Introduction: Since the publication of the International Guidelines (Borowitz, 2010; Illingworth, 2018), no study has assessed the long-term evolution of paroxysmal nocturnal hemoglobinuria (PNH) ...clones using high-resolution flow cytometry. The sole evaluation, performed by Sugimori et al, using a 2-color flow cytometry test, showed the disappearance of PNH clones in 24% of patients with bone marrow (BM) failure over 5 years (Sugimori, 2009). A diagnostic practice harmonization using high-resolution flow cytometry has spread in France since 2013 through an on-going inter-laboratory comparison program (Debliquis, 2015). Thus, our HPNAFC group has been able to initiate a French nation-wide multicenter prospective observational study.
Objective: We aimed to assess the evolution of PNH clones over a long term period using mostly high sensitivity test, which is required for minor clone assessment, with validated flow cytometry data.
Methods: All patients of any age with a PNH clone or GPI-deficient cells ≥0.01%, newly- or previously-diagnosed, detected in France from February 29th 2016, could be included in this Observatory, provided that the center had validated a PNH flow cytometry quality control. For each patient, the baseline assessment was always considered as the initial PNH clone detection, even if it occurred before the initiation of the Observatory. Thus, this strategy allowed the collection of cases with long-term follow-up. Referent cytometrist of each center included patients in the e-CRF available on the HPNAFC website providing clinical and biological information as well as flow cytometry raw data files. This study was approved by the national research ethics board.
Results: As of July 15th 2019, 48 participating flow cytometry laboratories across France have enrolled 356 patients with a PNH clone or GPI-deficient cells ≥ 0.01%. All cases have been carefully reviewed by the 2 principal investigators, who both thoroughly re-examined flow cytometry data and the e-CRF filling that led to the update of roughly one third of the submitted files. This enabled the validation of 200 patients at diagnosis, the remaining 156 being ongoing. One hundred and three of the 200 validated patients displayed at least one follow-up point (more than 3 months apart from the diagnosis) with a clone size determined at diagnosis (see flow chart figure 1A). For 8/103 patients, exchanges with centers are still ongoing. Thus, we were able to assess the evolution of PNH clones of 95 patients with 2 range: 1-8 follow-up points over a period of 4.1 0.3-14.2 years, corresponding to 200 validated follow-up points. The patient median age at diagnosis was 40 years old 10-85 with 3 pediatric cases (<18y) and a M/F sex ratio of 0.86. Diagnoses were made between 2003 and 2018 with clinical information available in 97% of cases: 19 patients (20%) had hemolytic anemia and most patients (n=73, 77%) displayed BM failure including aplastic anemia (n=62), myelodysplastic syndrome (n=7) and unexplained cytopenia(s) (n=4). No case of thrombosis was included. All patients with hemolytic anemia showed an increasing clone size over time including the two who were not treated with eculizumab (Figure 1B; median size at diagnosis on neutrophils: 81.3% vs median size over 5 years: 96.3%). The median clone size at diagnosis for patients with BM failure was 1.5% on neutrophils with a very wide range 0.01-97.87, almost half of them being less than 1%. When comparing the diagnosis point with the latest follow-up point, PNH clone size increased in 37 patients, decreased in 16 of them and remained stable in 20 cases (Figure 1C, D). Nine of the 37 patients reached a PNH clone size above 50% and 4 of them received a treatment by eculizumab in a median delay of 5 years 1.5-6.0. Interestingly, no patient showed spontaneous disappearance of PNH clones, pending the use of a high-resolution flow cytometry test. The only five patients with undetectable PNH clones (Figure 1D, red lines) were those who underwent BM transplantation.
Conclusion: This multicenter study based on robust flow cytometry analysis showed no disappearance of PNH clones, including minor ones, over a long period of time, regardless of the clinical manifestations, except for patients who underwent BM transplantation. Moreover, PNH clone size increased in half of patients with BM failure, justifying a long term PNH clone size monitoring, even in these patients.
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Le Garff-Tavernier:Alexion: Consultancy, Honoraria. Pruvot Debliquis:Alexion: Honoraria; Takeda: Honoraria; Pfizer: Honoraria; Gilead: Honoraria; Genzyme: Honoraria. Socie:Alexion: Consultancy. Peffault de Latour:Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding. Drenou:Alexion: Consultancy, Honoraria. Wagner Ballon:Alexion: Consultancy, Honoraria.
