First Interim Analysis of the French National Observatory of Paroxysmal Nocturnal Hemoglobinuria Clones

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 HPN^AFC). 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 9^th International Rare Disease Day (February 29^th 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 HPN^AFC website.

Results: As of June 30^th 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.