Serum thrombopoietin levels in patients undergoing ... - Nature

Bone Marrow Transplantation, (1997) 19, 771–775  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

Serum thrombopoietin levels in patients undergoing autologous peripheral blood stem cell transplantation C Shimazaki1, T Inaba2, H Uchiyama1 , T Sumikuma1, T Kikuta1, H Hirai1, Y Sudo1, N Yamagata1, E Ashihara1, H Goto1, S Murakami3, H Haruyama3, N Fujita2 and M Nakagawa1 1

Second Department of Medicine; 2Department of Clinical Laboratory Medicine, Kyoto Prefectural University of Medicine; and Department of Medicine, Shakaihoken Kyoto Hospital, Kyoto, Japan

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Summary: Recently, the ligand for c-mpl has been cloned and initial studies have shown it to be the platelet regulatory factor, thrombopoietin (TPO). To elucidate the role of TPO in the reconstitution of megakaryopoiesis and platelet production after stem cell transplantation, we measured serum TPO levels in nine patients undergoing autologous peripheral blood stem cell transplantation (PBSCT) and in healthy volunteers. Serum TPO levels significantly correlated with the degree of peripheral thrombocytopenia and a strong inverse correlation between serum TPO level and platelet count was observed (r = −0.700, P , 0.001). Serum TPO levels began to rise as the platelet count decreased after chemotherapy. TPO levels peaked at over 25.00 fmoles/ml between days 0 and 10; TPO levels then decreased gradually as the platelet count began to rise. One patient with multiple myeloma received purified CD34+ peripheral blood stem cells. No difference was observed in the kinetics of serum TPO levels between unfractionated and purified PBSCT. These observations suggest that TPO plays a critical role in the reconstitution of megakaryopoiesis and platelet production after PBSCT. Keywords: thrombopoietin; PBSCT; platelet

gated in patients receiving BMT and PBSCT.5–11 A strong inverse correlation between serum granulocyte colony-stimulating factor (G-CSF) level and absolute neutrophil count has been demonstrated,5–9 which suggests that G-CSF plays a critical role in the reconstitution of myeloid lineage progenitors after stem cell transplantation. However, of the growth factors studied, including interleukin-3 (IL-3), interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), none have been demonstrated to correlate with platelet recovery after stem cell transplantation.6,9–11 A possible exception was reported by Testa et al11 who showed that peak IL-6 levels are directly correlated with the peak of platelet recovery. Recently, the ligand for c-mpl has been cloned; in vitro and in vivo studies have shown that it stimulates both megakaryocytopoiesis and platelet production, suggesting that it is the long-sought platelet regulatory factor, thrombopoietin (TPO) itself.12–15 To elucidate the role of TPO in megakaryopoiesis and platelet recovery after stem cell transplantation, we measured serum TPO levels in patients undergoing autologous PBSCT using a recently established sensitive sandwich enzyme-linked immunosorbent assay (ELISA).16

Materials and methods Patients

High-dose chemotherapy followed by peripheral blood stem cell transplantation (PBSCT) has become an option for the treatment of high-risk hematological malignancies and solid tumors. 1–3 Following myeloablative chemotherapy, the recovery of hematopoiesis is dependent on stem cell self-renewal capacity and differentiation to lineage-committed progenitors, which undergo further differentiation and then maturation to morphologically recognizable precursor cells and terminal cells circulating in peripheral blood.4 Hematopoietic growth factors are thought to play a role in the engraftment of stem cells following bone marrow transplantation (BMT) or PBSCT. Thus far, serum levels of several growth factors have been investiCorrespondence: Dr C Shimazaki, Second Department of Medicine, Kyoto Prefectural University of Medicine, 465 Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602, Japan Received 15 July 1996; accepted 30 November 1996

