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A retrospective study of paroxysmal nocturnal hemoglobinuria in pediatric and adolescent patients

Blood Cells, Molecules, and Diseases, May 2017, Pages 45 - 50

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease, especially in children, characterized by intravascular hemolysis, thrombotic events, serious infections and bone marrow failure. We describe 16 patients who were diagnosed with PNH in childhood or adolescence. The time interval between the onset of symptoms and the PNH diagnosis and its treatment were compared in patients with classic PNH versus PNH associated with bone marrow disorder (PNH/BMD). A greater delay in diagnosis was observed in classic PNH compared to PNH/BMD patients. The first group of patients had higher levels of LDH, total bilirubin and absolute reticulocyte count and a bigger PNH clone size compared to PNH/BMD patients; also thrombotic events were observed only in the classic form of PNH. Conversely, PNH/BMD patients showed lower median levels of platelets. Apart from standard supportive measures, four patients with classic PNH received eculizumab whereas four patients with PNH/BMD underwent hematopoietic stem cell transplantation. Our series confirm that the most frequent presentation of PNH in the pediatric-adolescent age is PNH/BMD. The delay between the onset of symptoms and PNH diagnosis is relevant principally in the classic form. Moreover, our study showed that any case of unexpected thrombosis represents a criterium to perform a PNH screening.

Keywords: Paroxysmal nocturnal hemoglobinuria, Pediatric, adolescent, Intravascular hemolysis.

1. Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease characterized by intravascular hemolysis, thrombotic events, serious infections and bone marrow failure [1], [2], [3], and [4]. Usually, the age of onset is between 20 and 50 [5]. In children, PNH is extremely rare and predominantly affects teenagers [6], [7], [8], [9], [10], and [11]. The protein encoded by the PIG-A gene is necessary for the synthesis of glycosylphosphatidylinositol (GPI), which anchors various proteins on the cell surface of blood cells. This mutation leads to the loss of CD55/DAF (Decay Accelerating Factor) and CD59/MIRL (Membrane Inhibitor of Reactive Lysis), two inhibitors of the complement system. The loss of CD55 and CD59 causes an increased susceptibility of the affected cells to complement-mediated lysis [12] and [13]. PNH may develop without bone marrow disorder (BMD) or as a secondary condition to BMD, such as aplastic anemia (AA) or myelodysplastic syndrome (MDS). Depending on whether the clinical picture is dominated by hemolysis or by BMD, patients are described as having classic PNH or PNH associated to BMD (PNH/BMD) [14]. Diagnosis of PNH has significantly evolved over time. Prior to the past two decades the Ham test was used, based on the increased sensitivity of PNH-affected erythrocytes to complement-mediated lysis. Subsequently, flow cytometry (FCM) using antibodies directed against glycosylphosphatidylinositol-anchored proteins (GPI-APs) became the most sensitive and informative assay available for diagnosis of PNH [3], [14], [15], and [16]. The introduction into clinical practice of humanized monoclonal antibodies with anticomplement activity, such as eculizumab, resulted in remarkable improvement in controlling intravascular hemolysis, reducing thrombotic events, and improving overall survival [17]. Conversely, hematopoietic stem cell transplantation (HSCT), which in the past was considered the only curative therapy for severe classic PNH, is no longer considered the first choice therapeutic option due to its higher morbidity and mortality and is reserved for the form evolving to severe pancytopenia, myelodysplasia or leukemia [18], [19], [20], and [21]. Reports on pediatric PNH patient cohorts are limited; apart from case reports or small collections (2 to 4 patients) [22], [23], [24], [25], [26], [27], [28], [29], [30], and [31] the largest series of pediatric PNH have been reported by Ware et al. in 1991 [6], Van den Heuvel-Einbrink et al. in 2005 [7], Naithani et al. in 2008 [8], Curran et al. in 2011[9], Sreedharanunni et al. in 2016 [10] and Urbano-Ispizua et al. in 2016 [11] (Table 1). In our study, we described 16 patients who were diagnosed with PNH in childhood or adolescence focusing especially on the time delay between the onset of symptoms and the establishment of the PNH diagnosis, the PNH clone size, the clinical symptoms, the treatments and laboratory data of classic PNH compared to PNH/BMD.

Table 1

Review of pediatric patients with PNH diagnosis in the literature.

