Development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia

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1 short report Development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia Atsushi Narita, Hideki Muramatsu, Yusuke Okuno, Yuko Sekiya, Kyogo Suzuki, Motoharu Hamada, Shinsuke Kataoka, Daisuke Ichikawa, Rieko Taniguchi, Norihiro Murakami, Daiei Kojima, Eri Nishikawa, Nozomu Kawashima, Nobuhiro Nishio, Asahito Hama, Yoshiyuki Takahashi and Seiji Kojima Department of Paediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan Received 30 January 2017; accepted for publication 19 April 2017 Correspondence: Seiji Kojima, Department of Paediatrics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showaku, Nagoya, , Japan. Summary The clinical significance of paroxysmal nocturnal haemoglobinuria (PNH) in children with aplastic anaemia (AA) remains unclear. We retrospectively studied 57 children with AA between 1992 and During the follow-up, five patients developed clinical PNH, in whom somatic PIGA mutations were detected by targeted sequencing. The 10-year probability of clinical PNH development was 102% (95% confidence interval, %). Furthermore, the detection of minor PNH clones by flow cytometry at AA diagnosis was a risk factor for the subsequent development of clinical PNH. These patients with PNH clones at AA diagnosis should undergo periodic monitoring for potential clinical PNH development. Keywords: paroxysmal nocturnal haemoglobinuria, aplastic anaemia, PIGA, Children. Paroxysmal nocturnal haemoglobinuria (PNH), a non-malignant clonal disease of haematopoietic stem cells, results from somatic mutations in the phosphatidylinositol glycan class A gene (PIGA). PNH frequently manifests with bone marrow disorders, especially aplastic anaemia (AA), wherein minor PNH clones escape from immune-mediated destruction by pathogenic T cells. Approximately 40 53% of paediatric AA patients possess minor PNH clones (Scheinberg et al, 2010; Timeus et al, 2010; Narita et al, 2015). Previous studies conducted on children with AA demonstrated that the presence of minor PNH clones was associated with a favourable response to immunosuppressive therapy (Narita et al, 2015). Recent studies using next-generation sequencing revealed that somatic PIGA mutations in patients with AA are associated with better prognosis (Yoshizato et al, 2015). On the other hand, clinical PNH is a progressive and life-threatening disease driven by chronic haemolysis that leads to thrombosis, renal impairment, poor quality of life and death. Large studies in adults have reported that clinical PNH developed in 4 9% of patients with AA (de Planque et al, 1989; Najean & Haguenauer, 1990); however, the frequency of evolution to clinical PNH in children with AA has rarely been described. Here, we aimed to elucidate the pathological significance of clinical PNH during the course of paediatric AA. The primary cohort included 78 children newly diagnosed with acquired AA at our institution between 1992 and Among them, 21 patients underwent haematopoietic stem cell transplantation (HSCT) as first-line treatment within 1 year after AA diagnosis, and were thus excluded. No patient had evidence of clinical PNH at AA diagnosis. We retrospectively examined 57 children (35 boys and 22 girls). Forty-six patients received immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG) and ciclosporin A (CyA) as initial treatment for AA. Of these, 39 patients received 15 mg/kg/day horse ATG (lymphoglobulin, Genzyme, Cambridge, MA, USA) for 5 days and seven received mg/kg/day rabbit ATG (thymoglobulin, Sanofi, Paris, France) for 5 days. Treatment with CyA (6 mg/kg/day) was initiated on day 1 and continued for at least 180 days. The response to IST was evaluated according to previously described criteria (Narita et al, 2015). Patients with complete or partial response within 6 months of IST were defined as responders. Written informed consent was obtained from the parents of all patients. The study was approved by the Ethics Committee of the Nagoya University Graduate School of Medicine. Clinical PNH was defined as the presence of intravascular haemolysis or thrombosis and 5% PNH granulocytes or First published online 23 June 2017 doi: /bjh ª 2017 John Wiley & Sons Ltd

