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Spanish consensus statement for diagnosis and treatment of paroxysmal nocturnal haemoglobinuria
Med Clin (Barc). 2016;146(6):278.e1–278.e7
www.elsevier.es/medicinaclinica
Consensus statement
Spanish consensus statement for diagnosis and treatment of paroxysmal nocturnal haemoglobinuria夽 Ana Villegas a,∗ , Beatriz Arrizabalaga b , Santiago Bonanad c , Enrique Colado d , Anna Gaya e , ˜ f , Emilio Ojeda g , Alberto Orfao h , José-María Ribera i , Ataúlfo González a , Isidro Jarque c , Ramiro Núnez j e ˜ Vicente Vicente , Álvaro Urbano-Ispizua , Grupo de Trabajo de HPN de la Sociedad Espanola de Hematología y Hemoterapia a
Servicio de Hematología, Hospital Clínico San Carlos de Madrid, Madrid, Spain Servicio de Hematología, Hospital Cruces, Bilbao, Spain c Servicio de Hematología, Hospital La Fe, Valencia, Spain d Servicio de Hematología y Área de Gestión Clínica de Laboratorio de Medicina, Hospital Universitario Central de Asturias, Oviedo, Spain e Servicio de Hematología, Hospital Clínic, Barcelona, Spain f Servicio de Hematología, Hospital Virgen del Rocío, Sevilla, Spain g Servicio de Hematología, Hospital Puerta de Hierro, Madrid, Spain h Servicio de Hematología, Hospital Universitario de Salamanca, Salamanca, Spain i Servicio de Hematología Clínica, Instituto Catalán de Oncología-Hospital Germans Trias i Pujol, Badalona, Spain j Servicio de Hematología, Hospital Morales Meseguer, Murcia, Spain b
a r t i c l e
i n f o
Article history: Received 17 September 2015 Accepted 1 December 2015 Available online 16 June 2016 Keywords: Paroxysmal nocturnal haemoglobinuria Haematopoietic progenitor cells Mutation
a b s t r a c t Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of the haematopoietic progenitor cells due to a somatic mutation in the X-linked phosphatidylinositol glycan class A gene. The disease is characterised by intravascular haemolytic anaemia, propensity to thromboembolic events and bone marrow failure. Other direct complications of haemolysis include dysphagia, erectile dysfunction, abdominal pain, asthenia and chronic renal failure (65% of patients). The disease appears more often in the third decade of life and there is no sex or age preference. Detection of markers associated with glucosyl phosphatidyl inositol deficit by flow cytometry is currently used in the diagnosis of PNH. For years, transfusions have been the mainstay of therapy for PNH. A breakthrough in treatment has been the approval of the humanised monoclonal antibody eculizumab, which works by blocking the C5 complement protein, preventing its activation and therefore haemolysis. Several studies have confirmed that treatment with eculizumab avoids or decreases the need for transfusions, decreases the probability of thrombosis, improves the associated symptomatology and the quality of life in patients with PNH, showing an increase in survival. Because of rapid advances in the knowledge of the disease and its treatment, it may become necessary to adapt and standardise clinical guidelines for the management of patients with PNH. ˜ S.L.U. All rights reserved. © 2016 Elsevier Espana,
˜ para el diagnóstico y tratamiento de la hemoglobinuria Consenso espanol paroxística nocturna r e s u m e n Palabras clave: Hemoglobinuria paroxística nocturna Células progenitoras hematopoyéticas Mutación
La hemoglobinuria paroxística nocturna (HPN) es una enfermedad clonal de las células progenitoras hematopoyéticas originada por la mutación adquirida del gen fosfatidil-inositol-glicano del grupo A, situado en el brazo corto del cromosoma X. Se caracteriza por anemia hemolítica intravascular, tendencia a la trombosis y un componente variable de insuficiencia medular. Otras complicaciones derivadas de la
夽 Please cite this article as: Villegas A, Arrizabalaga B, Bonanad S, Colado E, Gaya A, González A, et al. Consenso espanol ˜ para el diagnóstico y tratamiento de la hemoglobinuria paroxística nocturna. Med Clin (Barc). 2016;146:278.e1–278.e7. ∗ Corresponding author. E-mail address: [email protected] (A. Villegas). ˜ S.L.U. All rights reserved. 2387-0206/© 2016 Elsevier Espana,
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hemólisis son disfagia, disfunción eréctil, dolores abdominales, astenia e insuficiencia renal crónica (un 65% de los pacientes). La enfermedad afecta por igual a ambos sexos y puede aparecer a cualquier edad, con una mayor incidencia en la tercera década de la vida. Actualmente, el diagnóstico se basa en la detección de poblaciones celulares con marcadores asociados al déficit de glucosil-fosfatidil-inositol mediante ˜ citometría de flujo. Durante anos, el pilar terapéutico de la HPN hemolítica era el soporte transfusional. Un gran avance en el tratamiento ha sido la aprobación del anticuerpo monoclonal humanizado eculizumab, que bloquea la proteína C5 del complemento impidiendo su activación, y por tanto, la hemólisis. Diversos estudios han confirmado que el tratamiento con eculizumab evita o disminuye el requerimiento transfusional, reduce la probabilidad de trombosis, mejora la sintomatología asociada y la calidad de vida de los pacientes con HPN, mostrando un aumento de la supervivencia. Este rápido avance en el conocimiento de la enfermedad y su tratamiento hace necesario adaptar y homogeneizar las directrices de actuación clínica en el manejo de pacientes con HPN. ˜ S.L.U. Todos los derechos reservados. © 2016 Elsevier Espana,
Introduction Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal disorder of haematopoietic progenitor cells that originates from the acquired mutation of the phosphatidylinositol glycan group A gene (PIGA-A), located in the short arm of chromosome X.1 As a result of this mutation, the glucosyl phosphatidyl inositol (GPI) anchor group does not synthesise, a necessary action for many membrane proteins to bind to the cell surface. Inside these proteins lies the membrane inhibitor of reactive lysis (MIRL) and the complement decay-accelerating factor DAF (CD55), both physiological inhibitors of complement activation. As a result of this deficiency, erythrocytes are more sensitive to the lytic action of complement, causing haemolysis and platelet activation, among other effects.2,3 The disease can appear at any age, with a higher incidence rate in the third decade of life; gender distribution is similar.2 Clinically, PNH is characterised by intravascular haemolysis, susceptibility to thrombosis and a variable component of bone marrow failure.4 Today PNH is considered a systemic disease – one which may affect several organs, especially the liver, kidney, central nervous system, lung and/or heart – because of the deficiency of nitric oxide (NO) and thrombotic events.4,5 Haemoglobinuria, a sign that names the disease, cannot be objectified; only 26% of cases initially show symptoms and 62% at some point throughout the course of the disease.3,6 Complications are a direct result of intravascular haemolysis and sequestration of NO by free haemoglobin. NO depletion produces peripheral vasoconstriction, occurring with dysphagia, erectile dysfunction, abdominal and chest pain and often with profound fatigue that prevents patients from carrying out a normal life. The most common complications are thromboses, the leading cause of death from PNH (40–67% depending on the type). Thromboses are mainly venous, and can be located in unusual places such as hepatic portal vein, splenic vein and mesenteric vein, inferior vena cava, central nervous system veins, skin veins or central retinal vein, although it should be noted that approximately 15% occur in blood circulation, especially in brain and coronary arteries.7–9 Pulmonary hypertension and renal failure may also be particularly serious complications of the disease.10,11 Occasionally, patients develop acute renal failure which is usually reversible but may require dialysis. 65% of patients have chronic renal failure, and severe chronic renal failure (stage 3 or higher) in 21% of cases.12 Pregnancy, raising the risk of thrombosis, complicates the natural course of the disease, causing an increase in maternal-foetal morbidity and mortality. It has been estimated that maternal mortality during pregnancy and the immediate post-partum period is 12–21%.13,14 There is also risk of miscarriage or premature birth.13,14