 

Reference N Age at diagnosis (years) PNH/BMD Classic PNH Thrombosis Treatment Outcome
Miller et al. [22] 2 7.5 and 14 1 1 1 Pdn NA
Kletzel et al. [23] 1 11 1 0 NA NA
Ware et al. [6] 26 0.8–21.4 15 5 8 Pdn/Andr/ATG 8 died, 18 alive
Wyatt et al. [24] 1 11 1 1 NA NA
Graham et al. [25] 1 12 1 1 HSCT Alive
Endo et al. [26] 1 10 1 HSCT NA
Lin et al. [27] 1 11 AA 1 Pdn Alive
Flotho et al. [28] 2 7 and 12 2 AA 2 CSA + G-CSF-HSCT Alive
Rizk et al. [29] 4 > 6 4 1 Pdn/Andr/CSA 1died, 3 NA
Wainwright et al. [30] 1 9.5 1 0 Pdn/Andr NA
Einbrink et al. [7] 11 9–17 7 AA-4MDS 0 2 Pdn/ATG/Andr- HSCT in 5 4 died, 7 alive
Gupta et al. [31] 1 8 AA 0 CSA NA
Naithani et al. [8] 18 11–18 9 AA 9 1 Pdn/Andr/CSA 14 alive, 4 NA
Curran et al. [9] 12 5–20 6AA-5MDS 1 6 ATG/Pdn/CSA- HSCT in 5- Eculizumab in 3 4 died, 8 alive
Sreedharanunni et al. [10] 9 < 12 9 AA 1 ATG/CSA/Andr 4NA, 5 alive
Urbano-Ispizua et al. [11] 99 < 18 77AA- 2MDS 20 2 ATG/CSA NA
Current study 16 8.7–28.4 5AA-5MDS 6 3 CSA/ATG-ALG/corticosteroids-HSCT in 5-
Eculizumab in 4
All alive

N: number; Pdn: prednisolone; Andr: androgens; HSCT: Hematopoietic stem cell transplantation; CSA: cyclosporine; ATG/ALG: antithymocyte globulin/antilymphocyte globulin; NA: not available.

2. Material and methods

Between January 2014 and November 2016, we performed a retrospective study among centers belonging to the Marrow Failure Working Group of the Italian Association of Pediatric Hematology Oncology (AIEOP) in order to collect demographic, clinical and laboratory data of pediatric patients with diagnosed PNH. Information about the study was also extended to Red Cell Diseases and Myelodysplastic Syndromes Working Groups. Case-patients were identified in 8 centers. Parent or patient (if applicable) written consents were obtained and data collection and processing was in accordance with the Italian law on patient confidentiality and good clinical practice. Demographic and clinical data were collected through a paper case report form including information on age, gender, date of diagnosis, symptoms, prophylaxis and treatments, complications events, and HSCT. Laboratory data included: lactate dehydrogenase (LDH) levels, absolute reticulocyte count, total bilirubin, hemoglobin levels, platelets count, absolute leucocyte and neutrophil count. Patients with classic PNH and PNH in the setting of another specified bone marrow disorder [14] were included in the study, but we excluded patients with subclinical PNH [14]. Diagnostic criteria for PNH were: a) positivity of the Ham test, or b) flow cytometry. The presence of a PNH clone (defined as a pool of cells with a deficit in expression of at least two GPI–linked proteins in two or more cellular populations) was assayed by flow cytometry on granulocytes, monocytes, and erythrocytes, and considered positive irrespective of its size. Moreover, data on cytogenetics were collected to assess any clonal evolution. Treatment and survival data are updated as at November 2016.

2.1. Flow cytometry

EDTA-anticoagulated peripheral venous blood sample was collected from each patient. The processing and acquisition of samples was performed within 24 h from blood collection. The processing of samples was done according to the Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria [16]. A combination of six reagents including FLAER, CD24, CD45, CD33, CD15, CD14 (7 centers) or of four antibodies CD66b, CD14, CD33 ,CD45 (1 center) was used for the identification of PNH clone on granulocytes and monocytes. CD59 antibody was used to identify the PNH clone on erythrocytes. At least 10.000, 30.000 and 200.000 events for monocytes, granulocytes and erythrocytes respectively, were acquired by flow cytometry. The presence of PNH-cell population was defined if at least 30 GPI-deficient events were recorded in a cluster pattern.

