Paroxysmal Nocturnal Hemoglobinuria Clones in Children with Acquired Aplastic Anemia: A Multicentric Study
Authors: Timeus Fabio Stefano , Crescenzio Nicoletta , Doria Alessandra , Foglia Luiselda , Pagliano Sara , Lorenzati Anna , Quarello Paola , Decimi Valentina , Svhan Johanna , Locatelli Franco , Ruggiero Antonio , Martire Baldassarre , Pillon Marta , Misuraca Aldo , Longoni Daniela , Dufour Carlo , Ramenghi Ugo , Saracco Paola 
Published: October 2019

Abstract
Abstract 1269

Introduction.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic disorder characterized by the clonal expansion of a PIG-A mutated stem cell and consequent defective synthesis of glycosil phosphatidyl-inositol-anchored proteins, complement-mediated hemolysis, increased incidence of thrombosis, bone marrow failure. PNH and acquired aplastic anemia (AA) are closely related and a reciprocal progression is possible. A relative resistance of the PNH stem cell to the immune-mediated damage can explain the PNH clonal expansion in AA. High resolution flow cytometry analysis (FCA) has revealed a high incidence of minor PNH clones in adult AA patients at diagnosis, predictive for some Authors of a favourable response to the immunosuppressive therapy (IST) (Maciejevki et al, 2001; Ishiyama et al, 2003; Sugimori et al, 2006). “Pure” PNH is a very rare disease in children. Only a few studies have so far evaluated longitudinally PNH clones in pediatric AA patients.

Materials and Methods.
Ninety AA patients diagnosed in 8 AIEOP (Italian Association of Pediatric Hematology-Oncology) Centers (age at diagnosis 1–20 years, median =10.8, 51 severe AA, 30 very severe AA, 9 non severe AA) were studied: forty-one since diagnosis, 25 during IST, 20 off therapy and 4 selected cases after hematopoietic stem cell transplantation (HSCT). Among the patients followed since diagnosis, 8 received an HLA matched sibling donor HSCT as first line therapy, whereas the other 33 patients were treated with IST according to EBMT protocols (anti-lymphocyte globulin/anti-thymocyte, ciclosporin ± granulocyte colony stimulating factor). The study started in 1998. Peripheral blood PNH cells were detected by lack of CD59 expression on granulocytes by a two-color FCA for CD59 (clone p282-FITC Becton-Dickinson) and CD11b (clone D12-PE Becton-Dickinson); at least 105 cells were analyzed, for a total of 1104 tests. The presence of a population CD11b+/CD59- > 0.15% was defined as abnormal; the cut off value was established in 1998 by evaluating 87 normal controls (PNH clones: median = 0.001%, mean+2SD=0.10%). Since 2009 FCA results were confirmed by more sensitive techniques with three or six-color sequential gating analysis for CD45/33/66b or CD45/33/15/24/14/FLAER.

Results.
A PNH+ clone was observed in 15 patients (36.6%) at diagnosis (clone size 0.17–10.4%), in 10 patients (40%) during IST (clone size 0.16–12.6%) and in 8 patients (40%) off-therapy (clone size 0.16–4.0%). The presence of a PNH+ clone at diagnosis did not predict a favourable response to IST, both in ALG and ATG-treated patients. In 33 patients (16 at diagnosis, 9 in IST, 8 off therapy), the presence of the PNH clone was sporadic or intermittent, whereas in 13 patients (9 at diagnosis, 3 in IST, 1 off therapy) the clone persisted for more than 3 following controls (follow up 6–60 months). Among the 26 PNH- patients at diagnosis, in 10 a PNH clone (clone size 0.16–1.7%) appeared later during IST. Among the 25 patients studied during IST, in one patient PNH clone appearance was associated with the tapering of cyclosporine (figure 1), in two with the relapse when off therapy. In one out of 4 patients treated with HSCT, a PNH clone appeared at time of relapse and disappeared after starting IST with cyclosporine (figure 2). A mild hemolysis was observed in the only 2 patients with a major PNH clone (clone size 12.6 and 10.4% respectively). No thrombotic events were reported.

Conclusions.
We have observed a significant incidence of minor PNH clones in pediatric AA at diagnosis, as reported in adults. Whereas previous studies in adults correlated the presence of pre-treatment minor PNH clones with a favourable response to IST, we do not confirm those observations both in the present multi-centre as in our previous single-centre study (Timeus et al, 2010), in agreement with Yoshida et al (2008) and Scheinberg et al (2010). The appearance of a PNH clone in a PNH- patient at diagnosis is described as uncommon (Sugimori et al, 2009), however in our series this was observed in 38% of previously PNH- patients. In AA the presence of PNH clones seems related to complex interactions between stem cells, immune-mediated damage and immunosuppressive therapy. A periodic screening for PNH clones in patients with AA is recommended, permitting modulation in IST, early identification of major PNH clones and prompt diagnosis of a frank PNH.

Disclosures:
Timeus: Alexion Pharma Italy s.r.l.: Research Funding. Dufour:Pfizer: Consultancy.