Nine patients with various types of malignancies were evaluated (Table 1). There were seven males and two females, age range 18 to 67 years. Diagnosis included nonHodgkin’s lymphoma (NHL) (four), acute myelogenous leukemia (AML) (two), acute lymphoblastic leukemia (ALL) (one), multiple myeloma (MM) (one) and small cell lung cancer (SCLC) (one). Transplant procedures The details of peripheral blood stem cell (PBSC) collection and PBSCT procedures have been previously described.17 Briefly, patients were treated with a regimen of high-dose cytosine arabinoside (Ara-C) (12 g/m2), high-dose cyclophosphamide (CY) (4 g/m2), or high-dose etoposide (VP16) (2 g/m2) followed by subcutaneous administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (filgrastim) 50 mg/m2. PBSC were collected

Serum TPO levels after PBSCT and BMT C Shimazaki et al

during leukocyte recovery using a CS3000 blood cell separator (Fenwal, Deerfield, IL, USA), and stored at −130°C. In the MM patient, CD34+ cells were separated from total PBSC using an Isolex system (Baxter Healthcare Immunotherapy Division, Round Lake, IL, USA). The pre-transplant conditioning regimen for AML consisted of busulfan (BU) (16 mg/kg) and CY (120 mg/kg); that for NHL consisted of CY (120 mg/kg), VP-16 (1500 mg/m2) and ranimustin (MCNU) (500 mg/m2); that for MM consisted of melphalan (L-PAM) (200 mg/m2); and that for SCLC consisted of CY (3000 mg/m2), VP-16 (1000 mg/m2) and carboplatin (CBDCA) (1200 mg/m2). PBSC were infused on day 0. All patients received rhG-CSF at a dose of 50 mg/m2 subcutaneously from day 1 until the neutrophil count exceeded 0.5 × 109/l. Serum samples After informed consent had been obtained, blood samples were drawn from the nine patients undergoing PBSCT. Samples were collected on day −8, and every 2–4 days thereafter until platelet recovery. Serum was separated by centrifugation shortly after collection and was frozen at −80°C until assayed.

microplate reader (International Reagents Corporation, Kobe, Japan). The absorbance of each sample was subtracted from that of each sample incubated with TN1. The average value of each standard or high TPO serum sample was subtracted from that of the blank for the standard. The sample concentration was calculated by regression analysis for the standard curve. This ELISA system was highly sensitive and specific for human TPO. The lower limit of detection was 0.09 fmoles/ml in serum and no significant cross-reactivity to other cytokines was observed. Statistical analysis A software program, StatView 4.0 (Abacus Concepts, Berkeley, CA, USA), was used for statistical analyses. The relationship between hematologic data and serum TPO levels was evaluated by Student’s t-test. A P value of ,0.05 was accepted as statistically significant. Correlation statistics were determined by Pearson’s correlation coefficient. Results Control subjects

TPO levels in serum were measured by a sandwich enzyme-linked immunosorbent assay (ELISA).16 Each well in a 96-well flat-bottomed microtiter plate (Maxisorp, Nunc, Roskilde, Denmark) was coated at 4°C overnight with 100 ml of TN1 (mouse anti-human TPO monoclonal antibody) (Kirin Pharmaceutical Research Laboratory, Gunma, Japan) at a concentration of 10 mg/ml in 50 mm carbonate buffer (pH 9.4). After washing twice with 20 mm Tris-HCl containing 0.5 m NaCl and 0.1% NaN3 (pH 7.5) (TBS), 200 ml of a blocking reagent (Super Block in TBS; Pierce, Rockford, IL, USA) was added to the wells and incubated at room temperature for 30 min. After the blocking reagent was aspirated, 100 ml of recombinant human (rh) TPO standard, serum test sample or blank were added to each well and reacted with the coated TN1 at room temperature overnight. After washing with 20 mm Tris-HCl containing 0.5 m NaCl, 0.05% Tween 20 and 0.1% NaN3 (T-TBS), 100 ml of biotinylated anti-rhTPO at a concentration of 500 ng/ml in dilution buffer (T-TBS containing 1% bovine serum albumin and 2% PEG 6000, pH 7.5) was added to each well and incubated at room temperature for 3 h. After washing with T-TBS, 100 ml of alkaline phosphatase-conjugated streptavidin (1 mU/ml in dilution buffer; Boehringer Mannheim, Mannheim, Germany) was added to each well, then incubated at room temperature for 1 h. The color was developed using an amplification system (Gibco BRL, Gaithersburg, MD, USA). After washing with TTBS, 50 ml of substrate solution was added to each well, and incubated at room temperature for 40 min. Then 50 ml of amplifier solution (NADPH-amplification system; Gibco) was added to each well and incubated at room temperature for 30 min. Color development was stopped by adding 50 ml of 0.3 m H2SO4. The color intensity was measured using the A 630 nm subtracted from the A492 nm on a