2.2. Analysis of PNH clone

Doublet discrimination was performed on FSC-H versus FSC-A plots to select single cell populations (data not shown). An initial gate was drawn on a CD45/SSC plot to include both granulocytes and monocytes (Fig. 1 A). Neutrophils and monocytes were identified as CD15positiveSSChigh cells (Fig. 1 B) and CD33brightSSClow cells (Fig. 1 D), respectively. The plots FLAER/CD24 (Fig. 1 C) and FLAER/CD14 (Fig. 1 E) were used to identify GPI-deficient neutrophils (FLAERnegative/CD24negtaive) or monocytes (FLAERnegative/CD14negative), respectively. Erythrocytes were identified by their physical characteristics on SSC-log/FSC-log plot (Fig. 1 F). The negative expression of CD59 identified a PNH clone on erythrocyte population (Fig. 1 G). For the identification of the PNH clone on granulocytes and monocytes without the FLAER staining, an initial gate was drawn on a CD45/SSC plot to include both granulocytes and monocytes. Neutrophils and monocytes were identified as CD33dim/positiveSSChigh cells and CD33brightSSClow cells respectively. The negative expression of CD66b on granulocytes and of CD14 on monocytes identified a PNH clone in these lineages (data not shown).

Fig. 1

Fig. 1

Gating strategy for identification of PNH clone in granulocytes, monocytes and erythrocytes.

Doublet discrimination was performed on FSC-H versus FSC-A plots to select single cell populations (data not shown). An initial gate was drawn on a CD45/SSC plot to include both granulocytes and monocytes (A). Neutrophils and monocytes were identified as CD15positiveSSChigh cells (B) and CD33brightSSClow cells (D), respectively. The plots FLAER/CD24 (C) and FLAER/CD14 (E) were used to identify GPI-deficient neutrophils (FLAERnegative/CD24negtaive) or monocytes (FLAERnegative/CD14negative), respectively. Erythrocytes were identified by their physical characteristics on SSC-log/FSC-log plot (F). The negative expression of CD59 identified a PNH clone on erythrocyte population (G).

 

2.3. Immunosuppressive therapy and response

The patients with PNH/AA were treated according the guidelines of AIEOP [32] with antithymocyte globulin/antilymphocyte globulin (ATG/ALG) plus cyclosporine (CSA), with or without corticosteroids. The treatment response was determined to be either “complete” or “partial” at 6 months post-ATG/ALG treatment. Complete response (CR) was defined by transfusion independence, absolute neutrophil count (ANC) ≥ 1.5 10e9/L, Hemoglobin (Hb) ≥ 11 g/dL and Platelet count (PLT) ≥ 100 10e9/L. Partial response (PR) was defined by transfusion independence and hematological values lower than those achieved in CR. The patients that did not achieve CR or PR were classified as non-responder [33].

3. Results

3.1. Patient characteristics

In the period from October 1989 to September 2016, we found that clinical characteristics of PNH were observed in 16 patients, 10 females and 6 males. The median age at diagnosis was 16.2 years (range: 8.7–28.4). Among 3 patients diagnosed after 18 years of age, 2 patients were followed and treated for severe aplastic anemia (SAA) for 20, and 8 years respectively, whereas one patient developed classic PNH at 21 years of age but was diagnosed and followed in a pediatric center. For 11 patients the median delay between the onset of symptoms and PNH diagnosis was 0.9 years (range: 0 days to 6.5 years) (Table 2). Five patients were not included: three patients did not present with any PNH-clinical associated symptoms, even if in one of them a PNH clone was found in peripheral blood six years before PNH diagnosis. The other two patients were not included because there were not available data on their symptoms. Six patients (38%) had a diagnosis of classic PNH, and 10 patients had PNH/BMD (63%), including 5 PNH patients with concomitant AA or hypoplastic anemia (PNH/AA) and 5 patients with MDS (PNH/MDS). All patients had normal karyotypes. FISH analysis performed in 8 patients (3 classic PNH, 2 PNH/AA and 3 PNH/MDS) did not show chromosomal abnormalities. Between classic PNH and PNH/BMD patients there was no difference in the median age both at the onset of symptoms and at diagnosis of PNH. However, a greater delay in diagnosis was observed in classic PNH patients compared to PNH/BMD (Table 2).

Table 2

Demographic and clinical characteristics of classic PNH and PNH/BMD patients.