The association with bone marrow failure, especially with aplastic anaemia, is widely recognised in all patients, preceding the diagnosis of PNH in many cases. Until 2007, haemolytic PNH patients were treated mainly with blood transfusions. A great therapeutic advance has been the monoclonal antibody, eculizumab, approved both by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). It was first authorised in Spain for use in adult patients with PNH on June 20, 2007, and in April 2013 the indication was extended to paediatric patients. Significantly, on March 30, 2015 the EMA changed the guideline criteria, to include patients with a high activity of the disease, defined as elevated haemolysis, together with one or more of the clinical symptoms associated with it (asthenia, haemoglobinuria, abdominal pain, dyspnoea, anaemia, thrombosis, dysphagia or erectile dysfunction). Eculizumab is a humanised monoclonal antibody that acts by blocking complement protein C5, preventing terminal complement activation and thus haemolysis.15,16 The use of eculizumab stabilises the haemoglobin and reduces transfusion requirements and symptoms associated with dysfunction of the smooth muscle, reducing fatigue and significantly improving quality of life.17 It has also been shown to reduce the relative risk of thromboembolism by 85% and the reduction in the frequency of cases treated with anticoagulants for previous occlusive vascular episodes is 94%.8 It also improves pulmonary hypertension and renal failure, especially in the early stages. Likewise, it has been used in women with PNH who are pregnant, with encouraging responses and without risk to the mother or the foetus.13,18,19 Recently, the largest series of monitored cases of pregnant patients with PNH has been published, demonstrating the safety and efficacy of eculizumab in this group of patients.20 At present, after more than 12 years of treatment, sufficient experience has been collected to confirm that treatment with eculizumab has changed the natural history of PNH. In a disease that had a short life expectancy compared to the general population, with a median survival of 10–15 years from diagnosis, there are data showing that treatment with eculizumab produces a significant increase in life expectancy, and its positive effect in improving the quality of life has been proven; it is medication that has a safe profile and which is generally well tolerated.21 Recently, a group of experts in PNH at a hospital in Leeds in England has published a retrospective study where eculizumab treatment improves the survival of patients with PNH relative to a control group of a healthy population of the same age and sex.22 Due to the rapid pace of advances in knowledge of the disease and its treatment, it has become necessary to standardise and adapt clinical practice guidelines in order to facilitate the monitoring of patients with classical or haemolytic PNH. In order to address this need, the Spanish PNH Working Group was created, sponsored by the Spanish Society of Haematology and
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Indications for the study of PHN populations • Haemolytic anaemia with negative Coombs' test.
• Hydroplastic-MDS RCMD: refractory cytopenia anaemia with multilineage dysplasia RAEB: refractory anaemia with excess blast RCMD-S: refractory cytopenia anaemia with
• Haemoglobin or haemosiderosis • Idiopathic thrombosis (venous or arterial) with 1 or more of the following criteria:
multilineage dysplasia with ringed sideroblasts,S1 syndrome.
c) Haemolysis a) Young b) Unusual locations d) Cytopenia
• Idiopathic cytopenia
• Dysphagia or abdominal pain with haemolysis • Medullary aplasia with 2 of the 3 following criteria:
Hb <10 g/dl Neutrophils <1.5 × 109L Platelet <150.00 L
a) Hb <10 g/dl b) Neutrophils <1.5 × 109/L c) Platelet <100 × 109/L
Consider or rule out PHN through a high sensitivity flow cytometry and clinical evaluation Repeat every 6 months
PHN clone +
PHN clone – In the case of aplasia, repeat cytometry every 6 months
Clone >0.001%
Monitoring Clinical history
Mandatory complimentary explorations
Intravascular haemolysis, 1. Laboratory analysis anaemia, dysfunction of smooth muscle, thrombosis. 2. Bone marrow aspirate and biopsy if necessary
Complimentary explorations recommended 1. HLA typing in a young patient. 2. Abdominal MRI
3. Thrombophilia study 4. Determining proBNP or NT-proBNP. 5. Imaging testsb
Indication of eculizumab in patients with any of the following criteria: • Haemolysis LDH> 1.5 × LSN + symptoms. • Thrombosis attributable to PNH. • CRF attributable to PNH or repeated episodes of RF. • Regular transfusions. • RF or PH with NYHA class III or IV. • Smooth muscle affectation: intense abdominal and lumbar pain or dysphagia. • Pregnancy.C Fig. 1. Algorithm diagnosis, monitoring and treatment of PNH. a Full, reticulocyte blood count, lactate dehydrogenase (LDH), indirect bilirubin, haptoglobin, plasma haemoglobin, haemoglobinuria, haemosidenuria, vitamin B12, serum erythropoietin, iron, folic acid, direct Coombs, creatinine, estimated glomerular filtration rate (eGFR), proteinuria. b Abdominal and cardiac ecodoppler, cranial MRI or angiography through helical computed tomography (annual or clinical changes). c Assess each case individually. BNP: B-type natriuretic peptide; Hb: haemoglobin; HLA: Human Leucocyte Antigen; PNH: paroxysmal nocturnal haemoglobinuria; PH: pulmonary hypertension; PI: pulmonary insufficiency; RF: renal failure; CRF: chronic renal failure; NYHA: New York Heart Association; NMR: nuclear magnetic resonance; MDS: myelodysplastic syndrome.
Haemotherapy, which published in 2011 in Medicina Clínica a first consensus document for management of PNH.23 New data released later that same year led the group to meet again in November 2011; the results of this meeting included an update of the consensus Guide in May 2012. Given the availability of new evidence a second meeting was held in December 2013 that led to another update of the guidelines in June, 2014.24 In January 2015 the Working Group met again in order to continue updating the document, specifically in regard to the following areas: the indication for the
study of PNH populations, detection of the PNH clone by flow cytometry, the monitoring of patients with PNH and treatment of the disease. Prior to the meeting, articles that had been published with new information related to these areas were identified through searches on Medline and Embase. The selection of articles to review was performed according to expert judgement. The classification of selected publications based on levels of evidence was conducted by the Centre of Evidence-Based Medicine, considering at level 1 randomised clinical trials; at level 2, cohort studies; at
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level 3, and case-control studies, and level 4, series of cases. Due to the idiosyncrasies of PNH, classified as an ultra-rare disease, the degree of recommendation is categorised based on experts’ clinical judgement and experience. Additionally, the update of this document has also considered information concerning the management of the disease in clinical practice recorded in the National and International PNH Registration database (PNH registry). During the meeting the selected evidence was presented and the recommendations contained therein were subsequently debated. In order to establish the degree of consensus for each of the recommendations, attendees expressed their level of agreement by voting, considering accepted recommendations as those where at least 66% of the participants agreed. For the December 2013 meeting, three haematologists who are experts in PNH were invited to attend as external evaluators for the review, discussion and validation of the recommendations proposed in the successive updates of the consensus document. This article exposes the recommendations contained in the group’s last meeting (May 2015) in regard to the four areas defined above (Fig. 1).
