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Experimental Hematology 2017;■■:■■–■■
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Persistent elevation of plasma thrombopoietin levels after treatment in severe aplastic anemia Xin Zhaoa,b, Xingmin Fenga, Zhijie Wua,b, Thomas Winklera, Ronan Desmonda, Matthew Olnesa, Bogdan Dumitriua, Danielle M. Townsleya, Cynthia E. Dunbara, and Neal S. Younga a
Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA; bInstitute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China (Received 16 August 2017; revised 11 September 2017; accepted 12 September 2017)
Although hematopoietic growth factors are found at high levels in aplastic anemia (AA) patients, little is known about their dynamic change over time after treatment. We examined plasma concentrations of hematopoietic growth factors sequentially in 55 severe AA patients, including 45 treatment-naive patients who had received immunosuppressive therapy (IST) or IST and eltrombopag, and 10 IST-refractory patients who had received eltrombopag only, focusing on thrombopoietin (TPO). TPO concentrations were much higher than normal in patients before treatment and then decreased in responders but not in nonresponders. We followed up on a cohort of nine patients who obtained stable complete remission for up to 7 years after IST and found that TPO levels declined gradually by 3 months after treatment, accompanying an increase in platelet counts, but stabilized at levels higher than normal. An inverse correlation was noted between TPO levels and platelet counts. The increased plasma TPO levels could be required to maintain normal platelet counts in remission and could also be attributed to reduced consumption by circulating platelets. Published by Elsevier Inc. on behalf of ISEH – Society for Hematology and Stem Cells. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
In acquired aplastic anemia (AA), bone marrow destruction is immune mediated and immunosuppressive therapy (IST) based on antithymocyte globulin (ATG) and cyclosporine (CSA) results in hematologic response in approximately 65% of patients [1]. Hematopoietic growth factors (HGFs) such as thrombopoietin (TPO), erythropoietin (EPO), and granulocyte-colony stimulating factor (G-CSF) are required for the proliferation and differentiation of hematopoietic stem cells into platelets, erythrocytes, and granulocytes, respectively. Blood levels of these factors in AA are much higher than in healthy controls, compensating for low blood counts [2–5]. Schrezenmeier et al. [3] measured TPO levels in 43 samples from AA patients at diagnosis before treatment and 26 samples after a partial or complete remission and observed XZ and XF contributed equally to this work. Offprint requests to: Xingmin Feng, PhD, Cell Biology Section, Hematology Branch, National Heart, Lung, and Blood Institute, 10 Center Drive, Room 3E-5216, Bethesda, MD 20892; E-mail:
[email protected]
sustained high TPO levels in patients even in remission. However, those posttreatment samples were only collected at one time point and it is unclear how TPO or other HGF levels change dynamically over time in remission and in other clinical status. Treatment with G-CSF and/or EPO has not generally been beneficial for AA patients [6,7]. Surprisingly eltrombopag, a TPO receptor agonist, improved response rates markedly in naive and treatment-refractory severe AA patients [8–10] despite markedly increased plasma TPO. Therefore, long-term follow-up of these growth factors is needed to understand the impact of environmental stresses on the hematopoietic system. Methods We measured HGF levels sequentially in severe AA patients undergoing various treatment regimens and manifesting a range of hematologic responses to determine longitudinal changes in HGFs in severe AA patients and their correlation with clinical response, focusing on TPO. Written informed consent from subjects was obtained in accordance with protocols approved by the institutional
0301-472X/Published by Elsevier Inc. on behalf of ISEH – Society for Hematology and Stem Cells. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.exphem.2017.09.006
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review board of the National Heart, Lung, and Blood Institute. Severe AA was defined as described previously [11]. Hematologic response was defined on the basis of improvement in peripheral blood counts according to our previously published studies [9]. A total of 55 patients including 24 females and 31 males at median age of 33 years (range 7–73 years) were enrolled in three clinical trials of severe AA (www.ClinicalTrials.gov numbers NCT00260689, NCT00922883 and NCT01623167). Forty-five treatment-naive patients received horse ATG and CSA (n = 18) or horse ATG/CSA plus eltrombopag (n = 27). Among them, 37 patients were hematologic responders and eight were nonresponders (five from the ATG group and three from the ATG plus eltrombopag group) at 6 months. Another cohort of 10 patients refractory to IST were treated with eltrombopag and achieved a partial hematologic response [8]. Peripheral blood samples were obtained at baseline pretreatment and 3, 6, 12, 24, 48, and 60 months after treatment using heparin or ethylenediamine tetraacetic acid as anticoagulants. A total of 47 age- and sex-matched healthy donors were included as controls. Plasma levels of G-CSF, EPO, and TPO were measured by magnetic multiplex assays (Luminex) as described previously [2]. Plasma albumin was depleted using an albumin depletion kit (Thermo Fisher Scientific, Waltham, MA) and 5 µL of albumin-depleted plasma was then subjected to Western blotting to measure the levels of TPO in AA patients and healthy controls. Statistical analyses were performed using Prism 6 (GraphPad Software, La Jolla, CA). The Mann–Whitney U test was used to evaluate the differences in HGF levels between the different groups. Linear regression analysis was performed for correlations of TPO levels with platelet counts or EPO levels with hemoglobin concentrations.
