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Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation
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Kaohsiung Journal of Medical Sciences (2017) xx, 1e6
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Original Article
Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation Hui-Ching Wang a,b, Ching-Yuan Kuo c, I-Ting Liu d, Tsai-Yun Chen d, Yu-Hsiang Chang e, Shyh-Jer Lin e, Shih-Feng Cho b, Yi-Chang Liu b, Ta-Chih Liu a,b, Sheng-Fung Lin a,b, Chao-Sung Chang f,g,* a
Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan b Division of Hematology and Oncology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan c Division of Hematology and Oncology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan d Section of Hematology and Oncology, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan e Division of Hematology and Oncology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan f School of Medicine, I-Shou University, Kaohsiung, Taiwan g Division of Hematology and Oncology, E-Da Hospital, Kaohsiung, Taiwan Received 1 December 2016; accepted 9 May 2017
KEYWORDS Paroxysmal nocturnal hemoglobinuria; Eculizumab; Hematopoietic stem cell transplantation
Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare acquired disorder. The aim of this study was to investigate the demographics, clinical manifestations, and outcomes of PNH patients in southern Taiwan. Data on PNH patients diagnosed over a 30-year period (1985e2015) were retrospectively collected from four tertiary medical centers in southern Taiwan. Blood samples were collected for hematologic panel testing and flow cytometry detection of PNH clones. Radiologic studies were performed to assess the frequency of complications. Twenty-four patients were enrolled in this study. The median duration of disease in the study participants was 10.8 years. The median granulocyte PNH clone size was 92.5% ( ra n ge , 1 . 3 % e99 . 8 %) , a n d t h e m ed i an la c t a t e d eh y d ro ge n as e ( L D H ) l ev el w a s 2920.2 1462.0 IU/L. The incidence of thromboembolism and impaired renal function was 16.7% and 29.2%, respectively. The primary treatment strategies included steroids (79.2%), androgens (42.0%), eculizumab (33.3%), immunosuppressants (16.7%), and anticoagulants (4.2%).
Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. School of Medicine, I-Shou University, Kaohsiung, Taiwan. E-mail address: [email protected] (C.-S. Chang). http://dx.doi.org/10.1016/j.kjms.2017.05.011 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
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H.-C. Wang et al. In eight patients treated with eculizumab, there was a marked reduction in the LDH levels of 14.89-folde1.63-fold that of the upper limit of normal; seven patients exhibited decreased transfusion requirements. Twenty-one patients were alive with regular follow-up at the time of publication. Our study demonstrates that PNH patients in southern Taiwan may exhibit different clinical characteristics and outcomes relative to patients in other countries. There was a trend toward a greater PNH granulocyte clone size, which may lead to more hemolysis. In our study, the percentage of patients with impaired renal function, but not the percentage of patients with thrombotic events, was higher than values reported worldwide and in the observational cross-sectional International PNH Registry. More large-scale studies with comprehensive data on the clinical response to different treatments are needed. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Introduction
Diagnostics of PNH
Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare clonal abnormality caused by a mutation of the PIG-A gene on the X chromosome [1]. The worldwide prevalence of PNH is estimated to be in the range of 1e5 cases per million individuals [2]. The functional somatic PIG-A mutation causes impaired biosynthesis of glycosylphosphatidylinositol (GPI), leading to the deficiency or diminished expression of the GPI-anchored complement regulatory proteins CD55 and CD59 on the cell membrane [3]. With respect to the clinical manifestations of PNH, the affected PNH cells exhibit increased sensitivity to complement lysis, leading to pancytopenia, hemolysis, and thrombosis. The conventional treatment options for PNH are limited to the use of supportive measures, such as blood transfusions and anticoagulation therapy. The novel complement inhibitor eculizumab, a humanized monoclonal antibody against complement C5, significantly improves the outcomes of patients with this disease [4,5]. As diagnostic tools improve and ongoing epidemiologic studies are conducted, some geographic and ethnic differences in the incidence of complications have been reported [6]. However, these studies lack the large case series reported in Taiwan. We conducted a multicenter study on the demographics, clinical manifestations, and outcomes of PNH over the last 30 years in southern Taiwan.
Before 2010, the diagnosis of PNH was established by clinical presentation, the direct and indirect Coombs test [7], and the Ham test [8] (or sugar water test [9]). After 2010, flow cytometric analysis using antibodies directed against GPI-AP were used for the diagnosis of PNH in Taiwan. For the detection of GPI-deficient granulocytes, a lyse-washstain technique was employed. Cells were analyzed in a CYTOMICS FC 500 (CXP Software) instrument (Beckman Coulter, Miami, Florida, USA). In red blood cells (RBC assay), an indirect immunofluorescence test with anti-CD59 FITC, anti-CD55 PE, and anti-CD235a CyChrome monoclonal antibodies was used in eighteen patients [10,11]. In fourcolor FLAER (Fluorescent Aerolysin) assay for white blood cells (WBCs), the samples from 21 patients were stained with anti-CD15 PC5, anti-CD45 PC7, anti-CD24 PE (Beckman Coulter, Miami, Florida, USA), and FLAER working solution (catalogue number FL2, Victoria, British Columbia, Canada; www.protoxbiotech.com) for 15 min at room temperature [11,12]. The minimum clone size detected was 0.1% for WBCs and 3% for RBCs. The results are expressed as the percentage of granulocytes negative for CD55/CD59 or FLAER/CD24 [13,14]. Previously diagnosed patients also underwent flow cytometric analysis retrospectively and met the minimal essential diagnostic criteria of PNH [5]. All patients received a bone marrow examination for concomitant myelodysplastic syndromes or aplastic anemia. The patient series was classified into three groups according to the clinical expression and PNH clone percentage: classic PNH, PNH in the setting of another specified bone marrow disorder (SBMD), and subclinical PNH, according to the Nakakuma classification as amended by Parker [6,15].