Table 1 Identification of deficient cells in glucosyl phosphatidyl inositol (GPI) by flow cytometry in diagnosis of PNH. Study variable
Recommendation
Type of sample Populations
Peripheral blood Step one Neutrophilic granulocytes and monocytes Step two Red blood cells Step one (granulocytes neutrophils and monocytes): FLAER and CD157 or FLAER, CD24 (or CD16) and CD14 Alternatively (if FLAER is not available): CD157PE, CD24 (or CD16) and CD14 Step two (red blood cells): CD59
GPI Markers
Additional information Additional cell identification markers
Combinations of markers to study leucocytea
Indications for the study of paroxysmal nocturnal haemoglobinuria populations The diagnosis of the PNH should be performed by flow cytometry. The study of PNH populations is indicated in patients with one or more of the following symptoms: • Haemolytic anaemia with negative Coombs’ test. • Haemoglobinuria. • Unexplained, venous or arterial thrombosis in patients who meet any of the following criteria: young patients, presence of thrombosis in unusual locations (intra-abdominal veins, Budd–Chiari syndrome, brain, skin, etc.), evidence of haemolysis and/or cytopenia. • Intermittent dysphagia or abdominal pain of unknown aetiology with evidence of haemolysis. • Medullary aplasia (at diagnosis and annually during monitoring). • Hypoplastic myelodysplastic syndrome. • Idiopathic and maintained cytopenias of uncertain significance. PNH clone detection by flow cytometry Currently, analysis is done by flow cytometry, in addition to screening diagnosis, to monitor and treat the disease (see Table 1). Sample type and cell populations of interest The study of PNH populations is done routinely in peripheral blood samples. A deficiency of GPI expression is initially identified in neutrophil granulocytes and monocytes. In cases where the presence of cells with GPI anchored proteins deficiency (PNH clone) have been demonstrated, a study of red cells is carried out in a second step. The analysis of the leucocyte for the detection of PNH populations is the best method available to properly assess the size of the deficient clone in GPI. Lymphocytes, because of their long half-life and variable expression of different GPI proteins, do not constitute an adequate population; whereas monocytes and neutrophils are suitable populations given their number, the correlation between the size of clone between themselves and that they can be easily assessed in parallel. The study in leucocytes is the technique of choice for detecting PNH clones, both for low
Combination of markers to study red blood cells Tests
Indications a
CD45 and CD10 or CD15 for neutrophil granulocytes CD45 and CD64 for monocytes CD235a for red blood cells 5-fluorescence (in a tube): FLAER CD157-PE CD45-PerCP CD64-APC CD10-APCH7 or FLAER CD157-PE CD45-PECy7 CD64-ECD CD10-PECy5 4-fluorescence (2 in a tubes): FLAER CD157 (or CD24)-PE CD45-PerCP CD10-APC and FLAER CD157 (or CD14)-PE CD45-PerCP CD10-APC or FLAER CD157 (or CD24)-PE CD45-PECy7 CD10-PECy5 FLAER CD157 (or CD14)-PE CD45-PECy7 CD64-PECy5 CD235a-FITC, CD59-PE (clone of reference: MEM43) Internal (positive and negative populations or with different fluorescence intensity for each marker) Screening diagnostic; monitoring of the disease and its treatment
Different combinations adapted to different flow cytometers.
and high sensitivity studies, and for monitoring patients with PNH clone.25,26
Markers associated with glycosyl phosphatidyl inositol There are several ways to create the neutrophilic granulocytes and monocytes selection strategy.25 FLAER is particularly useful for researching the deficit of expression of proteins associated to GPI in these populations, it is a fluorescent derivative of the bacterial toxin aerolysin that binds to GPI in different leucocyte populations, in combination with CD157 markers. Another option is to perform the analysis in these sub-populations with FLAER and CD24 (or CD16) and CD14 markers. If FLAER is not available, an alternative is to use CD157PE markers in combination with CD24 (or CD16) and CD14. The combination of CD45 and CD10 markers facilitates identification of neutrophilic granulocytes, whereas for monocytes CD45 and CD64 markers are used. In the case of detecting deficiencies in expression of proteins associated with GPI in neutrophilic granulocytes or monocytes, carrying out the second step is necessary which consists of evaluating the CD59 in red blood cells. CD59 expression is more informative than CD55 expression and other antigens such as CD58, as they are found in greater numbers on the surface of red blood cells. Although other options can work, using PE-conjugated antibodies for GPI molecules detection is recommended as there is more experience with that process. Simultaneous markers of CD55 and CD59 are not recommended, since they require careful titration of reagents to avoid agglutination between the red blood cells. In the case of performing highly sensitive techniques, using antigens for
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positive selection of red blood cells is essential, where there is only information of CD235a utility. Combinations of markers For the above mentioned first and second steps, leucocytes and erythrocytes detection respectively, different combinations of antibodies can be used according to the laboratory’s capabilities (see Table 1). The correct identification of sub-populations and GPI deficient clones can be performed using negative and positive controls or positive controls with different fluorescence intensity for each marker (see Table 1). Evaluation of patients once the diagnosis of paroxysmal nocturnal haemoglobinuria has been confirmed Clinical history PNH is a systemic disease with multiple clinical manifestations. For this reason, the patient’s medical history should be detailed so the presence of the most characteristic symptoms and signs of intravascular haemolysis can be detected, (dark urine, jaundice), anaemia, dysfunction of smooth muscles (severe asthenia, dysphagia, abdominal pain, chest pain and erectile dysfunction) and previous thrombosis (abdominal pain, dyspnoea, neurological deficit and chronic headache among others). Mandatory explorations • Laboratory tests include a complete blood count with haemoglobin, neutrophils, platelets, reticulocytes, biochemical study of lactate dehydrogenase (LDH) direct and indirect bilirubin, haptoglobin, plasma haemoglobin, haemoglobinuria, haemosiderinuria, ferric profile (sideremia, transferrin, saturation index transferrin and ferritin) levels of vitamin B12, folic acid, serum erythropoietin and direct Coombs test. An assessment of renal function through analysing plasma levels of creatinine should also be conducted, creatinine clearance, proteinuria and urinary sediment. • Bone marrow aspirate to carry out a morphological and iron staining study. To perform a differential diagnosis, including a cytogenetic study is also advisable. • Bone marrow biopsy if the analytical data suggests a bone marrow aplasia. • Thrombophilia studies. • Abdominal ultrasound with Doppler. • Doppler echocardiography: if there is evidence of pulmonary hypertension a pulmonary CT angiography must be requested. • Determining proBNP or NT-proBNP. • Magnetic resonance imaging (MRI) or cranial computed tomography (according to radiological criteria) in case of headache or other neurological symptoms. Other recommended explorations Depending on each case, performing other examinations that complement those listed above could be considered. In young patients, a Human Leucocyte Antigen (human leucocyte antigens) typing is recommended because of the possibility of an eventual stem cell transplantation. The radiological study by abdominal MRI is useful in assessing the degree of renal and hepatic iron deposit. Monitoring PNH patients require comprehensive monitoring with quarterly analytical reviews and renal function assessments (creatinine,
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creatinine clearance, sediment and proteinuria) every 6 months. Conducting an assessment of the clonal size by flow cytometry at 6 months of diagnosis is advisable to assess the patient’s stability, so that in such a case assessing the quantification of the clone annually can be valued. Annual reassessments, or if changes in clinical or biological behaviour of the disease are observed, are recommended according to the characteristics of each patient by performing thoracic and abdominal MRI, abdominal ultrasound with Doppler and Doppler echocardiography. Treatment of paroxysmal nocturnal haemoglobinuria The main objective of PNH treatment should be to reduce haemolysis and minimise the risk of complications given the systemic impact of the disease. Additionally, treatment of PNH may require supportive therapy including transfusions of concentrated red blood cells, folic acid and iron supplements, antithrombotic anticoagulant prophylaxis and treatment of thrombotic complications. Corticosteroids have been widely used for years in the treatment of PNH, however there is no clear evidence of their benefits. One great therapeutic advance has been the approval of the monoclonal antibody eculizumab, which controls haemolysis and the pathophysiological consequences of the disease. The only potentially curative treatment of PNH is allogeneic haematopoietic stem cell. However, despite the best results achieved with this procedure in recent years, it is still associated with high morbidity and mortality and is reserved for highly selected patients, especially those with very severe associated aplastic anaemia. Eculizumab Indications Data supporting the use of eculizumab in PNH patients for any of the following: indications is currently available: a) Patients with chronic intravascular haemolytic anaemia that shows LDH at 1.5 times the upper limit of normal and clinical symptoms due to haemolytic anaemia, which may manifest, among other symptoms, as a significant impact on quality of life. b) Patients with thrombosis attributable to PNH. c) Patients with chronic renal failure attributable to PNH or repeated episodes of acute renal failure. d) Patients with regular transfusional requirement due to haemolysis. e) Patients with lung failure who have dyspnoea and/or chest pain resulting in a limitation of normal activity (New York Heart Association class III or IV) and/or an established diagnosis of pulmonary hypertension when other causes of it have been excluded. f) Patients with involvement of smooth muscle who show recurrent episodes of severe pain (abdominal, lumbar or oesophageal spasm with a history of dysphagia) requiring hospitalisation or taking opioid analgesics regularly. Before these patients start treatment with eculizumab other causes must be ruled out. g) Pregnancy in a patient with PNH involves a high risk of thrombosis for mother and foetus, treatment with eculizumab may prevent these complications. However, it is recommended that its use should be assessed on an individual basis. The EMA recently approved an amendment of the indication criteria for treatment with eculizumab to include patients who have high disease activity, regardless of the history of transfusions. High disease activity is defined as elevated haemolysis together with one or more of associated clinical symptoms: fatigue, haemoglobinuria,
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Table 2 Dosage of eculizumab. Population
Initial phase
Maintenance phase
Adult patients (≥18 years old)
600 mg per week × 4
900 mg in the 5th week; then 900 mg every 14 ± 2 days
Paediatric patients with body weight 30–<40 kg
600 mg per week × 2
900 mg in the 3rd week; then 900 mg every two weeks. 600 mg in the 3rd week; then 600 mg every two weeks. 300 mg in the 2rd week; then 300 mg every two weeks. 300 mg in the 2nd week; then 300 mg every three weeks.