Serum TPO levels obtained from five healthy volunteers were 0.41 6 0.23 fmoles/ml (mean 6 s.d.). PBSCT patients Platelet recovery in the nine patients receiving PBSCT was rapid except for the two patients with AML and the median time to reach 50 × 109/l platelets was 16 days (Table 1). The relationship between the kinetics of serum TPO and platelet counts in the blood of seven patients with the fastest platelet recovery is shown in Figure 1. Serum TPO levels began to rise as the platelet count decreased after highdose chemotherapy. The TPO level peaked at over 25.00 fmoles/ml between days 0 and 10 and levels then gradually

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Measurement of serum level of TPO

Platelets (×109/l)

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10 50 0 –10

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Days after PBSCT Figure 1 Relationship between serum TPO level (I) and absolute platelet count (o) in seven patients undergoing peripheral blood stem cell transplantation (PBSCT). Serum TPO levels rose immediately after PBSCT, reaching a peak between days 0 and 10. TPO levels decreased as the platelet counts rose. Error bars indicate the standard error of the mean.

TPO (fmoles/ml)


Discussion

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This study demonstrated a significant correlation between the degree of thrombocytopenia post-transplantation and endogenous TPO production. A strong inverse correlation between serum TPO levels and circulating platelet counts was observed in patients undergoing PBSCT and purified CD34+ PBSCT. These observations suggest that TPO plays a critical role in the reconstitution of megakaryocytopoiesis and platelet production after stem cell transplantation. To date, various cytokines including IL-3,18 IL-6,19 IL1120 and LIF21 have been shown to stimulate the growth of megakaryocytic progenitors. However, in vitro studies have shown that the effects of these cytokines are less potent than those of TPO, and no direct correlation has been observed between the levels of these cytokines and platelet recovery after stem cell transplantation.6,9,10 Recently, Kuter and Rosenberg22 have determined the relationship between blood levels of TPO and changes in the circulating platelet mass in rabbits treated with busulfan. As the platelet mass declined, levels of TPO increased inversely and proportionally and peaked during the platelet nadir. Bernstein et al23 reported a significant inverse relationship between platelet counts and plasma TPO levels but not those of IL-3, IL-11 or LIF during the course of chemotherapy in patients with AML. In addition, Nichol et al24 demonstrated that the platelet nadir was always associated with the peak of serum TPO which returned towards baseline as platelet counts recovered in patients receiving PBSCT with or without bone marrow. These observations, together with ours, suggest that TPO is a major growth factor for platelets. Two mechanisms have been postulated concerning the regulation of serum TPO levels. One possible mechanism of TPO clearance from serum is receptormediated degradation of TPO by megakaryocytes and platelets.22,25,26 Based on the finding that platelets bear receptors for TPO, Kuter and Rosenberg 22 have proposed that TPO gene expression is constant and that serum levels are con-

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Platelets (×109/l) Figure 2 Inverse correlation between serum level of TPO and absolute platelet count (r = −0.70, P , 0.001).