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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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.
Introduction: Following the published International Guidelines (Borowitz, 2010), the French-Belgian biological workshop was initiated in 2010 by the French Paroxysmal Nocturnal Hemoglobinuria (PNH) ...group (named HPNAFC). This effort to harmonize the diagnostic practices has been pursued through the development of an inter-laboratory comparison program, involving more than 50 French-speaking centers since 2013 (Debliquis, 2015). Thus, the French National Observatory of PNH clones opened for 5 years on the 9th International Rare Disease Day (February 29th 2016), in order to collect all PNH clone cases diagnosed by French cytometrists.
Methods: The primary objective of the French National Observatory of PNH clones is to monitor all PNH clones ≥0.01%, along with two secondary objectives according to the PNH clone size: the evolution of minor clones <1% and the proportion and significance of type II and type III cells for clones ≥1%. All patients with a PNH clone ≥0.01% can be included, irrespective of age, provided that the center has validated a PNH flow cytometry quality control. For each patient, the inclusion time point always corresponds with the initial diagnosis date of the PNH clone, even if it was made before the opening of the Observatory, allowing the collection of all cases in the active French PNH file. This Observatory is based on a principle of non-opposition; inclusions are made by the referent cytometrist of each participating center by filling in the e-CRF form available on the HPNAFC website.
Results: As of June 30th 2017, 39 cytometry laboratories have registered across France. Of these, 24 centers have submitted 163 inclusion points with a PNH clone above 0.01%, each corresponding to a patient. The cases were reviewed by the 2 principal investigators, who carefully re-examined all of the flow cytometry data. This method enabled the validation and inclusion of 126 cases, with 37 ongoing ones due to missing clinical or biological data. The first extraction led to the preliminary analysis of these 126 cases and showed that patient median age at diagnosis was 45 years 11-87, with 6 pediatric cases, and a M:F sex ratio of 0.83. Diagnosis was made between 1988 and 2017 and clinical information at diagnosis was available for 92% of patients. More than half of them had aplastic anemia (58.7%). Other diagnosis included: hemolytic anemia (with or without aplastic anemia) (15.9%), unexplained cytopenia(s)(7.9%), myelodysplastic syndrome (6.3%), hemoglobinuria (4.8%) and 2 cases (1.6%) of recurrent thrombosis without hemolysis. Biological data showed a median hemoglobin value of 9.3 g/dL 4.7-16.5, 1.4 x109/L 0.1-11 neutrophils and 60 x109/L 2-356 platelets. The median clone size at diagnosis was 1.9% on granulocytes with a very wide range from 0.01% to 99.4%, most of them being less than 10% and mainly composed of type III cells (median type III clone size: 1.6%). A similar distribution of PNH clones was observed on monocytes, with a good correlation between clone sizes on both lineages. RBC analysis, which was performed for less than half of the patients showed a majority of minor clones and no correlation was observed between PNH clone size on granulocytes and RBC, due to an underestimation of the clone size on RBC. Regarding the clinical context, the median PNH clone size on granulocytes in case of aplastic anemia was 1%, yet some patients had very large clones, higher than 99%. Conversely, PNH clones from patients with hemolytic anemia were very large with a median size of 88%, but some patients had smaller clones of less than 20%. The 2 patients with thrombosis displayed a much smaller clone size, below 0.05%.
Conclusion: More than one year after its initiation, 126 confirmed patients were enrolled in the French National Observatory of PNH clones. Median PNH clone size was 1.9% on granulocytes and more than half of these patients had aplastic anemia. A good correlation was seen between clone sizes on granulocytes and monocytes, but not on granulocytes and RBCs. This collaborative work has established a strong network of motivated cytometrists as highlighted by the number of registered centers and the increasing inclusions since its recent opening. A comprehensive follow-up of a foreseeable larger number of PNH clones (so far 92 out of the 163 already have at least 1 follow up point) will lead to a better understanding of the physiopathology of minor PNH clones.
Drenou:Alexion: Consultancy, Honoraria. Pruvot Debliquis:Alexion: Consultancy, Honoraria. Letestu:Alexion: Consultancy, Honoraria. Chatelain:Alexion: Consultancy, Honoraria. Socié:Alexion Pharmaceuticals, Inc.: Consultancy. Peffault De Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding. Wagner Ballon:Alexion: Consultancy, Honoraria.
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