20–<30 kg
600 mg per week × 2
10–<20 kg
600 mg per week × 1
5–<10 kg
300 mg per week × 1
abdominal pain, dyspnoea, anaemia, thrombosis, dysphagia and/or erectile dysfunction. PNH registration (M07-001) was used to assess the efficacy of eculizumab in PNH patients without a history of red blood cell transfusions. These patients had high disease activity defined as high haemolysis (LDH ≥ 1.5 × LSN) and the presence of one or more associated clinical symptoms; fatigue, haemoglobinuria, abdominal pain, shortness of breath (dyspnoea), anaemia (haemoglobin < 10 g/dl), severe adverse vascular events (including thrombosis), dysphagia or erectile dysfunction. Vaccination against meningococcus with a conjugate vaccine that includes serotypes A, C, Y and W135, in addition to vaccination against serotype B (Bexero) is mandatory at least 2 weeks before starting treatment with eculizumab. Dosage Eculizumab is administered intravenously, at a weekly dose of 600 mg for 4 weeks. During the fifth week a dose of 900 mg is administered and thereafter doses of 900 mg are continuously administered approximately every 14 days (between 12 and 16 days). In patients with a body weight of less than 40 kg, dosage varies depending on the weight (see Table 2). Monitoring Evaluation and monitoring of the treatment is conducted by regular laboratory analysis including complete blood count, reticulocyte count and biochemical study of LDH. Conducting a study every three months is recommended to assess renal function and ferric profile (sideremia, transferrin, IST and ferritin). In case of iron overload, an abdominal MRI should be considered to analyse liver iron deposition, and repeated every 12 months. In addition to these parameters a general, direct Coombs test and an anti-C3 test every 3–6 months should also be conducted. Given the increased susceptibility to meningococcal sepsis in patients receiving eculizumab, prophylactic antibiotic treatment is recommended with oral penicillin at 400 mg/12 h with the prophylactic antibiotic protocol established for each centre. The possibility of a meningococcal infection not covered by vaccination should always be considered. Such that, in the case a patient being treated with eculizumab shows signs of headache, fever, nausea, stiff neck, back pain, rashes, confusion, muscle pain, sensitivity to light or other symptoms of illness, they should contact their haematologist immediately or the emergency department of their nearest hospital or health centre and show their security card. Criteria for continuity The benefits of treatment with eculizumab should be reviewed every 6 months according to the patient’s clinical and laboratory evolution. Additionally, patients who have not complied with their therapy instructions of that of preventive measures are excluded from treatment. However, before stopping treatment a
full assessment of the case and an evaluation of the pros and cons of the decision should be performed. Allogeneic haematopoietic Allogeneic haematopoietic is recommended for patients with PNH and associated severe bone marrow failure that meet the criteria for allogeneic haematopoietic transplantation of the Pethema-GETH group for medullary aplasia. National and international registration of paroxysmal nocturnal haemoglobinuria PNH registration aims to observe the natural evolution of patients with PNH, and the safety and efficacy of long term treatment with eculizumab. Given the rarity of this disease, centralising the data of patients with PNH in a national and international registration is advisable, so that its management and treatment can be furthered. The profile of patients to include in the register are those with a GPI-deficient clone, those with diagnosis of PNH in any of its variants and that are not in treatment, and patients diagnosed with PNH who are receiving eculizumab. Data collection is done in electronic notebooks through the website (https:www.webcrf.net/pnhregistry). Questionnaires must be completed at the time of inclusion in the registry and every 6 months. In Spain, the company responsible for its implementation is ICON ([email protected]). They can be contacted for information on freephone (0) 80042662000. Conflict of interest The authors declare no conflict of interest. Acknowledgements ˜ for The authors would like to thank Alexion Pharma Espana its support in the development of the consensus statement. Editorial services were provided by Ogilvy Healthworld Barcelona and ˜ funded by Alexion Pharma Espana. References 1. Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993;73:703–11. 2. Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural history of paroxysmal nocturnal hemoglobinuria. N Engl J Med. 1995;333:1253–8. 3. Parker C, Omine M, Richards S, Nishimura J, Bessler M, Ware R, et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood. 2005;106:3699–709. 4. Brodsky Brodsky RA. How I treat paroxysmal nocturnal hemoglobinuria. Blood. 2009;113:6522–7.
A. Villegas et al. / Med Clin (Barc). 2016;146(6):278.e1–278.e7 5. Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. JAMA. 2005;293:1653–62. 6. Schrezenmeier H, Muus P, Socie G, Szer J, Urbano-Ispizua A, Maciejewski JP, et al. Baseline characteristics and disease burden in patients in the International Paroxysmal Nocturnal Hemoglobinuria Registry. Haematologica. 2014;99:922–9. 7. Hall C, Richards S, Hillmen P. Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria (PNH). Blood. 2003;102:3587–91. 8. Hillmen P, Muus P, Duhrsen U, Risitano AM, Schubert J, Luzzatto L, et al. Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria. Blood. 2007;110:4123–8. 9. Hill A, Kelly RJ, Hillmen P. Thrombosis in paroxysmal nocturnal hemoglobinuria. Blood. 2013;121:4985–96, quiz 5105. 10. National Kidney Foundation. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis. 2002;39:S1–266. 11. Alcazar R, Egocheaga MI, Orte L, Lobos JM, Gonzalez Parra E, Alvarez Guisasola F, et al. SEN-SEMFYC consensus document on chronic kidney disease. Nefrologia. 2008;28:273–82. 12. Hillmen P, Elebute M, Kelly R, Urbano-Ispizua A, Hill A, Rother RP, et al. Long-term effect of the complement inhibitor eculizumab on kidney function in patients with paroxysmal nocturnal hemoglobinuria. Am J Hematol. 2010;85:553–9. 13. Kelly R, Arnold L, Richards S, Hill A, Bomken C, Hanley J, et al. The management of pregnancy in paroxysmal nocturnal haemoglobinuria on long term eculizumab. Br J Haematol. 2010;149:446–50. 14. De Guibert S, Peffault de Latour R, Varoqueaux N, Labussiere H, Rio B, Jaulmes D, et al. Paroxysmal nocturnal hemoglobinuria and pregnancy before the eculizumab era: the French experience. Haematologica. 2011;96:1276–83. 15. Hillmen P, Hall C, Marsh JC, Elebute M, Bombara MP, Petro BE, et al. Effect of eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2004;350:552–9. 16. Hillmen P, Young NS, Schubert J, Brodsky RA, Socie G, Muus P, et al. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2006;355:1233–43.