Participants and methods Data collection of the study population Between 1985 and 2015, there were 24 PNH cases, including 10 males and 14 females, from four medical centers in southern Taiwan. The collected clinical data included lactate dehydrogenase (LDH) levels, hemoglobin levels, platelet levels, transfusion requirements, thrombotic events (identified using major adverse vascular event categories), physician-reported renal dysfunction, and treatment modalities. Our study received Institutional Review Board approval (IRB number: KMUH-IRB-20140201, VGHKS 15-CT3-05, 105-6702C, B-BR-105-014-T).
Treatment of PNH The treatment of PNH included steroids, androgens, and immunosuppressive therapy (including azathioprine, antithymocyte globulin, and cyclosporine). Anticoagulation therapy was used in patients with thrombotic events. Eculizumab (Soliris; Alexion Pharmaceuticals, Inc., Cheshire, CT, USA) was approved in 2013 by the National Health Insurance of Taiwan for the treatment of PNH with >50%
Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
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Characteristics of PNH in Southern Taiwan
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granulocyte clone size and persistent hemolysis or thrombotic events. Eight patients were treated with eculizumab following approval. One patient underwent splenectomy, and one patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 2006.
Imaging and examination for complications All patients received abdominal sonography for chronic complications, including splenomegaly and cholelithiasis. Fourteen patients underwent cardiac sonography for pulmonary hypertension. Computed tomography (CT) radiology, Tc-99m RBC venography, or Doppler echo of the extremities was arranged if the patients developed symptoms of thrombosis. One patient underwent abdominal and cranial CT, and one patient received Tc-99m RBC venography and Doppler echo of the limbs.
Results The median age of the study population was 39.5 years (range, 12e70 years). The first patient was diagnosed in 1985. The mean follow-up time was 10.8 9.1 years. Twenty-four patients were divided into three clinical groups according to the Parker classification system: 14 patients (58.3%) belonged to the classic PNH group, and 10
Table 1
patients (41.7%) belonged to the SBMD group. None of the patients were classified as having subclinical PNH. The mean PNH granulocyte clone size was 72.3 37.9%, and the mean maximum lactate dehydrogenase (LDH) level was 2920.2 1462.0 IU/L (8.49-fold of the upper limit of normal). Nine cases (37.5%) had a medical history of aplastic anemia (AA), and one case (4.2%) was diagnosed with PNH and AA concomitantly. The clinical symptoms included fatigue and hemoglobinuria (18 cases, 75%), abdominal pain (12 cases, 50%), and dyspnea (15 cases, 62.5%). All patients received abdominal sonography; splenomegaly was observed in eight cases (33.3%). The hemolysis-related complications included renal insufficiency (7 cases, 29.2%), cholelithiasis (4 cases, 16.7%), and thrombotic events (4 cases, 16.7%; 2 episodes in 2 cases). The thromboembolic events occurred in four patients and included superficial and deep venous thrombosis in the lower limbs, splenic arterial infarction, arterial ischemic stroke, and myocardial infarction. None of the thrombotic events preceded the diagnosis of PNH. Fourteen patients received cardiac sonography; one case (7.1%) was diagnosed with pulmonary hypertension. Among the 14 female patients, one woman (7.1%) developed preeclampsia and preterm labor. We did not observe an association between LDH levels and PNH granulocyte clone size. Furthermore, there was no correlation between PNH granulocyte clone size and the incidence of thrombosis or other complications
Patient characteristics at the time of PNH diagnosis.