2 PNH red blood cells (RBCs), according to de Latour et al (2008). Flow cytometry (FCM) was used to detect PNH CD13 + /CD55 /CD59 granulocytes and PNH glycophorin A + /CD55 /CD59 RBCs. The presence of >0005% PNH granulocytes and/or >0010% PNH RBCs was defined as minor PNH clone positive (PNH + ) (Data S1). We performed targeted sequencing of bone marrow samples from patients with clinical PNH that were obtained at two time points: the time of AA diagnosis and after PNH development. The panels of 184 genes for targeted sequencing included most genes known to be mutated in myeloid malignancy and bone marrow failure syndromes (Muramatsu et al, 2017) (Table SI). Next-generation sequencing (NGS) was performed using a HiSeq 2500 (Illumina, San Diego, CA, USA), according to the manufacturer s instructions. The mean coverage of the target coding region was 4519 for targeted sequencing. We detected germline and somatic variants using our established pipeline (Suzuki et al, 2016). Identified somatic mutations were validated by polymerase chain reaction-based deep sequencing at the two time points with a variant allele frequency (VAF) detection threshold of >01%. We confirmed the absence of detected variations in two unrelated healthy individuals. Clinical characteristics of the 57 patients are shown in Table SII. The median patient age at AA diagnosis was 93 (range, ) years. AA was idiopathic in 51 patients and hepatitis-associated in six patients. A total of 43 patients were screened for PNH clones by FCM at the time of AA diagnosis, and minor PNH clones were identified in 21 patients (Table SIII). The median percentages of positive PNH granulocytes and positive PNH RBCs were 0061% (range, %) and 0046% (range, %), respectively. The response rate to IST was 53% at 6 months. Allogeneic HSCT was administered to 23 patients with refractory AA, two with secondary myelodysplastic syndrome (MDS), and one with clinical PNH. One patient died of Epstein Barr virus-associated lymphoproliferative disease 2 months after initiating IST. The median follow-up period was 123 months (range, months). During follow-up, five patients developed clinical PNH in adolescence (range, years old) (Table I). The median time between AA diagnosis and PNH development was 49 (range, 33 79) years. All five patients exhibited a response to IST before clinical PNH development. Gross haemoglobinuria was present in all patients with clinical PNH, but thrombosis was not observed. The size of PNH clones after clinical PNH development varied among patients: median PNH granulocytes and PNH RBCs were 430% (range, %) and 489% (range, %), respectively. Eight somatic PIGA mutations were detected in five patients with clinical PNH, with multiple PIGA mutations in two patients (Table SIV). None of five patients harboured gene mutations in other 183 associated genes (Table SI). Although minor PNH clones were detected at the time of AA diagnosis in all five patients, PIGA mutations were not detected by targeted Table I. Clinical characteristics of five patients with AA who developed clinical PNH. PNH clone size by FCM (%) Age at diagnosis (years) Symptoms AA diagnosis Clinical PNH diagnosis UPN AA PNH Gender Aetiology Severity IST Haemolysis Thrombosis Erythrocyte Neutrophil Erythrocyte Neutrophil Sucrose lysis test Ham s test Response to IST at 6 months M Idiopathic SAA ATG/CyA PR M Idiopathic NSAA ATG/CyA PR F Idiopathic VSAA ATG/CyA PR M Idiopathic VSAA ATG/CyA PR + ND ND F Idiopathic VSAA ATG/CyA CR + ND ND AA, aplastic anaemia; ATG, anti-thymocyte globulin; CR, complete response; CyA, ciclosporin A; F, female; FCM flow cytometry; IST, immunosuppressive therapy; M, male; ND, not done; NSAA, non-severe aplastic anaemia; PNH, paroxysmal nocturnal haemoglobinuria; PR, partial response; SAA, severe aplastic anaemia; UPN, unique patient number; VSAA, very severe aplastic anaemia. ª 2017 John Wiley & Sons Ltd 955