 

Total (n = 16) Classic PNH (n = 6) PNH/BMD (N = 10)
Median age at diagnosis, years (range) 16.2 (8.7–28.4) 16.5 (13.9–20.9) 16.1 (8.7–28.4)
Median age at presentation, years (range) (a) 15 (7.8–25.8) 15.1 (10.8–20.7) 12.6 (7.8–25.8)
Delay to diagnosis, years (range) (a) 0.9 (0–6.5) 1.4 (0.2–3.1) 0.9 (0–6.5)
Female, n 10 (62.5%) 4 (66.7%) 6 (60%)
 
Laboratory data, median (range)
Hb, g/dL (b) 8 (6.1–12.6) 7.7 (6.2–9.6) 8.5 (6.1–12.6)
WBC, × 10e9/L (b) 3.3 (0.8–6.6) 4.2 (2.3–6.6) 3.3 (0.8–6.2)
ANC, × 10e9/L (b) 1.7 (0.2–4.3) 2.1 (0.5–4.3) 1.7 (0.2–4.1)
PLT, × 10e9/L (b) 61 (11–269) 136 (49–296) 53 (11 − 202)
LDH × ULN (c) 2.4 (1.1–26.9) 3.2 (3.1–26.9) 1.3 (1.1.-6.1)
Reticulocytes, × 10e9/L (d) 126 (33–183,000) 221 (200–183,000) 101.3 (33–149)
Total Bilirubin, mg/dL (c) 1.5 (0.4–9.6) 2.2 (0.4–9.6) 1.1 (0.7–2.4)
 
PNH clone size (e)
Granulocyte clone size, %, median (range) 58.5 (2.5–95) 72 (50.8–95) 35.7 (2.5–88.4)
Monocyte, clone size, %, median (range) 65 (2.9–93.1) 84 (65–93.1) 32.7 (2.9–90.4)
Erythrocyte clone size, %, median (range) (f) 6.7 (0–69) 32.7 (1.5–69) 3.7 (0–15.4)
 
PNH symptoms
Fatigue, n 10 6 4 (3 PNH/AA, 1 PNH/MDS)
Hemoglobinuria, n 9 6 3 (2 PNH/AA, 1 PNH/MDS)
Abdominal pain 7 4 3 (2 PNH/AA, 1PNH/MDS)
Headache, n 4 2 2 PNH/AA
Backache, n 4 1 3 PNH/AA
Thrombotic events, n 3 3
Epileptic seizure, n 2 2
Ecchymosis/petechiae, n 2 2 (1PNH/AA, 1PNH/MDS)
Dysphagia, n 1 1
Chest pain, n 1 1PNH/AA

BMD: bone marrow disorder (5 patients with PNH/AA and 5 with PNH/MDS); Hb: hemoglobin; WBC: white blood cell count; ANC: absolute neutrophils count; PLT: platelets; LDH: lactate dehydrogenase; ULN: upper limit of normal. (a) Data evaluated on 11 patients, 5 PNH/BMD patients were not included because this data was not available; (b) data evaluated on 15 patients, 1 classic PNH patient was not included because this data was not available; (c) data evaluated on 14 patients, two patients including 1 PNH/BMD and 1 classic PNH were not included because this data was not available; (d) data evaluated on 8 patients, eight patients including 3 classic PNH and 5 PNH/BMD were not included because this data was not available. (c) Data evaluated on 14 patients, two patients including 1 PNH/BMD and 1 classic PNH were not included because this data was not available; (e) data evaluated on 15 patients, 1 classic PNH patient was not included because PNH diagnosis was established by Ham Test. (f) Date evaluated on 12 patients, three patients including 1 classic PNH and 2 PNH/BMD were not included because this data was not available.

3.2. PNH symptoms and adverse events

The main symptoms presented by patients during their clinical course included: fatigue (10/16), hemoglobinuria (9/16), abdominal pain (7/16), headache (4/16), backache (4/16), epileptic seizure (2/16), ecchymosis/petechiae (2/16), dysphagia (1/16), chest pain (1/16).

Three patients did not present with any PNH-clinical associated symptoms (Table 2). In general, the most recurring symptom was fatigue; in classic PNH patients fatigue, hemoglobinuria and abdominal pain were principally observed compared to PNH/BMD. In this latter group of patients we mainly observed backache.

Thrombotic events (venous and/or arterial) occurred in 3 classic PNH patients (Table 2). Notably 1 patient had multiple episodes of thrombosis in different sites: deep venous thrombosis, cerebral arterial occlusion and portal thrombosis. In this patient, thrombosis occurred after PNH diagnosis, whereas in the other 2 patients thrombotic events occurred before diagnosis.