Characteristics
Total (n Z 24)
A (n Z 7)
B (n Z 8)
C (n Z 3)
D (n Z 6)
Age, years, median (range) Female, n (%) Age at diagnosis, years, median (range) Disease duration, years, median (range) Parameter, median (range) Hemoglobin, g/L (5th, 95th percentile) Platelets, 109/L (5th, 95th percentile) Reticulocytes, % (5th, 95th percentile) Granulocyte clone size, %, median (5th, 95th percentile) Lactate dehydrogenase, ULN, median (5th, 95th percentile) Symptoms related to PNH Dyspnea (%) Hemoglobinuria (%) Abdominal pain (%) History of thrombotic events (%) History of impaired renal function (%) Treatment Steroids, n (%) Transfusion dependence, n (%) Androgens, n (%) Eculizumab, n (%) Immunosuppressive therapy, n (%)a Splenectomy, n (%) Allo-HSCT, n (%) Anticoagulation therapy, n (%)
39.5 (12e70) 14 (58.3) 37 (12e65) 8.5 (<1e29)
55 (33e70) 6 (85.7) 33 (20e52) 16 (<1e29)
42.5 (27e62) 4 (50.0) 42.5 (27e62) 3.5 (1e14)
21 (12e65) 2 (66.7) 21 (12e65) 4 (1e5)
39.5 (17e59) 2 (33.3) 39.5 (17e59) 20 (4e27)
74 (26.5, 115) 97 (18, 256.5) 5.1 (0.10, 10.50) 92.5 (1.43, 99.77)
70 150 5.1 89.2
74.5 81 8.7 99.2
74 111 5.6 89.7
62 36 3.15 50.9
8.49 (1.65, 18.98)
8.54
9.77
17.8
6.84
62.5 75.0 50.0 16.7 29.2
57.1 85.7 71.4 28.6 42.9
50 75 25 12.5 12.5
33.3 33.3 0 0 33.3
100 83.3 83.3 16.7 33.3
19 (79.2) 18 (75) 10 (41.7) 8 (33.3) 4 (16.7) 1 (4.2) 1 (4.2) 1 (4.2)
6 4 4 2 2 1 1 0
5 6 1 4 1 0 0 0
2 2 1 2 0 0 0 0
6 6 4 0 1 0 0 1
(85.7) (57.1) (57.1) (28.6) (28.6) (14.3) (14.3) (0)
(62.5) (75) (12.5) (50) (12.5) (0) (0) (0)
(66.7) (66.6) (33.3) (66.6) (0) (0) (0) (0)
(100) (100) (66.6) (0) (16.7) (0) (0) (16.7)
a
Immunosuppressive therapy included azathioprine, anti-thymocyte globulin (ATG) and cyclosporine. Groups AeD represent four tertiary medical centers in southern Taiwan.
Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
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Comparison with other studies of paroxysmal nocturnal hemoglobinuria.
First author, country (y) (reference)
Number Females Age at Additional PNH granulocyte PNH erythrocyte LDH at diagnosis, TE (%) Hemoglobinuria Prior Transfusion of patients (%) diagnosis (y) disorder clone size (%) clone size (%) xULN (range) /abdominal steroid dependence (AA or MDS) (%) pain (%) use (%) (%)
Hillmen, UK (1995) [16] 80 Socie, France (1996) [17] 220 Nishimura (2004) [18] USA 176
Kelly, USA (2011) [19] Lee, Korea (2013) [20] Munos-Linares, Spain (2013) [21] Schrezenmeier, international (2014) [22] Wang, Taiwan (2016) Mean of all patients
42 (16e75) 33 (6e82)
40.0/NA 30.0/5.0a
NA NA
NA NA
NA NA
39.0 6.0
26/NA NA/13.0
NA 21
NA 75b
56.2
30 (4e80)
29.0/5.1
209
43.5
45 (10e86)
79 301 56
50.0 50.0 35.7
37 (12e79) 37 (8e88) 32 (13e84)
CD59 (-): 68.6% CD55 (-): 71.6% 37.8/4.8 CD59 (-): 42.8% CD55 (-): 49.6% 29.1/1.3 96.4 (41.8e100) 40.2/6.3 48.8 (0e100) 67 (AA and MDS) NA
CD59 (-):45.0% CD55 (-): 39.5% CD59 (-): 37.8% CD55 (-): 41.1% 34.0 (2.9e100) 28.1 (0e99.8) NA
12.2
19.0
50.0/-
82.4
61.9b
8.2
6.0
33.5/-
46.9
40.2b
6.7 (1.36e23.95) 27.0 4.1 (0.2e36.3) 17.9 NA 35.0
63.0/30.0 56.1/46.8 60.0/48.0
NA 77.4 NA
81.3 59.1 82.1
1610
53.2
32 (3e87)
43.5/5.8
1.96 (0.65e10.32) 15.5
62.0/54.0
e
e
24 2755c
58.3 51.1
39.5 (12e70) 41.7/0 36.4 36.4/4.0
92.5 (1.3e99.8) 38.8 (3.9e95.2) 8.49 (1.55e20.79) 16.7 71.2 36.3 6.9 20.2
75.0/50.0 53.2/40.3
79.2 61.4
75.0 67.8
68.1 (0.01e100) e
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Japan
58.7 54.5
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AA, aplastic anemia; MDS, myelodysplastic syndrome; ULN, upper limits of normal; TE, thromboembolism. a The data includes acute leukemia. b There is no available data for transfusion-dependent patients; the data represents the percentage of patients who received transfusion as an initial treatment. c Eight studies were included with a total of 2755 patients.
H.-C. Wang et al.
Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
Table 2
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Characteristics of PNH in Southern Taiwan
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Fig. 1. Demographic and clinical characteristics of the patients: (a) clinical laboratory data, (b) percentage of patients reporting symptoms, and (c) percentage receiving treatment prior to enrollment.
