Multicentric MFI30 Study: Standardization of CD30 Expression By Flow Cytometry in Non-Hodgkin Lymphoma
Authors: Pruvot Debliquis Agathe , Baseggio Lucile , Jacob Marie-Christine , Bouyer Sabrina , Genevieve Franck , Guy Julien , Mayeur-Rousse Caroline , Chapuis Nicolas , Bennani Hind , Matthes Thomas , Drenou Bernard 
Published: October 2019

Abstract
Introduction
The CD30 antigen is expressed by a small number of cells in classical Hodgkin lymphoma (HL), by malignant cells in anaplastic large cell lymphoma (ALCL), and in several non-Hodgkin lymphomas (NHL). Brentuximab vedotin (BV), an anti-CD30 monoclonal antibody (Ab) conjugated to chemotherapy, showed its efficacy in HL and ALCL. However, a correlation between response to BV and CD30 expression in NHL is difficult to establish by immunohistochemistry (IHC) since the count of dimly positive cells is difficult to standardize. Flow cytometry (FCM) is a standardizable tool which allows the multiparametric characterization of malignant NHL cells and the quantification of proteins at the cell surface. The objective of this multicentric study is to standardize CD30 expression in NHL by quantitative FCM.
Materiel and methods
The nine centers used one or two cytometers: BD Biosciences (BD) n=7, Beckman Coulter (BC) n=4. The standardization was conducted using the Euroflow strategy, the same operating procedure and the same reagents. Abs recognizing different CD30 epitopes were: BerH83 (BD), HRS4 (BC) and a specific AC10 (Ancell) similar to BV. If tissue was available, pathologists locally used BerH2 (Dako) for IHC. The CD30 expression was normalized using Mean Fluorescence Intensity (MFI) to MFI CD4 expression and expressed as a percentage (nMFI30). Three cell lines (SUDHL4, K562 and L82) with differential expression of CD30 were shipped to the different laboratories in order to validate the centers using a robust statistical method. Then, NHL cases including anaplastic lymphomas, T lymphomas, and large B cell lymphomas were collected using a website www.mfi30.fr. All FCM Abs were used on available cells when nMFI30 >1% using Ancell Ab.
Results
On cell lines, all centers obtained similar results: z-scores between -2 to +2. Mean of nMFI30 were SUDHL4 0.1% (SD=0.1), K562 69.8% (SD=16.9), L82 292.9% (SD= 45.7) with BD Abs and 0.6% (SD=0.1), 229.9% (SD= 47.9) and 2053.8 (SD= 282) with BC Abs , respectively, demonstrating that BD and BC Abs are not equivalent.
Samples from 82 adult patients were included in the study: peripheral blood (n=39), lymph node suspensions (n=21), bone marrow (n=5) or different tissues (n=17, cerebrospinal, seroma, pleural and ascite fluid, spleen). The diagnoses included diffuse large B-cell lymphoma (DLCBL, n=33 with 4 DLBCL of central nervous system (CNS)), Sezary syndrome (SS, n=14), peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS, n=11), T-cell prolymphocytic leukaemia (T-PLL, n=8), enteropathy-associated T-cell lymphoma (EATL-1, n=2), angio-immunoblastic lymphoma (AITL, n=5), primary effusion lymphoma (PEL, n=1), plasmablastic lymphoma (PBL, n=1), B-lymphoblastic lymphoma (B-LBL, n=1), breast implant associated anaplastic large cell lymphoma (BIA-ALCL, n=1), anaplastic large cell lymphoma (ALCL, n=1), mantle cell lymphoma (n=1), T-cell large granular lymphocyte leukemia (T-LGLL, n=2) and chronic lymphoproliferative disorders of NK cells (CLPD-NK, n=1).
Four groups are defined: (1) a negative group (n=60) presents nMFI30 <1% using Ancell Ab. In this group, IHC is negative except in 1 case of PTCL-NOS; (2) a small highly positive group (n=4) presents nMFI30 >10% with the 3 FCM Abs and positive in IHC (2 ALCL, 1 DLBCL, 1 EATL). This group shows a more intense nMFI30 with BC Ab; (3) a dim positive group (n=8) presents nMFI30 between 1-10% with the 3 FCM Abs. In this group, IHC is negative in 2 out of the 3 tested cases; (4) a discordant group (n=10) presents nMFI30 >1% with at least 1 Ab and discordant with another one. In this group, 8 IHC were tested: 2 IHC positive <1% only with BD Ab, 5 IHC negative cases only >1% with Ancell Ab and 1 case shows a high discrepancy between 2 FCM Abs. Regarding lymphoma entities, T-PLL, DLBCL-CNS, and T-LGLL are always negative and the ALCL are positive. Seven out of the 33 DLBCL are in the positive or discordant groups (21%).
Conclusion
As previously reported in literature using IHC, CD30 is positive in all ALCL, in some DLCBL (20%) and only rarely in other NHL using FCM. Therefore these first results emphasize the feasibility of FCM in CD30 determination and quantification in NHL subtypes and mainly its multicentric standardization. The inclusion of FCM with relevant Abs in clinical trials using BV should be now validated in larger series to better understand clinical results.
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Disclosures
Pruvot Debliquis: Alexion: Consultancy, Honoraria; Takeda Oncology: Honoraria. Baseggio:Takeda Oncology: Honoraria. Jacob:Takeda Oncology: Honoraria. Drenou:Takeda Oncology: Honoraria, Research Funding; Alexion: Consultancy, Honoraria.