Serum thrombopoietin and erythropoietin levels in patients...

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Serum thrombopoietin and erythropoietin levels in patients with acute promyelocytic leukaemia during all‐trans retinoic acid treatment - Kinjo - 1999 - British Journal of Haematology - Wiley Online Library Free Access Serum thrombopoietin and erythropoietin levels in patients with acute promyelocytic leukaemia during all-trans retinoic acid treatment Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Endogenous serum thrombopoietin (TPO) and various cytokines including erythropoietin (EPO), interleukin (IL)-3, IL-6, IL-11, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and stem cell factor (SCF) levels were measured in five patients with acute promyelocytic leukaemia (APL) during all-trans retinoic acid (RA) treatment. During differentiation-inducing therapy, platelet counts slowly increased and reached a peak between days 29 and 46 (median day 35). Serum TPO levels increased parallel to the increasing platelet counts and reached a maximum level during the first 10–20 d of all-trans RA treatment. The circulating TPO levels then decreased in inverse correlation to the platelet counts. These unique changes in serum TPO levels revealed that TPO levels were not regulated by platelet or megakaryocyte mass in patients with APL during differentiation-inducing therapy, and it would appear that TPO levels are directly regulated by all-trans RA during the first 10–20 d of treatment. In addition, the change in circulating EPO levels and reticulocyte counts were similar to that of the TPO levels and platelet counts during all-trans RA treatment, suggesting a close relationship between TPO and EPO signalling. Acute promyelocytic leukaemia (APL) is characterized by the reciprocal 15;17 chromosomal translocation, which fuses the promyelocytic leukaemia (PML) and retinoic acid receptor (RAR)α gene (5). Several clinical studies have shown that nearly all patients who are treated by all-trans retinoic acid (RA) achieve complete remission through an induction of differentiation of the leukaemic cells (8; 3; 30; 11). This differentiation-inducing treatment is now standard therapy in patients with APL because of its safety and clinical usefulness. However, hyperleucocytosis and subsequently occurring life-threatening RA syndrome are well-known complications of the therapy (29). Recently, 20) reported transient and asymptomatic thrombocytosis in two cases of APL with platelet counts of  100 × 109/l during all-trans RA treatment. It was suggested (20) that interleukin (IL)-6 may play an important role in the pathogenesis of the thrombocytosis induced by all-trans RA. However, the exact mechanisms for the thrombocytosis are unknown. Recently, thrombopoietin (TPO), the ligand for c-mpl, has been isolated and cloned (1; 4; 19; 12). It stimulates both the proliferation and differentiation of megakaryocytic progenitor cells, and increases platelet production in vitro and in vivo (1; 4; 14; 19; 12). To address the mechanisms of thrombocytosis in patients with APL during differentiation-inducing therapy by retinoids, we measured both TPO and various thrombopoietic cytokine levels in five patients with APL using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) (26). 41 APL patients, diagnosed according to the French–American–British (FAB) system of classification (2) and as confirmed by the presence of the t(15;17) karyotype and PML/RARα fusion gene were treated with all-trans RA from May 1992 to October 1996 in our hospital. Out of 41 patients, five were investigated for measurement of serum TPO and other cytokines. The patients\' characteristics are summarized in Table I. All patients obtained a complete remission (CR) with all-trans RA 45 mg/m2/d administered orally. CR was defined as a normocellular bone marrow containing 5% blasts and peripheral blood counts showing 3 × 109/l leucocytes, 8 g/dl Hb, and 100 × 109/l platelets. To prevent RA syndrome followed by leucocytosis, three patients (nos. 1, 2 and 3) received chemotherapy consisting of daunorubicin (DNR) 40 mg/m2 i.v. for 2 d and behenoyl cytarabin (BHAC) 200 mg/m2 i.v. for 3 d at day 5 of the initial treatment. Serial serum samples for the evaluation of circulating cytokines were obtained and stored at −80°C until use. Table 1. Table I. Patient characteristics. * All-trans RA was given orally until clinical CR was confirmed according to the criteria as described in the Patients and Methods section. Serum TPO levels were measured by a sandwich ELISA as described elsewhere (26). Briefly, microtitre wells were coated overnight with 10 μg/ml mouse anti-rhTPO monoclonal antibodies (TN1 clone). TPO standards, serum samples, or blanks were added to