in our series. The detailed demographic and clinical characteristics of all patients are described in Table 1, and the subgroup analysis from four tertiary medical centers in southern Taiwan is presented separately. With respect to treatment, eighteen patients (75.0%) were transfusion-dependent. Nineteen patients (79.2%) received steroid therapy with prednisolone or dexamethasone, and 10 patients (41.7%) received androgen therapy. Eight patients (33.3%) were on eculizumab; one patient (4.2%) underwent splenectomy; four patients (16.7%) received immunosuppressive therapy, including azathioprine, anti-thymocyte globulin (ATG), and cyclosporine; and one patient (4.2%) underwent myeloablative conditioning allo-HSCT from an HLA-match unrelated donor. Treatment and prognostic outcomes are recorded in Table 1. Among the patients receiving steroid therapy, only three patients exhibited improved quality of life (QOL) and reduced LDH levels after treatment. Among patients receiving ATG and cyclosporine, none experienced a reduction in transfusion requirements or QOL improvements. One patient underwent splenectomy; hemolysis was not resolved though hemoglobin stabilization after the surgical intervention. All patients receiving eculizumab experienced decreased transfusion requirements and enhanced QOL. The mean LDH levels in these patients fell dramatically from 2859.3 1223.0 IU/L to 313.3 272.7 IU/L. No new thromboembolic events were recorded after eculizumab use, but impaired renal function in these patients was irreversible. Seven patients exhibited decreased transfusion requirements after eculizumab. One patient received allo-HSCT, and improvement of both anemia and hemolysis was noted without regular transfusion after allo-HSCT. Twenty-one patients were still alive at the time of publication, but two patients had died of severe sepsis, and one patient was lost to follow-up.
Discussion This study investigated the clinical characteristics, treatment strategies, and clinical outcomes of PNH in southern Taiwan. We detected possible differences among the clinical characteristics of our patients relative to those in other countries. Compared to previous studies [16e22] (Table 2), the male and female incidence, the percentage of patients
with AA, and the age at diagnosis were similar. With respect to the PNH clone size, which was available in only four studies [18e20], the PNH erythrocyte clone size (including type II and type III erythrocytes) was similar among different countries. However, the PNH granulocyte clone size was higher in Western countries and lower in Asian studies. Notably, our study reports an extremely high PNH granulocyte clone size, which differs from the results of other Asian studies. Compared with patients in the International PNH Registry, a worldwide and observational crosssectional analysis of patients enrolled through June 30, 2012 [22], our patients exhibited unique demographics and clinical characteristics (Fig. 1). Our patients exhibit larger PNH granulocyte clone sizes and more severe thrombocytopenia and anemia, which may explain 62.5% of the patients in our study suffered from dyspnea. Higher LDH levels indicated that our patients developed more profound hemolysis than did patients in other countries. Importantly, the incidence of kidney impairment was higher in our patients compared with patients in the International PNH Registry, but there was no difference in the incidence thrombotic events. Hence, most of our patients did not receive anticoagulants. Most of our patients received steroid therapy after diagnosis, and the change in the hemoglobin level and hemolysis response was not satisfactory. Interestingly, one case with pancytopenia was diagnosed with concurrent AA and PNH, and the bone marrow biopsy revealed less than 1% cellularity with 27.5% PNH granulocyte clones in FLAER and multiparameter flow cytometry. In our study, we found that the patient outcomes remained poor and that the frequency of transfusion remained high after conventional treatments, including steroids, androgens, and immunosuppressive therapy. Splenectomy exhibited limited benefit in our patients. However, after receiving eculizumab, eight patients in our study demonstrated stabilization of their hemoglobin levels and reduction in intravascular hemolysis. QOL improved with decreased requirements for transfusion. Transplantation is the only curative treatment for PNH and is effective in eradicating abnormal PNH clones [23]. One patient underwent matched unrelated donor allo-HSCT before the eculizumab era, and the patient’s intravascular hemolysis and anemia improved significantly after transplantation. The main limitation of our study is the
Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
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6 relatively small sample size, which may reflect the extremely low prevalence rate and under-diagnosis of PNH. Physicians often overlook atypical symptoms in patients, and the available detection methods, such as FLAER and multiparameter flow cytometry, are not widely used. More studies should be conducted with larger sample sizes and different ethnic groups to validate and extend our findings. In conclusion, our study provides comprehensive clinical information of PNH patients in southern Taiwan with a follow-up period of up to 30 years. Our findings demonstrate that our patients tend to exhibit higher PNH clone sizes and greater kidney injury compared with patients in the International PNH Registry. Anti-complement therapy has produced promising responses in several studies, but HSCT remains the only PNH treatment to produce a definitive cure.
H.-C. Wang et al.
[11]
[12]
[13]
[14]
[15]
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neutrophils from aplastic anaemia patients. Br J Haematol 1995;90:728e30. Fores R, Bautista G, Steegmann JL, Javier Penalver F, Cabrera R, Ferna ´ndez MN, et al. De novo smoldering paroxysmal nocturnal hemoglobinuria: a flow cytometric diagnosis. Haematologica 1997;82:695e7. Brodsky RA, Mukhina GL, Li S, Nelson KL, Chiurazzi PL, Buckley JT, et al. Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 2000;114:459e66. Sutherland DR, Kuek N, Davidson J, Barth D, Chang H, Yeo E, et al. Diagnosing PNH with FLAER and multiparameter flow cytometry. Cytom B Clin Cytom 2007;72:167e77. 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. Cytom B Clin Cytom 2010;78: 211e30. Nakakuma H, Kawaguchi T, Horikawa K, Hidaka M, Nagakura S, Iwamoto N, et al. Proposal for a classification of the clinical stages of paroxysmal nocturnal hemoglobinuria. Blood 1995; 86:2051e2. Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV. Natural history of paroxysmal nocturnal hemoglobinuria. N Engl J Med 1995;333:1253e8. Socie G, Mary JY, de Gramont A, Rio B, Leporrier M, Rose C, et al. Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. French Society of Haematology. Lancet 1996;348:573e7. Nishimura J, Kanakura Y, Ware RE, Shichishima T, Nakakuma H, Ninomiya H, et al. Clinical course and flow cytometric analysis of paroxysmal nocturnal hemoglobinuria in the United States and Japan. Medicine 2004;83:193e207. 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;117:6786e92. Lee JW, Jang JH, Kim JS, Yoon SS, Lee JH, Kim YK, et al. Clinical signs and symptoms associated with increased risk for thrombosis in patients with paroxysmal nocturnal hemoglobinuria from a Korean registry. Int J Hematol 2013;97:749e57. Munoz-Linares C, Ojeda E, Fores R, Pastrana M, Cabero M, Morillo D, et al. Paroxysmal nocturnal hemoglobinuria: a single Spanish center’s experience over the last 40 yr. Eur J Haematol 2014;93:309e19. 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;99:922e9. Bemba M, Guardiola P, Garderet L, Devergie A, Ribaud P, Esperou H, et al. Bone marrow transplantation for paroxysmal nocturnal haemoglobinuria. Br J Haematol 1999;105:366e8.