Results and discussion Concentrations of EPO and G-CSF were under the level of detection in healthy controls, but were greatly elevated in AA patients before treatment initiation (Fig. 1A). G-CSF declined rapidly to the normal range by 6 months in both responders and nonresponders (Fig. 1A). There was no correlation between G-CSF concentration and peripheral absolute neutrophil counts at baseline or after response. EPO concentrations decreased by 3 months after treatment initiation (p = 0.0065 at 3 months; p < 0.0001 at 6 months), but levels in nonresponders remained higher at 6, 12, and 24 months compared with those in responders (p = 0.001, p = 0.0015, and p = 0.038, respectively; Figs. 1A and 1B); there was a negative correlation between EPO and hemoglobin levels (r2 = 0.3126, p < 0.0001; Fig. 1C). In the cohort of ISTrefractory patients who achieved clinical response to eltrombopag, EPO and G-CSF levels decreased at 3–4 months after initiation of eltrombopag treatment (Fig. 1D). Plasma TPO concentrations in healthy controls were 315.9 ± 12.7 pg/mL (range 180–506 pg/mL). The levels were greatly elevated in severe AA patients before treatment (2747.0 ± 133.2 pg/mL, range 818–5322 pg/mL; p < 0.0001 vs. healthy controls; Fig. 2A). TPO concentrations decreased by 6 months in responders (p = 0.0013 at 6 months; p < 0.0001 at 12 and 24 months, respectively), but remained far above the normal range until 24 months (p < 0.0001 vs. healthy controls). There was no decrease of TPO levels
in nonresponders at 6, 12, and 24 months. For IST-refractory patients who had achieved clinical response to eltrombopag, plasma TPO remained at high levels for up to 5 years after eltrombopag treatment (Fig. 2B). Elevated TPO levels in severe AA patients were confirmed using Western blotting (Fig. 2C). To determine whether TPO levels decreased to the normal range after longer period of remission, we followed a cohort of nine patients who obtained stable complete remission with near normalization of blood counts for up to 7 years after IST. These patients had platelet counts of 102–314 × 109/L (median, 164 × 109/L) by 6 months and at all later time points and megakaryocytes were either mildly decreased or normal in number on follow-up bone marrow examinations. TPO levels in these complete responders had declined by 3 months after treatment accompanying an increase in platelet counts, but stabilized at levels higher than those of healthy controls even during prolonged follow-up (Fig. 2D). An inverse correlation was found between TPO levels and platelet counts (Fig. 2E; r2 = 0.6371, p < 0.0001), in agreement with a previous report [3]. There is a close relationship between thrombopoiesis and stem cell activity; for example, delayed or inadequate platelet recoveries are the most sensitive indicators of inadequate numbers of transplanted stem cells [12]. In Schrezenmeier et al.’s and our data, most complete responders’ platelet counts were near the lower limit of the normal range, and in some patients’ bone marrow, megakaryocytes remained mildly decreased, indicating a reduced hematopoietic potential that persists even in complete remission patients. Higher levels of TPO (p < 0.0001) were found in complete responders than in healthy controls with similar platelet counts (150–300 × 109/L; Fig. 2E, square). Therefore, the increased plasma TPO levels could be required to maintain normal platelet counts in remission and attributed to the reduced consumption by circulating platelets. TPO is constitutively produced by human liver and kidney [13]. Platelets and megakaryocyte mass regulate circulating TPO levels through TPO–TPO receptor (myeloproliferative leukemia protein [MPL])-mediated internalization and degradation [14]. In severe AA, reduced receptor-mediated removal of the hormone from circulation may be responsible for elevation of plasma TPO concentration due to thrombocytopenia and especially reduced megakaryocyte mass. Low platelet levels induce TPO mRNA expression, mainly