3.3. PNH clone

The presence of a PNH clone was assayed by peripheral blood flow cytometry on granulocyte, monocyte and erythrocyte populations (Table 2). The PNH clone size on granulocytes ranged from 2.5% to 95% (median: 58.5%). The PNH clone size in monocytes ranged from 2.9% to 93.1% (median 65%). The PNH clone size in erythrocytes (n = 12, data about three patients were not included because the screening of erythrocytes was not done) ranged from 0% to 69% (median: 6.7%). The median clone size on granulocytes, monocytes and erythrocytes were larger in classic PNH compared to PNH/BMD patients (72%, 84%, 32.7% vs 35.7%, 32.7% and 3.7%, respectively).

3.4. Hematologic laboratory data

No difference was observed in the median value of absolute leucocyte and neutrophils count, nor in the median hemoglobin levels between classic PNH and PNH/BMD patients (Table 2); conversely, the median value of platelet count, LDH levels, total bilirubin and absolute reticulocyte count was higher in classic PNH compared to PNH/BMD (Table 2).

3.5. Therapy

Eleven out of 16 patients received different immunosuppressive therapy (IST) (Table 3). Five PNH/AA patients were treated with ATG/ALG and CSA, four of them also received corticosteroids along with ATG/ALG/CSA. Two PNH/AA patients achieved CR whereas the other three patients achieved PR. Four PNH/AA patients, two patients that achieved PR and two patients that achieved CR, are currently maintained with CSA. Three PNH/MDS patients received IST: two patients with an initial diagnosis of AA and subsequently re-evaluated as MDS were initially treated with CSA along with ATG/ALG the first one and the other one with CSA, ATG/ALG and corticosteroids. One patient achieved PR and is currently maintained with CSA, whereas the other patient underwent HSCT. The third PNH/MDS patient was treated with CSA and then underwent HSCT. Two classic PNH patients were treated with corticosteroids and another patient with CSA and corticosteroids as symptomatic treatment of PNH disease. Three classic PNH patients received anticoagulant therapy after a thrombotic event; despite that, one of these patients had multiple thrombotic events during therapy. All these three patients and another classic PNH patient received eculizumab: one patient was 14 years old when he started eculizumab, whereas the other three patients were 18, 21 and 33. These patients are currently maintained with eculizumab. For three of them, eculizumab was combined with anticoagulant therapy and both treatments are still ongoing. In the fourth patient, who had a history of epileptic seizures, eculizumab was combined with levitiracetam and both treatments are still ongoing. One classic PNH and 4 PNH/MDS patients underwent HSCT. One PNH/MDS patient received prophylactic anticoagulant therapy during HSCT. Supportive therapy with red blood cells (RBCs) transfusions was used in 11 patients and six of them also received platelets (PLTs) transfusions, while growth factors were administered to 4 PNH/BMD and 1 classic PNH (Table 3).

Table 3

Patient treatments.

 

Patients # Diagnosis Immunosuppressive therapy Anticoagulation therapy Transfusion Growth factors HSCT Eculizumab Outcome
1 CLASSIC PNH Yesb RBCs Eculizumabb Alive
2 PNH/AA (ATG/ALG), CSA, corticosteroids Alive
3 CLASSIC PNH Alive
4 PNH/AA (ATG/ALG), CSAa, corticosteroids RBCs Alive
5 PNH/AA (ATG/ALG), CSAa Alive
6 PNH/AA (ATG/ALG), CSAa, corticosteroids RBCs + PLTs G-CSF Alive
7 CLASSIC PNH Corticosteroids Yesb RBCs Erythropoietin Eculizumabb Alive
8 PNH/MDS CSA Yes RBCs + PLTs Erythropoietin/G-CSF Yes Alive
9 CLASSIC PNH Corticosteroids Yesb Eculizumabb Alive
10 PNH/AA (ATG/ALG), CSAa, corticosteroids RBCs + PLTs G-CSF Alive
11 CLASSIC PNH CSA, corticosteroids RBCs Yes Alive
12 PNH/MDS (ATG/ALG), CSAa RBCs + PLTs G-CSF Alive
13 PNH/MDS RBCs + PLTs Yes Alive
14 CLASSIC PNH RBCs Eculizumabc Alive
15 PNH/MDS RBCs + PLTs Yes Alive
16 PNH/MDS (ATG/ALG), CSA, corticosteroids Yes Alive

a Patients are currently maintained with CSA.

b In these patients eculizumab was combined with anticoagulant therapy and both treatments are still ongoing.

c In this patient eculizumab was combined with levitiracetam and both treatments are still ongoing.