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Kaohsiung Journal of Medical Sciences (2017) xx, 1e6
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Original Article
Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation Hui-Ching Wang a,b, Ching-Yuan Kuo c, I-Ting Liu d, Tsai-Yun Chen d, Yu-Hsiang Chang e, Shyh-Jer Lin e, Shih-Feng Cho b, Yi-Chang Liu b, Ta-Chih Liu a,b, Sheng-Fung Lin a,b, Chao-Sung Chang f,g,* a
Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan b Division of Hematology and Oncology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan c Division of Hematology and Oncology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan d Section of Hematology and Oncology, Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan e Division of Hematology and Oncology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan f School of Medicine, I-Shou University, Kaohsiung, Taiwan g Division of Hematology and Oncology, E-Da Hospital, Kaohsiung, Taiwan Received 1 December 2016; accepted 9 May 2017
KEYWORDS Paroxysmal nocturnal hemoglobinuria; Eculizumab; Hematopoietic stem cell transplantation
Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare acquired disorder. The aim of this study was to investigate the demographics, clinical manifestations, and outcomes of PNH patients in southern Taiwan. Data on PNH patients diagnosed over a 30-year period (1985e2015) were retrospectively collected from four tertiary medical centers in southern Taiwan. Blood samples were collected for hematologic panel testing and flow cytometry detection of PNH clones. Radiologic studies were performed to assess the frequency of complications. Twenty-four patients were enrolled in this study. The median duration of disease in the study participants was 10.8 years. The median granulocyte PNH clone size was 92.5% ( ra n ge , 1 . 3 % e99 . 8 %) , a n d t h e m ed i an la c t a t e d eh y d ro ge n as e ( L D H ) l ev el w a s 2920.2 1462.0 IU/L. The incidence of thromboembolism and impaired renal function was 16.7% and 29.2%, respectively. The primary treatment strategies included steroids (79.2%), androgens (42.0%), eculizumab (33.3%), immunosuppressants (16.7%), and anticoagulants (4.2%).
Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. School of Medicine, I-Shou University, Kaohsiung, Taiwan. E-mail address: [email protected] (C.-S. Chang). http://dx.doi.org/10.1016/j.kjms.2017.05.011 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
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H.-C. Wang et al. In eight patients treated with eculizumab, there was a marked reduction in the LDH levels of 14.89-folde1.63-fold that of the upper limit of normal; seven patients exhibited decreased transfusion requirements. Twenty-one patients were alive with regular follow-up at the time of publication. Our study demonstrates that PNH patients in southern Taiwan may exhibit different clinical characteristics and outcomes relative to patients in other countries. There was a trend toward a greater PNH granulocyte clone size, which may lead to more hemolysis. In our study, the percentage of patients with impaired renal function, but not the percentage of patients with thrombotic events, was higher than values reported worldwide and in the observational cross-sectional International PNH Registry. More large-scale studies with comprehensive data on the clinical response to different treatments are needed. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Introduction
Diagnostics of PNH
Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare clonal abnormality caused by a mutation of the PIG-A gene on the X chromosome [1]. The worldwide prevalence of PNH is estimated to be in the range of 1e5 cases per million individuals [2]. The functional somatic PIG-A mutation causes impaired biosynthesis of glycosylphosphatidylinositol (GPI), leading to the deficiency or diminished expression of the GPI-anchored complement regulatory proteins CD55 and CD59 on the cell membrane [3]. With respect to the clinical manifestations of PNH, the affected PNH cells exhibit increased sensitivity to complement lysis, leading to pancytopenia, hemolysis, and thrombosis. The conventional treatment options for PNH are limited to the use of supportive measures, such as blood transfusions and anticoagulation therapy. The novel complement inhibitor eculizumab, a humanized monoclonal antibody against complement C5, significantly improves the outcomes of patients with this disease [4,5]. As diagnostic tools improve and ongoing epidemiologic studies are conducted, some geographic and ethnic differences in the incidence of complications have been reported [6]. However, these studies lack the large case series reported in Taiwan. We conducted a multicenter study on the demographics, clinical manifestations, and outcomes of PNH over the last 30 years in southern Taiwan.