each well and incubated overnight at room temperature. After washing, 500 ng/ml biotinylated anti-rhTPO F(ab′ )2 antibody was added and incubated for 3 h. Streptavidin–alkaline phosphatase conjugate was then added, and the plates were incubated for 1 h. The colour was developed using an amplification system (Gibco BRL, Gaithersburg, Md.). The use of the avidin-biotin technique and an amplification system contributes to the extraordinarily high sensitivity (detection limit 0.09 fmol/ml) of this assay. The intra- and inter-assay variation ranged from 3.0% to 4.9% and from 5.9% to 6.1%, respectively. The ELISA showed no cross-reactivity with a variety of blood components and cytokines (26). It has been determined that several cytokines have thrombopoietic activity (13). We therefore measured the serum levels of IL-3, IL-6, IL-11, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), stem cell factor (SCF), and erythropoietin (EPO) using ELISA kits purchased from R & D systems (Mineapolis, Min.). The cytokine levels circulating in the serum were assayed in triplicate according to the manufacturer\'s instructions. The statistical analysis of the relationship between the platelet counts and serum cytokine levels during the all-trans RA treatment was performed using Friedman\'s nonparametric test for significance (6). Spearman\'s rank correlation test was used in the calculation correlations among serum TPO, EPO and platelet counts. These analyses were performed with StatView 4.0 software. Serum TPO, various cytokine levels, platelet and reticulocyte counts were simultaneously and sequentially measured after informed consent was obtained in five patients with APL during all-trans RA treatment. All patients obtained CR at a median of 43 d (range 28–56 d) of all-trans RA therapy, and three patients (nos. 1, 2 and 3) received chemotherapy for the prevention of RA syndrome (Table I). Disseminated intravascular coagulation (DIC) was present at diagnosis in all patients, and resolved within the first 10 d. The median platelet count was 106.4 × 109/l (range 27–222 × 109/l) on admission. During the treatment course, platelet counts exhibited a specific pattern of change characterized by a slow increase beginning with the initiation of treatment and reaching a peak on days 29–46 (median day 35), following a progressive increase from around day 20 (Figs 1 and 2). . Serum TPO, EPO levels and circulating platelet (PLT), reticulocyte (RET) counts in a patient with APL (patient 2) during all-trans RA treatment. Serum TPO and EPO levels were measured sequentially before and after differentiation-inducing therapy by all-trans RA as described in the Patients and Methods section. . Serum TPO and EPO levels in five patients with APL. Patients were treated with all-trans RA 45 mg/m2 daily and obtained a clinical CR. Data represent the mean of each point, and the SD was within 10% of the mean. The decrease in TPO levels after 16 d of all-trans RA treatment and increase in platelet counts were inversely correlated. As shown in Table I and Fig 1, the pre-treatment platelet count in patient 2 was 33 × 109/l; this increased to a maximum level of 336 × 109/l on day 30 of all-trans RA treatment. The serum TPO level was 4.56 fmol/ml at the start of differentiation-inducing therapy. The levels increased parallel to the platelet count during therapy and peaked at day 21, reaching a maximum value of 10.40 fmol/ml. The serum TPO levels transiently decreased after platelet transfusion, and the TPO level then significantly decreased and returned to less than baseline levels (Fig 1). All patients exhibited similar patterns for their platelet counts and serum TPO levels during therapy (Fig 2). Circulating TPO levels before treatment were between 2.60 and 9.07 fmol/ml (median 5.47 fmol/ml). The TPO levels increased and peaked at a median of 16 d after the all-trans RA treatment. The highest level of serum TPO was a median of 20.92 fmol/ml. After this point, TPO levels dropped and returned to baseline levels with a significant increase in platelet counts. These decreases in serum TPO levels and increases in platelet counts were inversely correlated (P 0.05) (Fig 2). We simultaneously measured the serum levels of IL-3, IL-6, IL-11, SCF, GM-CSF, G-CSF and EPO in five patients with APL during all-trans RA therapy. Serum EPO levels transiently increased and reached a maximum level at a median of day 15.6 (range 10–20 d). The highest mean serum level of EPO in all patients was 24.54 mU/ml (range 2.41–53.0 mU/ml) (Figs 1 and 2). The EPO levels then decreased and returned to baseline levels. Also, there was a inverse relationship between EPO levels, and increasing platelet and reticulocyte counts in all patients (P = 0.0399) (Fig 2). In addition, serum