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17. Brodsky RA, Young NS, Antonioli E, Risitano AM, Schrezenmeier H, Schubert J, et al. Multicenter phase 3 study of the complement inhibitor eculizumab for the treatment of patients with paroxysmal nocturnal hemoglobinuria. Blood. 2008;111:1840–7. 18. Marasca R, Coluccio V, Santachiara R, Leonardi G, Torelli G, Notaro R, et al. Pregnancy in PNH: another eculizumab baby. Br J Haematol. 2010;150: 707–8. 19. Danilov AV, Smith H, Craigo S, Feeney DM, Relias V, Miller KB. Paroxysmal nocturnal hemoglobinuria (PNH) and pregnancy in the era of eculizumab. Leuk Res. 2009;33:e4–5. 20. Kelly RJ, Hochsmann B, Szer J, Kulasekararaj A, de Guibert S, Roth A, et al. Eculizumab in pregnant patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2015;373:1032–9. 21. Hillmen P, Muus P, Roth A, Elebute MO, Risitano AM, Schrezenmeier H, et al. Long-term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol. 2013;162: 62–73. 22. Kelly RJ, Hill A, Arnold LM, Brooksbank GL, Richards SJ, Cullen M, et al. Long-term treatment with eculizumab in paroxysmal nocturnal hemoglobinuria: sustained efficacy and improved survival. Blood. 2011;117:6786–92. 23. Urbano-Ispizua A, Gaya A, Colado E, Lopez M, Arrizabalaga B, Vicente V, et al. [Diagnosis and treatment of nocturnal paroxysmal hemoglobinuria]. Med Clin (Barc). 2011;136:121–7. 24. Villegas A, Arrizabalaga B, Bonanad S, Colado E, Gaya A, González A, et al. Guía ˜ para diagnóstico y tratamiento de Hemoglobinclínica HPN. Consenso espanol uria Paroxística Nocturna. 2014 [accessed May 2015]. Available from: http://www.sehh.es/en/documents/guides-and-documents/2605-guias-deconsenso-sobre-diagnostico-y-tratamiento-de-la-hemoglobinuria-paroxisticanocturna-hpn-actualizacion-2014.html 25. Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010;78:211–30. 26. Sutherland DR, Illingworth A, Keeney M, Richards SJ. High-sensitivity detection of PNH red blood cells, red cell precursors, and white blood cells. Curr Protoc Cytom. 2015;72:6 37 1–629.
www.elsevier.es/medicinaclinica
Consensus statement
Spanish consensus statement for diagnosis and treatment of paroxysmal nocturnal haemoglobinuria夽 Ana Villegas a,∗ , Beatriz Arrizabalaga b , Santiago Bonanad c , Enrique Colado d , Anna Gaya e , ˜ f , Emilio Ojeda g , Alberto Orfao h , José-María Ribera i , Ataúlfo González a , Isidro Jarque c , Ramiro Núnez j e ˜ Vicente Vicente , Álvaro Urbano-Ispizua , Grupo de Trabajo de HPN de la Sociedad Espanola de Hematología y Hemoterapia a
Servicio de Hematología, Hospital Clínico San Carlos de Madrid, Madrid, Spain Servicio de Hematología, Hospital Cruces, Bilbao, Spain c Servicio de Hematología, Hospital La Fe, Valencia, Spain d Servicio de Hematología y Área de Gestión Clínica de Laboratorio de Medicina, Hospital Universitario Central de Asturias, Oviedo, Spain e Servicio de Hematología, Hospital Clínic, Barcelona, Spain f Servicio de Hematología, Hospital Virgen del Rocío, Sevilla, Spain g Servicio de Hematología, Hospital Puerta de Hierro, Madrid, Spain h Servicio de Hematología, Hospital Universitario de Salamanca, Salamanca, Spain i Servicio de Hematología Clínica, Instituto Catalán de Oncología-Hospital Germans Trias i Pujol, Badalona, Spain j Servicio de Hematología, Hospital Morales Meseguer, Murcia, Spain b
a r t i c l e
i n f o
Article history: Received 17 September 2015 Accepted 1 December 2015 Available online 16 June 2016 Keywords: Paroxysmal nocturnal haemoglobinuria Haematopoietic progenitor cells Mutation
a b s t r a c t Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of the haematopoietic progenitor cells due to a somatic mutation in the X-linked phosphatidylinositol glycan class A gene. The disease is characterised by intravascular haemolytic anaemia, propensity to thromboembolic events and bone marrow failure. Other direct complications of haemolysis include dysphagia, erectile dysfunction, abdominal pain, asthenia and chronic renal failure (65% of patients). The disease appears more often in the third decade of life and there is no sex or age preference. Detection of markers associated with glucosyl phosphatidyl inositol deficit by flow cytometry is currently used in the diagnosis of PNH. For years, transfusions have been the mainstay of therapy for PNH. A breakthrough in treatment has been the approval of the humanised monoclonal antibody eculizumab, which works by blocking the C5 complement protein, preventing its activation and therefore haemolysis. Several studies have confirmed that treatment with eculizumab avoids or decreases the need for transfusions, decreases the probability of thrombosis, improves the associated symptomatology and the quality of life in patients with PNH, showing an increase in survival. Because of rapid advances in the knowledge of the disease and its treatment, it may become necessary to adapt and standardise clinical guidelines for the management of patients with PNH. ˜ S.L.U. All rights reserved. © 2016 Elsevier Espana,
˜ para el diagnóstico y tratamiento de la hemoglobinuria Consenso espanol paroxística nocturna r e s u m e n Palabras clave: Hemoglobinuria paroxística nocturna Células progenitoras hematopoyéticas Mutación
La hemoglobinuria paroxística nocturna (HPN) es una enfermedad clonal de las células progenitoras hematopoyéticas originada por la mutación adquirida del gen fosfatidil-inositol-glicano del grupo A, situado en el brazo corto del cromosoma X. Se caracteriza por anemia hemolítica intravascular, tendencia a la trombosis y un componente variable de insuficiencia medular. Otras complicaciones derivadas de la
夽 Please cite this article as: Villegas A, Arrizabalaga B, Bonanad S, Colado E, Gaya A, González A, et al. Consenso espanol ˜ para el diagnóstico y tratamiento de la hemoglobinuria paroxística nocturna. Med Clin (Barc). 2016;146:278.e1–278.e7. ∗ Corresponding author. E-mail address: [email protected] (A. Villegas). ˜ S.L.U. All rights reserved. 2387-0206/© 2016 Elsevier Espana,
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hemólisis son disfagia, disfunción eréctil, dolores abdominales, astenia e insuficiencia renal crónica (un 65% de los pacientes). La enfermedad afecta por igual a ambos sexos y puede aparecer a cualquier edad, con una mayor incidencia en la tercera década de la vida. Actualmente, el diagnóstico se basa en la detección de poblaciones celulares con marcadores asociados al déficit de glucosil-fosfatidil-inositol mediante ˜ citometría de flujo. Durante anos, el pilar terapéutico de la HPN hemolítica era el soporte transfusional. Un gran avance en el tratamiento ha sido la aprobación del anticuerpo monoclonal humanizado eculizumab, que bloquea la proteína C5 del complemento impidiendo su activación, y por tanto, la hemólisis. Diversos estudios han confirmado que el tratamiento con eculizumab evita o disminuye el requerimiento transfusional, reduce la probabilidad de trombosis, mejora la sintomatología asociada y la calidad de vida de los pacientes con HPN, mostrando un aumento de la supervivencia. Este rápido avance en el conocimiento de la enfermedad y su tratamiento hace necesario adaptar y homogeneizar las directrices de actuación clínica en el manejo de pacientes con HPN. ˜ S.L.U. Todos los derechos reservados. © 2016 Elsevier Espana,