Before 2010, the diagnosis of PNH was established by clinical presentation, the direct and indirect Coombs test [7], and the Ham test [8] (or sugar water test [9]). After 2010, flow cytometric analysis using antibodies directed against GPI-AP were used for the diagnosis of PNH in Taiwan. For the detection of GPI-deficient granulocytes, a lyse-washstain technique was employed. Cells were analyzed in a CYTOMICS FC 500 (CXP Software) instrument (Beckman Coulter, Miami, Florida, USA). In red blood cells (RBC assay), an indirect immunofluorescence test with anti-CD59 FITC, anti-CD55 PE, and anti-CD235a CyChrome monoclonal antibodies was used in eighteen patients [10,11]. In fourcolor FLAER (Fluorescent Aerolysin) assay for white blood cells (WBCs), the samples from 21 patients were stained with anti-CD15 PC5, anti-CD45 PC7, anti-CD24 PE (Beckman Coulter, Miami, Florida, USA), and FLAER working solution (catalogue number FL2, Victoria, British Columbia, Canada; www.protoxbiotech.com) for 15 min at room temperature [11,12]. The minimum clone size detected was 0.1% for WBCs and 3% for RBCs. The results are expressed as the percentage of granulocytes negative for CD55/CD59 or FLAER/CD24 [13,14]. Previously diagnosed patients also underwent flow cytometric analysis retrospectively and met the minimal essential diagnostic criteria of PNH [5]. All patients received a bone marrow examination for concomitant myelodysplastic syndromes or aplastic anemia. The patient series was classified into three groups according to the clinical expression and PNH clone percentage: classic PNH, PNH in the setting of another specified bone marrow disorder (SBMD), and subclinical PNH, according to the Nakakuma classification as amended by Parker [6,15].
Participants and methods Data collection of the study population Between 1985 and 2015, there were 24 PNH cases, including 10 males and 14 females, from four medical centers in southern Taiwan. The collected clinical data included lactate dehydrogenase (LDH) levels, hemoglobin levels, platelet levels, transfusion requirements, thrombotic events (identified using major adverse vascular event categories), physician-reported renal dysfunction, and treatment modalities. Our study received Institutional Review Board approval (IRB number: KMUH-IRB-20140201, VGHKS 15-CT3-05, 105-6702C, B-BR-105-014-T).
Treatment of PNH The treatment of PNH included steroids, androgens, and immunosuppressive therapy (including azathioprine, antithymocyte globulin, and cyclosporine). Anticoagulation therapy was used in patients with thrombotic events. Eculizumab (Soliris; Alexion Pharmaceuticals, Inc., Cheshire, CT, USA) was approved in 2013 by the National Health Insurance of Taiwan for the treatment of PNH with >50%
Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
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Characteristics of PNH in Southern Taiwan
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granulocyte clone size and persistent hemolysis or thrombotic events. Eight patients were treated with eculizumab following approval. One patient underwent splenectomy, and one patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 2006.
Imaging and examination for complications All patients received abdominal sonography for chronic complications, including splenomegaly and cholelithiasis. Fourteen patients underwent cardiac sonography for pulmonary hypertension. Computed tomography (CT) radiology, Tc-99m RBC venography, or Doppler echo of the extremities was arranged if the patients developed symptoms of thrombosis. One patient underwent abdominal and cranial CT, and one patient received Tc-99m RBC venography and Doppler echo of the limbs.
Results The median age of the study population was 39.5 years (range, 12e70 years). The first patient was diagnosed in 1985. The mean follow-up time was 10.8 9.1 years. Twenty-four patients were divided into three clinical groups according to the Parker classification system: 14 patients (58.3%) belonged to the classic PNH group, and 10
Table 1
patients (41.7%) belonged to the SBMD group. None of the patients were classified as having subclinical PNH. The mean PNH granulocyte clone size was 72.3 37.9%, and the mean maximum lactate dehydrogenase (LDH) level was 2920.2 1462.0 IU/L (8.49-fold of the upper limit of normal). Nine cases (37.5%) had a medical history of aplastic anemia (AA), and one case (4.2%) was diagnosed with PNH and AA concomitantly. The clinical symptoms included fatigue and hemoglobinuria (18 cases, 75%), abdominal pain (12 cases, 50%), and dyspnea (15 cases, 62.5%). All patients received abdominal sonography; splenomegaly was observed in eight cases (33.3%). The hemolysis-related complications included renal insufficiency (7 cases, 29.2%), cholelithiasis (4 cases, 16.7%), and thrombotic events (4 cases, 16.7%; 2 episodes in 2 cases). The thromboembolic events occurred in four patients and included superficial and deep venous thrombosis in the lower limbs, splenic arterial infarction, arterial ischemic stroke, and myocardial infarction. None of the thrombotic events preceded the diagnosis of PNH. Fourteen patients received cardiac sonography; one case (7.1%) was diagnosed with pulmonary hypertension. Among the 14 female patients, one woman (7.1%) developed preeclampsia and preterm labor. We did not observe an association between LDH levels and PNH granulocyte clone size. Furthermore, there was no correlation between PNH granulocyte clone size and the incidence of thrombosis or other complications
Patient characteristics at the time of PNH diagnosis.