in the bone marrow [13]. These two mechanisms need not be mutually exclusive in regulating circulating TPO levels. TPO and MPL play a key role in regulating hematopoietic stem cell maintenance in steady-state adult bone marrow and in promoting stem cell expansion after transplantation [15]. The addition of eltrombopag to IST achieved markedly higher hematologic response rates among patients with severe AA than did IST alone in a historical cohort; eltrombopag increased bone marrow cellularity, CD34+ cell number, and early hematopoietic progenitors [9]. Eltrombopag does not compete with TPO for MPL because it binds in the membrane-spanning region outside of the ligand-binding
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Figure 1. Longitudinal plasma EPO and G-CSF levels in severe AA patients. (A) EPO and G-CSF levels in treatment-naive patients (n = 37). Blue circles represent responders; red circles represent nonresponders. *p < 0.05; **p < 0.01; ***p < 0.001. (B) Follow-up of EPO levels and hemoglobin concentrations in complete responders to IST (n = 9). (C) Correlation of plasma EPO levels with hemoglobin concentrations in severe AA patients. (D) EPO and G-CSF levels in IST-refractory patients (n = 10). Error bar indicates mean and SEM for hemoglobin levels in (B) and median and interquartile in others.
pocket of MPL. Eltrombopag activates signaling through the Janus-associated kinase–signal transducers and activators of transcription (JAK–STAT) and mitogen-activated protein kinase (MAPK) pathways [16]. Notably, eltrombopag evades the blockade of MPL signaling that occurs in the presence of interferon-γ [17]. Patients with severe AA are at risk of progression to clonal marrow dysfunction. Any hematopoietic cytokines, particularly one that affects hematopoietic stem
and progenitors directly via the MPL receptor, could affect proliferation or self-renewal and the emergence of abnormal clones. The short-term (6 months) addition of eltrombopag to IST for the initial treatment of severe AA is not associated with a higher risk of clonal progression compared with historic IST [9], but long-term consequences of high plasma concentrations of endogenous TPO, including stem cell exhaustion and clonal evolution, are unknown.
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Figure 2. Changes in plasma TPO levels over time in severe AA patients. TPO levels in treatment-naive (A) and IST-refractory patients (B). Blue circles represent responders; red circles represent nonresponders. *p < 0.05; **p < 0.01; ***p < 0.001. (C) Different levels of plasma TPO between severe AA patients (n = 10) and healthy donors (n = 8) validated by Western blotting (TPO antibody from Thermo Fisher Scientific). The intensities of bands were quantified and depicted in the bar graph. (D) Follow-up of TPO levels and platelet counts in complete responders to IST (n = 9). (E) Correlation of plasma TPO levels with platelet counts. Red dots and black dots represent complete responders and nonresponders from treatment-naive group, respectively; blue dots represent IST-refractory patients; green dots represent healthy controls. Dots within the square were compared for TPO levels between patients and healthy controls. Patient’s samples from different time points at baseline and after treatment were all included in this figure. Error bar indicates mean and SEM for platelet count in (D) and median and interquartile in others.
Acknowledgments We thank the staff of the Center of Human Immunology for technical support and all patients who donated the samples to this study. This work was supported by the Intramural Research Program of the National Institutes of Health at the National
Heart, Lung, and Blood Institute. The National Heart, Lung, and Blood Institute receives research funding to support clinical development of eltrombopag. Conflict of interest disclosure The authors declare no competing financial interests.
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