outcomes 5 years after response torituximab therapy in children and adults with immune thrombocytopenia. Blood.2012;119(25):5989-5995.3. Zwaginga JJ, van der Holt B, Te Boekhorst PA, et al; Dutch HOVON 64 studygroup. Multi-center randomized open label phase II trial on three rituximab dosingschemes in immune thrombocytopenia patients. Haematologica. 2015;100(3):e90-e92.4. 2011 Clinical Practice Guideline on the Evaluation and Management of ImmuneThrombocytopenia (ITP). The American Society of Hematology 2011 Evidence-Based Practice Guideline for Immune Thrombocytopenia. Washington, DC:American Society of Hematology; 2011.5. Porcelijn L, Folman CC, Bossers B, et al. The diagnostic value of thrombopoietinlevel measurements in thrombocytopenia. Thromb Haemost. 1998;79(6):1101-1105.6. von dem Borne AE, Verheugt FW, Oosterhof F, von Riesz E, de la Rivière AB,Engelfriet CP. A simple immunofluorescence test for the detection of plateletantibodies. Br J Haematol. 1978;39(2):195-207.7. Kiefel V, Santoso S, Weisheit M, Müeller-Eckhardt C. Monoclonal antibody–specific immobilization of platelet antigens (MAIPA): a new tool for theidentification of platelet-reactive antibodies. Blood. 1987;70(6):1722-1726.8. Rodeghiero F, Stasi R, Gernsheimer T, et al. Standardization of terminology,definitions and outcome criteria in immune thrombocytopenic purpura of adultsand children: report from an international working group. Blood. 2009;113(11):2386-2393.9. Stasi R. Rituximab in autoimmune hematologic diseases: not just a matter ofB cells. Semin Hematol. 2010;47(2):170-179.10. Roche PA, Furuta K. The ins and outs of MHC class II-mediated antigenprocessing and presentation. Nat Rev Immunol. 2015;15(4):203-216.11. Shlomchik MJ, Craft JE, Mamula MJ. From T to B and back again: positivefeedback in systemic autoimmune disease. Nat Rev Immunol. 2001;1(2):147-153.12. Martin F, Chan AC. B cell immunobiology in disease: evolving concepts fromthe clinic. Annu Rev Immunol. 2006;24:467-496.13. Cooper N, Stasi R, Cunningham-Rundles S, Cesarman E, McFarland JG,Bussel JB. Platelet-associated antibodies, cellular immunity and FCGR3agenotype influence the response to rituximab in immune thrombocytopenia. Br JHaematol. 2012;158(4):539-547.14. Stasi R, Cooper N, Del Poeta G, et al. Analysis of regulatory T-cell changes inpatients with idiopathic thrombocytopenic purpura receiving B cell-depletingtherapy with rituximab. Blood. 2008;112(4):1147-1150.15. Mahévas M, Patin P, Huetz F, et al. B cell depletion in immune thrombocytopeniareveals splenic long-lived plasma cells. J Clin Invest. 2013;123(1):432-442.16. Audia S, Samson M, Mahévas M, et al. Preferential splenic CD8(1) T-cellactivation in rituximab-nonresponder patients with immune thrombocytopenia.Blood. 2013;122(14):2477-2486.17. Chow L, Aslam R, Speck ER, et al. A murine model of severe immunethrombocytopenia is induced by antibody- and CD81 T cell-mediatedresponses that are differentially sensitive to therapy. Blood. 2010;115(6):1247-1253.18. Arthur CM, Patel SR, Sullivan HC, et al. CD81 T cells mediate antibody-independent platelet clearance in mice. Blood. 2016;127(14):1823-1827.19. Choi PY, Roncolato F, Badoux X, Ramanathan S, Ho SJ, Chong BH. A noveltriple therapy for ITP using high-dose dexamethasone, low-dose rituximab, andcyclosporine (TT4). Blood. 2015;126(4):500-503.DOI 10.1182/blood-2016-11-751719© 2017 by The American Society of HematologyBLOOD, 22 JUNE 2017 x VOLUME 129, NUMBER 25 LETTERS TO BLOOD 3391For personal use only.on August 9, 2018. by guest  www.bloodjournal.orgFrom online May 3, 2017 originally publisheddoi:10.1182/blood-2016-11-7517192017 129: 3389-3391  Jan ZwagingaLeendert Porcelijn, Elly Huiskes, Martin Schipperus, Bronno van der Holt, Masja de Haas and Jaap nonresponsiveness to rituximab treatment in ITP patientsLack of detectable platelet autoantibodies is correlated with http://www.bloodjournal.org/content/129/25/3389.full.htmlUpdated information and services can be found at: (250 articles)Thrombocytopenia     (797 articles)Platelets and Thrombopoiesis     (4802 articles)Clinical Trials and Observations    Articles on similar topics can be found in the following Blood collectionshttp://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:  Copyright 2011 by The American Society of Hematology; all rights reserved.of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American SocietyFor personal use only.on August 9, 2018. by guest  www.bloodjournal.orgFrom 

Lack of detectable platelet autoantibodies is correlated with nonresponsiveness to rituximab treatment in ITP patients

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Lack of detectable platelet autoantibodies is correlated with nonresponsiveness to rituximab treatment in ITP patients

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