Introduction Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal disorder of haematopoietic progenitor cells that originates from the acquired mutation of the phosphatidylinositol glycan group A gene (PIGA-A), located in the short arm of chromosome X.1 As a result of this mutation, the glucosyl phosphatidyl inositol (GPI) anchor group does not synthesise, a necessary action for many membrane proteins to bind to the cell surface. Inside these proteins lies the membrane inhibitor of reactive lysis (MIRL) and the complement decay-accelerating factor DAF (CD55), both physiological inhibitors of complement activation. As a result of this deficiency, erythrocytes are more sensitive to the lytic action of complement, causing haemolysis and platelet activation, among other effects.2,3 The disease can appear at any age, with a higher incidence rate in the third decade of life; gender distribution is similar.2 Clinically, PNH is characterised by intravascular haemolysis, susceptibility to thrombosis and a variable component of bone marrow failure.4 Today PNH is considered a systemic disease – one which may affect several organs, especially the liver, kidney, central nervous system, lung and/or heart – because of the deficiency of nitric oxide (NO) and thrombotic events.4,5 Haemoglobinuria, a sign that names the disease, cannot be objectified; only 26% of cases initially show symptoms and 62% at some point throughout the course of the disease.3,6 Complications are a direct result of intravascular haemolysis and sequestration of NO by free haemoglobin. NO depletion produces peripheral vasoconstriction, occurring with dysphagia, erectile dysfunction, abdominal and chest pain and often with profound fatigue that prevents patients from carrying out a normal life. The most common complications are thromboses, the leading cause of death from PNH (40–67% depending on the type). Thromboses are mainly venous, and can be located in unusual places such as hepatic portal vein, splenic vein and mesenteric vein, inferior vena cava, central nervous system veins, skin veins or central retinal vein, although it should be noted that approximately 15% occur in blood circulation, especially in brain and coronary arteries.7–9 Pulmonary hypertension and renal failure may also be particularly serious complications of the disease.10,11 Occasionally, patients develop acute renal failure which is usually reversible but may require dialysis. 65% of patients have chronic renal failure, and severe chronic renal failure (stage 3 or higher) in 21% of cases.12 Pregnancy, raising the risk of thrombosis, complicates the natural course of the disease, causing an increase in maternal-foetal morbidity and mortality. It has been estimated that maternal mortality during pregnancy and the immediate post-partum period is 12–21%.13,14 There is also risk of miscarriage or premature birth.13,14
The association with bone marrow failure, especially with aplastic anaemia, is widely recognised in all patients, preceding the diagnosis of PNH in many cases. Until 2007, haemolytic PNH patients were treated mainly with blood transfusions. A great therapeutic advance has been the monoclonal antibody, eculizumab, approved both by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). It was first authorised in Spain for use in adult patients with PNH on June 20, 2007, and in April 2013 the indication was extended to paediatric patients. Significantly, on March 30, 2015 the EMA changed the guideline criteria, to include patients with a high activity of the disease, defined as elevated haemolysis, together with one or more of the clinical symptoms associated with it (asthenia, haemoglobinuria, abdominal pain, dyspnoea, anaemia, thrombosis, dysphagia or erectile dysfunction). Eculizumab is a humanised monoclonal antibody that acts by blocking complement protein C5, preventing terminal complement activation and thus haemolysis.15,16 The use of eculizumab stabilises the haemoglobin and reduces transfusion requirements and symptoms associated with dysfunction of the smooth muscle, reducing fatigue and significantly improving quality of life.17 It has also been shown to reduce the relative risk of thromboembolism by 85% and the reduction in the frequency of cases treated with anticoagulants for previous occlusive vascular episodes is 94%.8 It also improves pulmonary hypertension and renal failure, especially in the early stages. Likewise, it has been used in women with PNH who are pregnant, with encouraging responses and without risk to the mother or the foetus.13,18,19 Recently, the largest series of monitored cases of pregnant patients with PNH has been published, demonstrating the safety and efficacy of eculizumab in this group of patients.20 At present, after more than 12 years of treatment, sufficient experience has been collected to confirm that treatment with eculizumab has changed the natural history of PNH. In a disease that had a short life expectancy compared to the general population, with a median survival of 10–15 years from diagnosis, there are data showing that treatment with eculizumab produces a significant increase in life expectancy, and its positive effect in improving the quality of life has been proven; it is medication that has a safe profile and which is generally well tolerated.21 Recently, a group of experts in PNH at a hospital in Leeds in England has published a retrospective study where eculizumab treatment improves the survival of patients with PNH relative to a control group of a healthy population of the same age and sex.22 Due to the rapid pace of advances in knowledge of the disease and its treatment, it has become necessary to standardise and adapt clinical practice guidelines in order to facilitate the monitoring of patients with classical or haemolytic PNH. In order to address this need, the Spanish PNH Working Group was created, sponsored by the Spanish Society of Haematology and
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Indications for the study of PHN populations • Haemolytic anaemia with negative Coombs' test.
• Hydroplastic-MDS RCMD: refractory cytopenia anaemia with multilineage dysplasia RAEB: refractory anaemia with excess blast RCMD-S: refractory cytopenia anaemia with
• Haemoglobin or haemosiderosis • Idiopathic thrombosis (venous or arterial) with 1 or more of the following criteria:
multilineage dysplasia with ringed sideroblasts,S1 syndrome.
c) Haemolysis a) Young b) Unusual locations d) Cytopenia
• Idiopathic cytopenia
• Dysphagia or abdominal pain with haemolysis • Medullary aplasia with 2 of the 3 following criteria:
Hb <10 g/dl Neutrophils <1.5 × 109L Platelet <150.00 L
a) Hb <10 g/dl b) Neutrophils <1.5 × 109/L c) Platelet <100 × 109/L
Consider or rule out PHN through a high sensitivity flow cytometry and clinical evaluation Repeat every 6 months
PHN clone +
PHN clone – In the case of aplasia, repeat cytometry every 6 months
Clone >0.001%
Monitoring Clinical history
Mandatory complimentary explorations
Intravascular haemolysis, 1. Laboratory analysis anaemia, dysfunction of smooth muscle, thrombosis. 2. Bone marrow aspirate and biopsy if necessary
Complimentary explorations recommended 1. HLA typing in a young patient. 2. Abdominal MRI
3. Thrombophilia study 4. Determining proBNP or NT-proBNP. 5. Imaging testsb
Indication of eculizumab in patients with any of the following criteria: • Haemolysis LDH> 1.5 × LSN + symptoms. • Thrombosis attributable to PNH. • CRF attributable to PNH or repeated episodes of RF. • Regular transfusions. • RF or PH with NYHA class III or IV. • Smooth muscle affectation: intense abdominal and lumbar pain or dysphagia. • Pregnancy.C Fig. 1. Algorithm diagnosis, monitoring and treatment of PNH. a Full, reticulocyte blood count, lactate dehydrogenase (LDH), indirect bilirubin, haptoglobin, plasma haemoglobin, haemoglobinuria, haemosidenuria, vitamin B12, serum erythropoietin, iron, folic acid, direct Coombs, creatinine, estimated glomerular filtration rate (eGFR), proteinuria. b Abdominal and cardiac ecodoppler, cranial MRI or angiography through helical computed tomography (annual or clinical changes). c Assess each case individually. BNP: B-type natriuretic peptide; Hb: haemoglobin; HLA: Human Leucocyte Antigen; PNH: paroxysmal nocturnal haemoglobinuria; PH: pulmonary hypertension; PI: pulmonary insufficiency; RF: renal failure; CRF: chronic renal failure; NYHA: New York Heart Association; NMR: nuclear magnetic resonance; MDS: myelodysplastic syndrome.