Characteristics
Total (n Z 24)
A (n Z 7)
B (n Z 8)
C (n Z 3)
D (n Z 6)
Age, years, median (range) Female, n (%) Age at diagnosis, years, median (range) Disease duration, years, median (range) Parameter, median (range) Hemoglobin, g/L (5th, 95th percentile) Platelets, 109/L (5th, 95th percentile) Reticulocytes, % (5th, 95th percentile) Granulocyte clone size, %, median (5th, 95th percentile) Lactate dehydrogenase, ULN, median (5th, 95th percentile) Symptoms related to PNH Dyspnea (%) Hemoglobinuria (%) Abdominal pain (%) History of thrombotic events (%) History of impaired renal function (%) Treatment Steroids, n (%) Transfusion dependence, n (%) Androgens, n (%) Eculizumab, n (%) Immunosuppressive therapy, n (%)a Splenectomy, n (%) Allo-HSCT, n (%) Anticoagulation therapy, n (%)
39.5 (12e70) 14 (58.3) 37 (12e65) 8.5 (<1e29)
55 (33e70) 6 (85.7) 33 (20e52) 16 (<1e29)
42.5 (27e62) 4 (50.0) 42.5 (27e62) 3.5 (1e14)
21 (12e65) 2 (66.7) 21 (12e65) 4 (1e5)
39.5 (17e59) 2 (33.3) 39.5 (17e59) 20 (4e27)
74 (26.5, 115) 97 (18, 256.5) 5.1 (0.10, 10.50) 92.5 (1.43, 99.77)
70 150 5.1 89.2
74.5 81 8.7 99.2
74 111 5.6 89.7
62 36 3.15 50.9
8.49 (1.65, 18.98)
8.54
9.77
17.8
6.84
62.5 75.0 50.0 16.7 29.2
57.1 85.7 71.4 28.6 42.9
50 75 25 12.5 12.5
33.3 33.3 0 0 33.3
100 83.3 83.3 16.7 33.3
19 (79.2) 18 (75) 10 (41.7) 8 (33.3) 4 (16.7) 1 (4.2) 1 (4.2) 1 (4.2)
6 4 4 2 2 1 1 0
5 6 1 4 1 0 0 0
2 2 1 2 0 0 0 0
6 6 4 0 1 0 0 1
(85.7) (57.1) (57.1) (28.6) (28.6) (14.3) (14.3) (0)
(62.5) (75) (12.5) (50) (12.5) (0) (0) (0)
(66.7) (66.6) (33.3) (66.6) (0) (0) (0) (0)
(100) (100) (66.6) (0) (16.7) (0) (0) (16.7)
a
Immunosuppressive therapy included azathioprine, anti-thymocyte globulin (ATG) and cyclosporine. Groups AeD represent four tertiary medical centers in southern Taiwan.
Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
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Comparison with other studies of paroxysmal nocturnal hemoglobinuria.
First author, country (y) (reference)
Number Females Age at Additional PNH granulocyte PNH erythrocyte LDH at diagnosis, TE (%) Hemoglobinuria Prior Transfusion of patients (%) diagnosis (y) disorder clone size (%) clone size (%) xULN (range) /abdominal steroid dependence (AA or MDS) (%) pain (%) use (%) (%)
Hillmen, UK (1995) [16] 80 Socie, France (1996) [17] 220 Nishimura (2004) [18] USA 176
Kelly, USA (2011) [19] Lee, Korea (2013) [20] Munos-Linares, Spain (2013) [21] Schrezenmeier, international (2014) [22] Wang, Taiwan (2016) Mean of all patients
42 (16e75) 33 (6e82)
40.0/NA 30.0/5.0a
NA NA
NA NA
NA NA
39.0 6.0
26/NA NA/13.0
NA 21
NA 75b
56.2
30 (4e80)
29.0/5.1
209
43.5
45 (10e86)
79 301 56
50.0 50.0 35.7
37 (12e79) 37 (8e88) 32 (13e84)
CD59 (-): 68.6% CD55 (-): 71.6% 37.8/4.8 CD59 (-): 42.8% CD55 (-): 49.6% 29.1/1.3 96.4 (41.8e100) 40.2/6.3 48.8 (0e100) 67 (AA and MDS) NA
CD59 (-):45.0% CD55 (-): 39.5% CD59 (-): 37.8% CD55 (-): 41.1% 34.0 (2.9e100) 28.1 (0e99.8) NA
12.2
19.0
50.0/-
82.4
61.9b
8.2
6.0
33.5/-
46.9
40.2b
6.7 (1.36e23.95) 27.0 4.1 (0.2e36.3) 17.9 NA 35.0
63.0/30.0 56.1/46.8 60.0/48.0
NA 77.4 NA
81.3 59.1 82.1
1610
53.2
32 (3e87)
43.5/5.8
1.96 (0.65e10.32) 15.5
62.0/54.0
e
e
24 2755c
58.3 51.1
39.5 (12e70) 41.7/0 36.4 36.4/4.0
92.5 (1.3e99.8) 38.8 (3.9e95.2) 8.49 (1.55e20.79) 16.7 71.2 36.3 6.9 20.2
75.0/50.0 53.2/40.3
79.2 61.4
75.0 67.8
68.1 (0.01e100) e
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Japan
58.7 54.5
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AA, aplastic anemia; MDS, myelodysplastic syndrome; ULN, upper limits of normal; TE, thromboembolism. a The data includes acute leukemia. b There is no available data for transfusion-dependent patients; the data represents the percentage of patients who received transfusion as an initial treatment. c Eight studies were included with a total of 2755 patients.