HSCT: hematopoietic stem cell transplantation; RBCs: red blood cells; PLTs: Platelets; CSA: cyclosporine; ATG/ALG: antithymocyte globulin/antilymphocyte globulin.

Compared to PNH/BMD patients, classic PNH patients were principally treated with anticoagulant therapy, eculizumab, and RBCs transfusions. Conversely, PNH/BMD patients mainly received IST, growth factors, RBCs and PLTs transfusions and HSCT.

4. Discussion

PNH is a rare disease and its onset usually occurs in adulthood, being rare in children [5], [6], [7], [8], [9], [10], and [11]. One main difference between adult and pediatric patients is the presenting symptoms, with classic PNH being more frequently reported in adult series than in pediatric series.

The BMD form of PNH has been observed in 24–33% of adult cases [34] and [35] whereas, in children, the rate seems to be higher. Were et al. [6] reported 58% of child or adolescent cases with BMD at diagnosis; Van den Heuvel-Eibrink et al. [7] described 11 pediatric PNH patients, including 7 with AA and 4 with MDS; Naithani et al. [8] reported 18 pediatric PNH patients, of which 9 with AA; Curran et al. [9] described 12 pediatric PNH patients, 6 patients with AA, and 5 with MDS; Sreedharanunni et al. [10] described 70 children of Indian origin affected by AA. A GPI-negative population was found in 9 patients. This finding is confirmed by the recent study of Urbano-Ispizua et al. [11] that showed an incidence of almost 80% of PNH/BMD in pediatric patients recruited in the international PNH Registry Study. In agreement with most reports, we found BMD in a majority of patients (63%) at the time of PNH diagnosis (Table 1).

One of the most critical points is the timely diagnosis of PNH. Delay in diagnosis has been commonly observed in our series where we reported a median interval time of 0.9 years between the beginning of symptoms and PNH diagnosis. This interval time was higher in classic PNH patients than for PNH/BMD patients probably because the classic form is so rare in children. Moreover the sensitization to screen the PNH clone in patients with BMD led to an early diagnosis of PNH compared to classic PNH patients.

Thrombosis is the most common cause of mortality in PNH [36] and may occur at any site; however, venous thrombosis is more common than arterial. Thrombotic events have also been reported in pediatric PNH patients [6], [7], [8], [9], [10], [11], [24], [25], [28], and [29]. Consistently, we observed three classic PNH patients displaying thrombotic events, including one patient with recurrent episodes in multiple sites. While the mechanism of thrombosis in PNH is multifactorial, a correlation between clone size and thrombotic risk has been established in adults [37]. Although we found a bigger median clone size and thrombotic events in classic PNH patients compared to PNH/BMD groups, no significant correlation was observed between clone size and thrombosis probably due to the small number of cases in our study.

In line with intravascular hemolysis, patients with classic PNH had higher levels of LDH, total bilirubin and absolute reticulocyte count as compared to PNH/BMD patients. Conversely, PNH/BMD patients showed lower median levels of PLTs. On the basis of clinical picture, PNH/BMD patients were principally treated with immunosuppressive therapy, growth factors, RBC and PLT transfusions, and HSCT whereas classic PNH patients received RBC transfusions, eculizumab and anticoagulant therapy. Eculizumab, a humanized monoclonal antibody that inhibits terminal complement activation was approved in 2007 for treatment of PNH patients > 18 years [38]. Many studies have demonstrated that eculizumab provided a rapid and sustained reduction of hemolysis, fatigue, transfusion requirements and risk of thrombotic events [39], [40], [41], and [42]. Data on the use of eculizumab in children are scarce, because of the rarity of the disease. Recently, Reiss et al. [43] in a multi-centre phase I/II study published a work that described the efficacy and safety of eculizumab in children with PNH. In our patient group three patients started eculizumab at 21, 33 and 18 years old years old, while another one started the drug at 14 years old. The reason for starting eculizumab was a thrombotic event for three patients, associated with high transfusion requirement in one patient, and severe hemolysis and hemoglobinuria in the other patient. The treatment has been ongoing for 4, 11, 9 and 2 years, respectively, with clinical benefit and no adverse effects reported.