Haemotherapy, which published in 2011 in Medicina Clínica a first consensus document for management of PNH.23 New data released later that same year led the group to meet again in November 2011; the results of this meeting included an update of the consensus Guide in May 2012. Given the availability of new evidence a second meeting was held in December 2013 that led to another update of the guidelines in June, 2014.24 In January 2015 the Working Group met again in order to continue updating the document, specifically in regard to the following areas: the indication for the
study of PNH populations, detection of the PNH clone by flow cytometry, the monitoring of patients with PNH and treatment of the disease. Prior to the meeting, articles that had been published with new information related to these areas were identified through searches on Medline and Embase. The selection of articles to review was performed according to expert judgement. The classification of selected publications based on levels of evidence was conducted by the Centre of Evidence-Based Medicine, considering at level 1 randomised clinical trials; at level 2, cohort studies; at
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level 3, and case-control studies, and level 4, series of cases. Due to the idiosyncrasies of PNH, classified as an ultra-rare disease, the degree of recommendation is categorised based on experts’ clinical judgement and experience. Additionally, the update of this document has also considered information concerning the management of the disease in clinical practice recorded in the National and International PNH Registration database (PNH registry). During the meeting the selected evidence was presented and the recommendations contained therein were subsequently debated. In order to establish the degree of consensus for each of the recommendations, attendees expressed their level of agreement by voting, considering accepted recommendations as those where at least 66% of the participants agreed. For the December 2013 meeting, three haematologists who are experts in PNH were invited to attend as external evaluators for the review, discussion and validation of the recommendations proposed in the successive updates of the consensus document. This article exposes the recommendations contained in the group’s last meeting (May 2015) in regard to the four areas defined above (Fig. 1).
Table 1 Identification of deficient cells in glucosyl phosphatidyl inositol (GPI) by flow cytometry in diagnosis of PNH. Study variable
Recommendation
Type of sample Populations
Peripheral blood Step one Neutrophilic granulocytes and monocytes Step two Red blood cells Step one (granulocytes neutrophils and monocytes): FLAER and CD157 or FLAER, CD24 (or CD16) and CD14 Alternatively (if FLAER is not available): CD157PE, CD24 (or CD16) and CD14 Step two (red blood cells): CD59
GPI Markers
Additional information Additional cell identification markers
Combinations of markers to study leucocytea
Indications for the study of paroxysmal nocturnal haemoglobinuria populations The diagnosis of the PNH should be performed by flow cytometry. The study of PNH populations is indicated in patients with one or more of the following symptoms: • Haemolytic anaemia with negative Coombs’ test. • Haemoglobinuria. • Unexplained, venous or arterial thrombosis in patients who meet any of the following criteria: young patients, presence of thrombosis in unusual locations (intra-abdominal veins, Budd–Chiari syndrome, brain, skin, etc.), evidence of haemolysis and/or cytopenia. • Intermittent dysphagia or abdominal pain of unknown aetiology with evidence of haemolysis. • Medullary aplasia (at diagnosis and annually during monitoring). • Hypoplastic myelodysplastic syndrome. • Idiopathic and maintained cytopenias of uncertain significance. PNH clone detection by flow cytometry Currently, analysis is done by flow cytometry, in addition to screening diagnosis, to monitor and treat the disease (see Table 1). Sample type and cell populations of interest The study of PNH populations is done routinely in peripheral blood samples. A deficiency of GPI expression is initially identified in neutrophil granulocytes and monocytes. In cases where the presence of cells with GPI anchored proteins deficiency (PNH clone) have been demonstrated, a study of red cells is carried out in a second step. The analysis of the leucocyte for the detection of PNH populations is the best method available to properly assess the size of the deficient clone in GPI. Lymphocytes, because of their long half-life and variable expression of different GPI proteins, do not constitute an adequate population; whereas monocytes and neutrophils are suitable populations given their number, the correlation between the size of clone between themselves and that they can be easily assessed in parallel. The study in leucocytes is the technique of choice for detecting PNH clones, both for low
Combination of markers to study red blood cells Tests
Indications a
CD45 and CD10 or CD15 for neutrophil granulocytes CD45 and CD64 for monocytes CD235a for red blood cells 5-fluorescence (in a tube): FLAER CD157-PE CD45-PerCP CD64-APC CD10-APCH7 or FLAER CD157-PE CD45-PECy7 CD64-ECD CD10-PECy5 4-fluorescence (2 in a tubes): FLAER CD157 (or CD24)-PE CD45-PerCP CD10-APC and FLAER CD157 (or CD14)-PE CD45-PerCP CD10-APC or FLAER CD157 (or CD24)-PE CD45-PECy7 CD10-PECy5 FLAER CD157 (or CD14)-PE CD45-PECy7 CD64-PECy5 CD235a-FITC, CD59-PE (clone of reference: MEM43) Internal (positive and negative populations or with different fluorescence intensity for each marker) Screening diagnostic; monitoring of the disease and its treatment
Different combinations adapted to different flow cytometers.
and high sensitivity studies, and for monitoring patients with PNH clone.25,26
Markers associated with glycosyl phosphatidyl inositol There are several ways to create the neutrophilic granulocytes and monocytes selection strategy.25 FLAER is particularly useful for researching the deficit of expression of proteins associated to GPI in these populations, it is a fluorescent derivative of the bacterial toxin aerolysin that binds to GPI in different leucocyte populations, in combination with CD157 markers. Another option is to perform the analysis in these sub-populations with FLAER and CD24 (or CD16) and CD14 markers. If FLAER is not available, an alternative is to use CD157PE markers in combination with CD24 (or CD16) and CD14. The combination of CD45 and CD10 markers facilitates identification of neutrophilic granulocytes, whereas for monocytes CD45 and CD64 markers are used. In the case of detecting deficiencies in expression of proteins associated with GPI in neutrophilic granulocytes or monocytes, carrying out the second step is necessary which consists of evaluating the CD59 in red blood cells. CD59 expression is more informative than CD55 expression and other antigens such as CD58, as they are found in greater numbers on the surface of red blood cells. Although other options can work, using PE-conjugated antibodies for GPI molecules detection is recommended as there is more experience with that process. Simultaneous markers of CD55 and CD59 are not recommended, since they require careful titration of reagents to avoid agglutination between the red blood cells. In the case of performing highly sensitive techniques, using antigens for
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positive selection of red blood cells is essential, where there is only information of CD235a utility. Combinations of markers For the above mentioned first and second steps, leucocytes and erythrocytes detection respectively, different combinations of antibodies can be used according to the laboratory’s capabilities (see Table 1). The correct identification of sub-populations and GPI deficient clones can be performed using negative and positive controls or positive controls with different fluorescence intensity for each marker (see Table 1). Evaluation of patients once the diagnosis of paroxysmal nocturnal haemoglobinuria has been confirmed Clinical history PNH is a systemic disease with multiple clinical manifestations. For this reason, the patient’s medical history should be detailed so the presence of the most characteristic symptoms and signs of intravascular haemolysis can be detected, (dark urine, jaundice), anaemia, dysfunction of smooth muscles (severe asthenia, dysphagia, abdominal pain, chest pain and erectile dysfunction) and previous thrombosis (abdominal pain, dyspnoea, neurological deficit and chronic headache among others). Mandatory explorations • Laboratory tests include a complete blood count with haemoglobin, neutrophils, platelets, reticulocytes, biochemical study of lactate dehydrogenase (LDH) direct and indirect bilirubin, haptoglobin, plasma haemoglobin, haemoglobinuria, haemosiderinuria, ferric profile (sideremia, transferrin, saturation index transferrin and ferritin) levels of vitamin B12, folic acid, serum erythropoietin and direct Coombs test. An assessment of renal function through analysing plasma levels of creatinine should also be conducted, creatinine clearance, proteinuria and urinary sediment. • Bone marrow aspirate to carry out a morphological and iron staining study. To perform a differential diagnosis, including a cytogenetic study is also advisable. • Bone marrow biopsy if the analytical data suggests a bone marrow aplasia. • Thrombophilia studies. • Abdominal ultrasound with Doppler. • Doppler echocardiography: if there is evidence of pulmonary hypertension a pulmonary CT angiography must be requested. • Determining proBNP or NT-proBNP. • Magnetic resonance imaging (MRI) or cranial computed tomography (according to radiological criteria) in case of headache or other neurological symptoms. Other recommended explorations Depending on each case, performing other examinations that complement those listed above could be considered. In young patients, a Human Leucocyte Antigen (human leucocyte antigens) typing is recommended because of the possibility of an eventual stem cell transplantation. The radiological study by abdominal MRI is useful in assessing the degree of renal and hepatic iron deposit. Monitoring PNH patients require comprehensive monitoring with quarterly analytical reviews and renal function assessments (creatinine,
