Multicentric MFI30 study: standardization of CD30 expression by flow cytometry in Non-Hodgkin lymphoma Lucile Baseggio1, Agathe Debliquis2, , Marie-Christine Jacob3, Sabrina Bouyer4, Hind Bennani5, Nicolas Chapuis6, Francine Garnache Ottou7, Franck Genevieve8, Julien Guy9, Véronique Harrivel10, Remi Letestu11, Caroline Mayeur-Rousse12 , Bernard Drenou2 1 Laboratoire d'Hématologie Cellulaire, Groupement Hospitalier Sud/Hospices Civils de Lyon, France, 2 Laboratoire d’Hématologie, Groupe Hospitalier de la Région Mulhouse Sud Alsace (GHRMSA), France, 3 Laboratoire d'Immunologie, Centre Hospitalier Universitaire de Grenoble, France, 4 Service d'Hématologie Biologique, Centre Hospitalier Universitaire de Poitiers, France 5 Laboratoire de biologie, Hopital Foch Suresnes, France, 6 Service d'Hématologie Biologique, Hopital Cochin APHP Paris, France, 7Laboratoire Hématologie, UMR1098, EFS BFC , Université de Franche Comté, Besançon, France, 8 Laboratoire d'Hématologie, Centre Hospitalier Universitaire d’Angers, France, 9 Service d'Hématologie biologique, Centre Hospitalier Universitaire de Dijon, France, 10 Laboratoire d'hématologie, C HU Amiens-Picardie, Amiens, France, 11Laboratoire d'hématologie, Hôpital Avicenne, Bobigny, France, 12Laboratoire d'Hématologie, Centre Hospitalier Universitaire de Strasbourg, France INTRODUCTION The Brentuximab vedotin (BV), an anti-CD30 monoclonal antibody (Ab) conjugated to chemotherapy, has shown its efficacy in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) that typically expressed the CD30. However, the response to BV is difficult to correlate to CD30 expression defined by immunohistochemistry (IHC). The objective of this multicentric study is to standardize and evaluate CD30 expression by flow cytometry (FCM) in malignant T and B-cell proliferation. MATERIALS and METHODS Among 18 centers participated to the first step of standardization, 12 included cases (n=155) : 5 ALCL, 70 B-NHL including 64 DLBCL, 80 malignant T-cell proliferations (24 Sezary, 26 PTCL-NOS, 14 T-PLL, 8 AITL, 2 EATL, 6 T / NK-LGL) from various samples (74 bloods, 43 lymph node suspensions, 10 bone marrows, 28 others) (www.mfi30.fr). BD cytometers Biosciences (BD, n = 9) or Beckman Coulter (BC, n = 9) are used with the Euroflow strategy. Abs recognizing different CD30 epitopes are tested: BerH83 (BD), HRS4 (BC) and AC10 (Ancell) which recognizes same epitope as BV. The mean fluorescence intensity (MFI) of CD30 is normalized in % compared to CD4 (nMFI30). Abs BC and BD are tested if only nMFI30> 1% with Ab Ancell. In 85 cases IHC has been also realized with the clone BerH2 (Dako). RESULTS 1/First step = standardization (18 centers) Using 3 cell lines (SUDHL4, K562 and L82) with different expression of CD30, FCM protocol was standardized and validated (robust statistics). PE On 3 cell lines, all the centers obtain similar results (z-score between -2 and +2). without Abs Anti-CD30 PE 2/Second step = inclusion (12 centers, 155 cases) Negative group Discordant group Dim group Positive group n=109 n=19 n=16 n=11 FCM results has allowed to define 4 groups : -a high positive group (n = 11) with nMFI30> 5% with the 3 Abs tested; -a dim positive group (n = 16) with nMFI30 between 1 and 5% with the 3 Abs tested; -a discordant group (n = 19) with nMFI30> 1% with Ab Ancell and <1% with another Ab; -a negative group (n = 109) with nMFI30 <1% with Ab Ancell. When tested, IHC is positive in all cases of high positive group (10), in 4 out of 7 in the dim positive group, 10 out of 16 in the discordant group, and in 7 out of 52 in the negative group (2 AITL, 1 PTCL-NOS and 4 DLBCL). This last discordance could be explained by CD30 expression of non-tumoral cells present in microenvironment or by intracytoplasmic staining. 10/38 DLBCL are positive by IHC (26%) and 21/64 (33%) are in positive or discordant groups by FCM. 6/13 Sezary are positive by IHC (46%) and 7/24 (29%) are in positive or discordant groups by FCM. All ALCL are positive by both techniques. All T-PLLs, DLBCL-CNS and LGL are negative. EATL case: High positive group (FCM) DLCBL case: dim positive group (FCM) CD30 Ancell CD30 Ancell CD30 CD30 IHC concordance IHC discorcordance CONCLUSION Multicenter standardization of CD30 is achievable by FCM, using in particular the AC10 and making it therefore a powerful tool for clinical trials to extend the treatment of BV to various NHL. Poster presented at: Printing supported by: 159--P Biology and Pathology DOI: 10.3252/pso.eu.15ICML.2019 15ICML Lucile Baseggio.