In conclusion, our series confirms that the most frequent presentation of PNH in the pediatric-adolescent age group is PNH/BMD and less frequently the classic form. Moreover, delay from the beginning of symptoms to diagnosis is still relevant in the classic form. Early detection is critical to improve the management and the patient's prognosis. To this end, the diagnosis of PNH should be considered in any patient with persistent or recurrent episodes of intravascular hemolysis associated to hyperbilirubinemia and anemia and in any case of unexpected thrombosis. Moreover, considering that in pediatric patients the PNH clone is frequently associated with BMD, we suggest to include the PNH screening in any patient with these disorders.

The experience with eculizumab is very limited but preliminary results show that the treatment is as effective in children and adolescents as in adults.

List of centers (number of patients)

Verona (4); Rome (2); Turin (2); Padua (2); Palermo (2); Pescara (2); Bologna (1); Monza (1).

Author contributions

Simone Cesaro and Angela Mercuri: Conception and design, collection and interpretation of data, manuscript writing.

Piero Farruggia, Fabio Timeus, Laura Lombardi, Maria Elena Cantarini, Paola Corti, Daniela Onofrillo, Maria Caterina Putti, Marta Pillon, Gloria Tridello, Massimiliano De Bortoli, Anna Pegoraro: Approval of the study, collection of data, and final approval of the manuscript.

Conflicts of interest

Authors declare no conflict of interests.

Funding

This study was in partly funded with a grant to A.M. by the non-profit organization ABEO (Parent Association of Pediatric Hematology–Oncology Children of Verona) (Grant number 2-2015).

Acknowledgements

I would like to thank Omar Perbellini, M.D., for his support in the flow cytometry analysis and Mr. Rodney Seddom for the review of the english style of the manuscript.

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Footnotes

a U.O.C Oncoematologia Pediatrica-AOUI Verona, P.le L.A. Scuro 10, 37134 Verona, Italy

b Pediatric Hematology and Oncology Unit, Oncology Department, A.R.N.A.S. Ospedali Civico, Di Cristina e Benfratelli, Piazza Nicola Leotta 4, 90127 Palermo, Italy

c S.C. Oncoematologia Pediatrica e Centro Trapianti-Presidio Infantile Regina Margherita-A.O.U. Città della Salute e della Scienza, Piazza Polonia 94, 10126 Torino, Italy

d UOC Ematologia-Dipart. di Biotecnologie Cellulari ed Ematologia-Policlinico Umberto I Università “ Sapienza”di Roma, via Benevento 6, 00161 Roma, Italy

e UOS di Oncoematologia Pediatrica, Dipartimento di Ematologia, Medicina Trasfusionale e Biotecnologie-Ospedale Spirito Santo, via Fonte Romana 8, 65123 Pescara, Italy

f Oncoematologia Pediatrica- Azienda Ospedaliera di Padova, Via N. Giustiniani 3, 35128 Padova, Italy

g Oncologia ed Ematologia Pediatrica, U.O. Pediatria- Pession, Dipartimento per la salute della donna, del bambino e delle malattie urologiche, Azienda Ospedaliero-Universitaria Sant’Orsola-Malpighi, via Massarenti 11, 40138 Bologna, Italy

h Clinica Pediatrica-Università Milano Bicocca, Fondazione Monza e Brianza Bambino Mamma, via Pergolesi 33, 20900 Monza, Italy

Corresponding author at: U.O.C Oncoematologia Pediatrica-AOUI Verona, P.le L.A. Scuro 10, 37134 Verona, Italy.

Comment by Jeff Szer
This paper describes a small series of an ultra-rare acquired clonal but non-malignant disorder PNH, in the paediatric population. It points out the differences in diagnostic delay between patients with classical (overtly haemolytic) PNH and those in whom a PNH clone was identified in the course of workup of a patient with another bone marrow disorder (such as aplastic anaemia). The data presented are useful and additional to those provided by the global PNH registry in which this Italian group has not participated. This paper should be considered along with the recent paper by Urbano-Ispizua A et al (Different clinical characteristics of paroxysmal nocturnal hemoglobinuria in pediatric and adult patients. Haematologica March 2017 102: e76-e79) in which the characteristics of 99 children were compared with over 2000 adult patients in that registry and found to have significantly different disease characteristics to adults. Both of these papers remind us that children are not just small adults.