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creatinine clearance, sediment and proteinuria) every 6 months. Conducting an assessment of the clonal size by flow cytometry at 6 months of diagnosis is advisable to assess the patient’s stability, so that in such a case assessing the quantification of the clone annually can be valued. Annual reassessments, or if changes in clinical or biological behaviour of the disease are observed, are recommended according to the characteristics of each patient by performing thoracic and abdominal MRI, abdominal ultrasound with Doppler and Doppler echocardiography. Treatment of paroxysmal nocturnal haemoglobinuria The main objective of PNH treatment should be to reduce haemolysis and minimise the risk of complications given the systemic impact of the disease. Additionally, treatment of PNH may require supportive therapy including transfusions of concentrated red blood cells, folic acid and iron supplements, antithrombotic anticoagulant prophylaxis and treatment of thrombotic complications. Corticosteroids have been widely used for years in the treatment of PNH, however there is no clear evidence of their benefits. One great therapeutic advance has been the approval of the monoclonal antibody eculizumab, which controls haemolysis and the pathophysiological consequences of the disease. The only potentially curative treatment of PNH is allogeneic haematopoietic stem cell. However, despite the best results achieved with this procedure in recent years, it is still associated with high morbidity and mortality and is reserved for highly selected patients, especially those with very severe associated aplastic anaemia. Eculizumab Indications Data supporting the use of eculizumab in PNH patients for any of the following: indications is currently available: a) Patients with chronic intravascular haemolytic anaemia that shows LDH at 1.5 times the upper limit of normal and clinical symptoms due to haemolytic anaemia, which may manifest, among other symptoms, as a significant impact on quality of life. b) Patients with thrombosis attributable to PNH. c) Patients with chronic renal failure attributable to PNH or repeated episodes of acute renal failure. d) Patients with regular transfusional requirement due to haemolysis. e) Patients with lung failure who have dyspnoea and/or chest pain resulting in a limitation of normal activity (New York Heart Association class III or IV) and/or an established diagnosis of pulmonary hypertension when other causes of it have been excluded. f) Patients with involvement of smooth muscle who show recurrent episodes of severe pain (abdominal, lumbar or oesophageal spasm with a history of dysphagia) requiring hospitalisation or taking opioid analgesics regularly. Before these patients start treatment with eculizumab other causes must be ruled out. g) Pregnancy in a patient with PNH involves a high risk of thrombosis for mother and foetus, treatment with eculizumab may prevent these complications. However, it is recommended that its use should be assessed on an individual basis. The EMA recently approved an amendment of the indication criteria for treatment with eculizumab to include patients who have high disease activity, regardless of the history of transfusions. High disease activity is defined as elevated haemolysis together with one or more of associated clinical symptoms: fatigue, haemoglobinuria,
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Table 2 Dosage of eculizumab. Population
Initial phase
Maintenance phase
Adult patients (≥18 years old)
600 mg per week × 4
900 mg in the 5th week; then 900 mg every 14 ± 2 days
Paediatric patients with body weight 30–<40 kg
600 mg per week × 2
900 mg in the 3rd week; then 900 mg every two weeks. 600 mg in the 3rd week; then 600 mg every two weeks. 300 mg in the 2rd week; then 300 mg every two weeks. 300 mg in the 2nd week; then 300 mg every three weeks.
20–<30 kg
600 mg per week × 2
10–<20 kg
600 mg per week × 1
5–<10 kg
300 mg per week × 1
abdominal pain, dyspnoea, anaemia, thrombosis, dysphagia and/or erectile dysfunction. PNH registration (M07-001) was used to assess the efficacy of eculizumab in PNH patients without a history of red blood cell transfusions. These patients had high disease activity defined as high haemolysis (LDH ≥ 1.5 × LSN) and the presence of one or more associated clinical symptoms; fatigue, haemoglobinuria, abdominal pain, shortness of breath (dyspnoea), anaemia (haemoglobin < 10 g/dl), severe adverse vascular events (including thrombosis), dysphagia or erectile dysfunction. Vaccination against meningococcus with a conjugate vaccine that includes serotypes A, C, Y and W135, in addition to vaccination against serotype B (Bexero) is mandatory at least 2 weeks before starting treatment with eculizumab. Dosage Eculizumab is administered intravenously, at a weekly dose of 600 mg for 4 weeks. During the fifth week a dose of 900 mg is administered and thereafter doses of 900 mg are continuously administered approximately every 14 days (between 12 and 16 days). In patients with a body weight of less than 40 kg, dosage varies depending on the weight (see Table 2). Monitoring Evaluation and monitoring of the treatment is conducted by regular laboratory analysis including complete blood count, reticulocyte count and biochemical study of LDH. Conducting a study every three months is recommended to assess renal function and ferric profile (sideremia, transferrin, IST and ferritin). In case of iron overload, an abdominal MRI should be considered to analyse liver iron deposition, and repeated every 12 months. In addition to these parameters a general, direct Coombs test and an anti-C3 test every 3–6 months should also be conducted. Given the increased susceptibility to meningococcal sepsis in patients receiving eculizumab, prophylactic antibiotic treatment is recommended with oral penicillin at 400 mg/12 h with the prophylactic antibiotic protocol established for each centre. The possibility of a meningococcal infection not covered by vaccination should always be considered. Such that, in the case a patient being treated with eculizumab shows signs of headache, fever, nausea, stiff neck, back pain, rashes, confusion, muscle pain, sensitivity to light or other symptoms of illness, they should contact their haematologist immediately or the emergency department of their nearest hospital or health centre and show their security card. Criteria for continuity The benefits of treatment with eculizumab should be reviewed every 6 months according to the patient’s clinical and laboratory evolution. Additionally, patients who have not complied with their therapy instructions of that of preventive measures are excluded from treatment. However, before stopping treatment a
full assessment of the case and an evaluation of the pros and cons of the decision should be performed. Allogeneic haematopoietic Allogeneic haematopoietic is recommended for patients with PNH and associated severe bone marrow failure that meet the criteria for allogeneic haematopoietic transplantation of the Pethema-GETH group for medullary aplasia. National and international registration of paroxysmal nocturnal haemoglobinuria PNH registration aims to observe the natural evolution of patients with PNH, and the safety and efficacy of long term treatment with eculizumab. Given the rarity of this disease, centralising the data of patients with PNH in a national and international registration is advisable, so that its management and treatment can be furthered. The profile of patients to include in the register are those with a GPI-deficient clone, those with diagnosis of PNH in any of its variants and that are not in treatment, and patients diagnosed with PNH who are receiving eculizumab. Data collection is done in electronic notebooks through the website (https:www.webcrf.net/pnhregistry). Questionnaires must be completed at the time of inclusion in the registry and every 6 months. In Spain, the company responsible for its implementation is ICON ([email protected]). They can be contacted for information on freephone (0) 80042662000. Conflict of interest The authors declare no conflict of interest. Acknowledgements ˜ for The authors would like to thank Alexion Pharma Espana its support in the development of the consensus statement. Editorial services were provided by Ogilvy Healthworld Barcelona and ˜ funded by Alexion Pharma Espana. References 1. Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993;73:703–11. 2. Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural history of paroxysmal nocturnal hemoglobinuria. N Engl J Med. 1995;333:1253–8. 3. Parker C, Omine M, Richards S, Nishimura J, Bessler M, Ware R, et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood. 2005;106:3699–709. 4. Brodsky Brodsky RA. How I treat paroxysmal nocturnal hemoglobinuria. Blood. 2009;113:6522–7.
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