TPO and EPO levels in patients with APL during all-trans RA treatment changed in a similar manner and correlated with each other (r = −0.581, P = 0.0019) (Fig 3). Serum levels of IL-3, IL-6, IL-11, GM-CSF, G-CSF and SCF remained unchanged during differentiation-inducing therapy, and there was no relationship between these cytokine levels and platelet counts in any patients (data not shown). . Relationship between serum TPO and EPO levels in five APL patients during all-trans RA treatment. Endogenous TPO levels correlated with EPO levels during treatment (r = 0.581, P = 0.0019). TPO is an essential cytokine for the primary regulation of megakaryopoiesis and platelet production in vivo and in vitro (13). However, the exact regulatory mechanism of TPO production is not clearly understood. Researchers have hypothesized that TPO levels are regulated by platelet mass (18) or by megakaryocyte mass in the bone marrow (25). TPO levels change reciprocally to platelet counts in patients with cyclic thrombocytopenia or in those undergoing bone marrow transplantation after myeloablative therapy (21; 10; 16). Also, there was a significant inverse correlation between platelet counts and TPO concentrations in aplastic anaemia patients (24). In addition, platelet transfusion induced a decrease in endogenous TPO levels in the thrombocytopenic rabbit (18). These results suggest that the platelet mass may play a direct role in regulating the circulating levels of TPO. In contrast, serum TPO levels recorded in patients with immune thrombocytopenic purpura (ITP) and myelodysplastic syndrome (MDS) have been only slightly higher than normal (17; 7). These results suggest that megakaryocyte mass affects the circulating TPO levels. Recent clinical studies have reported that transient thrombocytosis occurred in patients with APL during all-trans RA treatment (20; 15). However, the mechanisms of thrombocytosis and the regulatory mechanisms of TPO have not been examined in APL patients undergoing all-trans RA treatment. Therefore we sequentially measured the endogenous TPO as well as other cytokines including EPO in five patients with APL during all-trans RA treatment by a sensitive ELISA. TPO levels increased in parallel with platelet counts during the first 10–20 d of all-trans RA treatment. Platelet counts slowly and then progressively increased during the treatment, and all patients obtained a clinical CR. Whether improvement of DIC in the first 10 d of the all-trans RA treatment is responsible for the rise in platelet counts is unclear. TPO has a prolonged serum half-life of about 20–30 h and remains in the circulation for about 5–6 d (27). Therefore it is reasonable that the peak of platelet counts during the treatment course on days 29–46 (median day 35) followed a progressive increase from day 20. It has been reported that all-trans RA potentiates the megakaryocyte colony in vitro and in vivo after administration to APL patients (28). The number of CFU-meg has been reported to be significantly higher in APL patients treated with all-trans RA compared to non-APL AML patients treated with chemotherapy (28). However, all-trans RA has only stimulatory effects on purified CD34+ cells (23; 28), and the megakaryocyte colony-stimulating activity of retinoids might be due to an increased production of cytokines by bone marrow stromal cells. Taken together, our results and previous investigations suggest that all-trans RA may stimulate the production of endogenous TPO from bone marrow stromal cells in APL patients during the first 10–20 d of treatment, resulting in the promotion of platelet production. These results seem to suggest that TPO may play an important role in the pathogenesis of transient thrombocytosis induced by all-trans RA in APL. Because the serum TPO levels are regulated by a feedback mechanism that depends on platelet mass, TPO levels decrease and are inversely related to the platelet counts. Interestingly, the serum EPO levels changed in a similar manner to the TPO levels during the all-trans RA treatment. Also, circulating platelets and reticulocytes were changed in a similar manner. There is a close relationship between erythropoiesis and megakaryopoiesis, and progenitor cells from both lineages share common features, including surface antigens, signalling mediators, transcription factors, and so on (22). EPO has been observed to enhance CFU-meg colony growth (9), and EPO and TPO share structural homology and exhibit qualitatively similar biologic activity (13). Our data, showing a similar regulation pattern between TPO and EPO in patients with APL during all-trans RA treatment, also suggest that these cytokines have many biological features in common. 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