H.-C. Wang et al.
Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
Table 2
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Characteristics of PNH in Southern Taiwan
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Fig. 1. Demographic and clinical characteristics of the patients: (a) clinical laboratory data, (b) percentage of patients reporting symptoms, and (c) percentage receiving treatment prior to enrollment.
in our series. The detailed demographic and clinical characteristics of all patients are described in Table 1, and the subgroup analysis from four tertiary medical centers in southern Taiwan is presented separately. With respect to treatment, eighteen patients (75.0%) were transfusion-dependent. Nineteen patients (79.2%) received steroid therapy with prednisolone or dexamethasone, and 10 patients (41.7%) received androgen therapy. Eight patients (33.3%) were on eculizumab; one patient (4.2%) underwent splenectomy; four patients (16.7%) received immunosuppressive therapy, including azathioprine, anti-thymocyte globulin (ATG), and cyclosporine; and one patient (4.2%) underwent myeloablative conditioning allo-HSCT from an HLA-match unrelated donor. Treatment and prognostic outcomes are recorded in Table 1. Among the patients receiving steroid therapy, only three patients exhibited improved quality of life (QOL) and reduced LDH levels after treatment. Among patients receiving ATG and cyclosporine, none experienced a reduction in transfusion requirements or QOL improvements. One patient underwent splenectomy; hemolysis was not resolved though hemoglobin stabilization after the surgical intervention. All patients receiving eculizumab experienced decreased transfusion requirements and enhanced QOL. The mean LDH levels in these patients fell dramatically from 2859.3 1223.0 IU/L to 313.3 272.7 IU/L. No new thromboembolic events were recorded after eculizumab use, but impaired renal function in these patients was irreversible. Seven patients exhibited decreased transfusion requirements after eculizumab. One patient received allo-HSCT, and improvement of both anemia and hemolysis was noted without regular transfusion after allo-HSCT. Twenty-one patients were still alive at the time of publication, but two patients had died of severe sepsis, and one patient was lost to follow-up.
Discussion This study investigated the clinical characteristics, treatment strategies, and clinical outcomes of PNH in southern Taiwan. We detected possible differences among the clinical characteristics of our patients relative to those in other countries. Compared to previous studies [16e22] (Table 2), the male and female incidence, the percentage of patients
with AA, and the age at diagnosis were similar. With respect to the PNH clone size, which was available in only four studies [18e20], the PNH erythrocyte clone size (including type II and type III erythrocytes) was similar among different countries. However, the PNH granulocyte clone size was higher in Western countries and lower in Asian studies. Notably, our study reports an extremely high PNH granulocyte clone size, which differs from the results of other Asian studies. Compared with patients in the International PNH Registry, a worldwide and observational crosssectional analysis of patients enrolled through June 30, 2012 [22], our patients exhibited unique demographics and clinical characteristics (Fig. 1). Our patients exhibit larger PNH granulocyte clone sizes and more severe thrombocytopenia and anemia, which may explain 62.5% of the patients in our study suffered from dyspnea. Higher LDH levels indicated that our patients developed more profound hemolysis than did patients in other countries. Importantly, the incidence of kidney impairment was higher in our patients compared with patients in the International PNH Registry, but there was no difference in the incidence thrombotic events. Hence, most of our patients did not receive anticoagulants. Most of our patients received steroid therapy after diagnosis, and the change in the hemoglobin level and hemolysis response was not satisfactory. Interestingly, one case with pancytopenia was diagnosed with concurrent AA and PNH, and the bone marrow biopsy revealed less than 1% cellularity with 27.5% PNH granulocyte clones in FLAER and multiparameter flow cytometry. In our study, we found that the patient outcomes remained poor and that the frequency of transfusion remained high after conventional treatments, including steroids, androgens, and immunosuppressive therapy. Splenectomy exhibited limited benefit in our patients. However, after receiving eculizumab, eight patients in our study demonstrated stabilization of their hemoglobin levels and reduction in intravascular hemolysis. QOL improved with decreased requirements for transfusion. Transplantation is the only curative treatment for PNH and is effective in eradicating abnormal PNH clones [23]. One patient underwent matched unrelated donor allo-HSCT before the eculizumab era, and the patient’s intravascular hemolysis and anemia improved significantly after transplantation. The main limitation of our study is the
Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011
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6 relatively small sample size, which may reflect the extremely low prevalence rate and under-diagnosis of PNH. Physicians often overlook atypical symptoms in patients, and the available detection methods, such as FLAER and multiparameter flow cytometry, are not widely used. More studies should be conducted with larger sample sizes and different ethnic groups to validate and extend our findings. In conclusion, our study provides comprehensive clinical information of PNH patients in southern Taiwan with a follow-up period of up to 30 years. Our findings demonstrate that our patients tend to exhibit higher PNH clone sizes and greater kidney injury compared with patients in the International PNH Registry. Anti-complement therapy has produced promising responses in several studies, but HSCT remains the only PNH treatment to produce a definitive cure.
H.-C. Wang et al.
[11]
[12]
[13]
[14]
[15]
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Please cite this article in press as: Wang H-C, et al., Distinct clinical characteristics of paroxysmal nocturnal hemoglobinuria in patients in Southern Taiwan: A multicenter investigation, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.05.011