3 sequencing. After identifying PIGA mutations in samples at clinical PNH, focused deep sequencing identified mutations at AA diagnosis in a patient with very small allele frequencies. Univariate analysis identified the presence of minor PNH clones at AA diagnosis, aetiology, IST treatment and the response to IST as risk factors for the development of clinical PNH (Table SV). Altogether, the 10-year probability of clinical PNH development was 102% (95% confidence interval [CI], %) (Fig 1A). Among 43 patients screened for PNH clones at AA diagnosis, the 10-year cumulative incidence of clinical PNH was significantly higher in patients with minor PNH clones than in those without minor PNH clones [29% (95% CI, 10 51%) vs. 0% (95% CI, 0 0%); P = 0015] (Fig 1B). In addition, patients who developed clinical PNH harboured significantly higher percentages of positive PNH granulocytes [median (range); 0324% (0 051%) vs. 0% (0 4451%), P = 0017] and positive PNH RBCs [median (range); 0291% ( %) vs. 0% (0 0840%), P = 0004] compared to patients without clinical PNH (Figure S1). PNH has been noted as a late complication in AA patients with long-term follow-up. Several studies indicated that 4 9% of adult patients with AA developed clinical PNH (de Planque et al, 1989; Najean & Haguenauer, 1990). Although several case series of childhood PNH have been published, the association between PNH and AA in children has been rarely addressed (Ware et al, 1991; Curran et al, 2012). Here we investigated the cumulative incidence of clinical PNH in children with AA and found that the 10-year probability of clinical PNH development was 10%. Curran et al (2012) reported that 12 of 60 children with AA and MDS developed clinical PNH; however, the exact cumulative incidence of clinical PNH in their cohort was not reported. Our study showed that the presence of minor PNH clones at AA diagnosis might be a risk factor for subsequently developing clinical PNH. These results are compatible with those of a previous adult study wherein all eight patients with AA who developed clinical PNH harboured minor PNH clones from the time of AA diagnosis (Sugimori et al, 2009). They also showed that PNH clones rarely emerge in the course of follow-up of PNH-negative patients. In patients with AA with minor PNH clones at the time of AA diagnosis, measuring PNH clones during the observation period may be useful for predicting the subsequent clinical PNH development. Mechanisms leading to the clonal expansion and dominance of PNH stem cells are incompletely understood. A study in a mouse model showed that PIGA mutation alone does not account for the dominance of mutant stem cells (Murakami et al, 1999). In classic PNH, the evolution of a PNH clone may be associated with additional somatic mutational events such as NRAS, JAK2, TET2 and ASXL1 mutations; however, additional mutations were absent in >50% of patients (Mortazavi et al, 2000; Shen et al, 2014). In contrast, in patients with AA who subsequently develop clinical PNH, minor PNH clones carrying only PIGA mutations may exert Fig 1. Cumulative incidence of clinical paroxysmal nocturnal haemoglobinuria. (A) Cumulative incidence of clinical paroxysmal nocturnal haemoglobinuria (PNH) in patients with aplastic anaemia (AA). The 10-year probability of developing clinical PNH was 102% (95% CI, %). (B) Cumulative incidence of clinical PNH in AA patients with and without minor PNH clones at the time of diagnosis of AA. The 10- year cumulative incidence of clinical PNH in patients with and without minor PNH clones at diagnosis of AA was 29% (95% CI, 10 51%) and 0% (95% CI, 0 0%), respectively. 956 ª 2017 John Wiley & Sons Ltd

4 clonal dominance over mutation-negative haematopoietic stem cells by escaping continuous autoimmune attacks. Our findings are in line with such a hypothesis because we detected only PIGA mutations and no other concomitant somatic mutations through targeted deep sequencing. Another possibility is that additional somatic mutations may not be frequent in childhood PNH because the prevalence of somatic mutation increases with age in patients with AA (Yoshizato et al, 2015). This study has some limitations because it was conducted retrospectively and screening for PNH clones at diagnosis was not performed in 14 patients. Despite these limitations, our results will certainly help researchers and clinicians to understand this rare disease better. In conclusion, the percentage of children with AA who eventually developed clinical PNH was comparable to that reported in previous adult series. The presence of minor PNH clones at AA diagnosis was a risk factor for subsequently developing clinical PNH. Further studies are necessary in order to reveal the mechanisms of PNH clone expansion in children with AA. Acknowledgements The authors would like to thank Ms. Yoshie Miura, Ms. Yuko Imanishi, and Ms. Hiroe Namizaki for their valuable assistance. The authors acknowledge the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine for technical support in cell sorting and next-generation sequencing. The authors acknowledge the Human Genome Centre, the University of Tokyo, for providing super-computing resources ( Competing interests SK was supported by a grant from Sanofi K.K. Author contributions AN, HM and YO performed laboratory analyses, gathered clinical information, designed and conducted the research, analysed data, and wrote the paper. YS, KS, MH, SK, DI, RT, NM and DK performed laboratory analyses; EN, KK, NN, AH and YT gathered clinical information; SK directed the research and wrote the paper. Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Detection of PNH-type cells and Statistical methods. Table SI. The panel of targeted sequencing. Table SII. Patient characteristics. Table SIII. Univariate analysis of development of clinical PNH from AA. Table SIV. Detected mutations by targeted sequencing and deep sequencing. Table SV. Univariate analysis of minor PNH clones at AA diagnosis. Fig S1. The frequencies of minor PNH clones in patients with and without clinical PNH. References Curran, K.J., Kernan, N.A., Prockop, S.E., Scaradavou, A., Small, T.N., Kobos, R., Castro-Malaspina, H., Araten, D., DiMichele, D., O Reilly, R.J. & Boulad, F. (2012) Paroxysmal nocturnal hemoglobinuria in pediatric patients. 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Therapeutic Advances in Treatment of Aplastic Anemia. Seiji Kojima MD. PhD.

Therapeutic Advances in Treatment of Aplastic Anemia Seiji Kojima MD. PhD. Department of Pediatrics Nagoya University Graduate School of Medicine Chairman of the Severe Aplastic Anemia Working Party Asia-Pacific