decreased as the platelet count began to rise (Figure 1). Serum TPO levels significantly correlated with the degree of peripheral thrombocytopenia and an inverse correlation was observed between serum levels of TPO and the absolute platelet count (r = −0.70, P , 0.001) (Figure 2). In the patient with myeloma who received purified CD34+ PBSC, the times required for neutrophil and platelet recovery were similar to those for unfractionated PBSCT; the times to reach 0.5 × 109/l neutrophils and 50 × 109/l platelets were 12 and 18 days, respectively. In this patient, the serum TPO level reached a significantly elevated level on day 0, and high levels continued until the platelet count began to rise. The kinetics of serum TPO levels were similar in patients receiving unfractionated PBSCT. In the two patients with AML, who showed delayed platelet recovery, serum TPO levels rose immediately after PBSCT and high levels continued until the platelet count began to rise.

Table 1 Case

1 2 3 4 5 6 7 8 9a

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Clinical data and serum TPO level after peripheral blood stem cell transplantation Age/Sex

67/M 42/F 18/M 20/M 47/F 34/M 40/M 36/M 31/M

Diagnosis

SCLC NHL NHL NHL NHL AML AML ALL MM

No. of cells infused MNC (×108 /kg)

CFU-GM (×105/kg)

1.1 4.0 2.3 3.0 2.6 2.5 1.9 3.2 1.7b

3.7 15.2 5.6 21.0 22.4 6.4 3.0 14.6 1.4

TPO peak value (fmoles/ml)

27.00 59.00 31.50 48.25 34.81 43.00 35.75 40.62 27.25

Days to recovery ANC . 0.5 (×109/l)

Platelets . 50 (×109/l)

9 9 17 9 11 14 11 10 12

10 9 12 15 16 40 63 18 18

M = male; F = female; SCLC = small cell lung cancer; NHL = non-Hodgkin’s lymphoma; AML = acute myelogenous leukemia; MM = multiple myeloma; ALL = acute lymphoblastic leukemia; MNC = mononuclear cells; CFU-GM = colony forming units-granulocyte macrophage; TPO = thrombopoietin; ANC = absolute neutrophil count. a Purified CD34+ PBSCT. b Number of CD34+ cells (×106/kg).


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trolled by the platelet mass through uptake and metabolism. Higher platelet counts would result in increased catabolism of TPO, leading to a lower serum TPO level. Similar receptor-mediated clearance mechanisms have been proposed for G-CSF, 27,28 M-CSF29 and GM-CSF.30 Another potential mechanism is feedback regulation at the level of gene expression, as seen with erythropoietin.31 McCarty et al32 have reported that TPO mRNA cannot be detected in the bone marrow and spleen of monkeys during steady state but does appear during a thrombocytopenic state. No data have been reported on the site of TPO production in humans. Further study is required to elucidate these hypotheses. In this study, no differences in the kinetics of serum TPO levels were observed between patients undergoing PBSCT and purified CD34+ PBSCT although the number of patients receiving purified CD34+ PBSCT is small. As for the kinetics of G-CSF, the increase in serum G-CSF levels in PBSCT patients starts immediately following graft infusion partly because endogenous G-CSF is derived from an increased number of infused monocytes present in PBSC grafts. 33 In contrast, TPO has been reported to be produced by liver and kidney but not monocytes.32,34 These reports are consistent with our observations that no kinetic differences in serum TPO levels were demonstrated between PBSCT and purified CD34+ PBSCT. The present study demonstrates that TPO is an important cytokine for the reconstitution of megakaryocytic lineage cells after stem cell transplantation. Considering the potential clinical application of this cytokine in patients undergoing stem cell transplantation, 35 further study is warranted to clarify the mechanisms regulating TPO in humans. References 1 Kessinger A, Armitage JO. The evolving role of autologous peripheral stem cell transplantation following high-dose therapy for malignancies. Blood 1991; 77: 211–213. 2 Gale RP, Henon P, Juttner C. Blood stem cell transplants come of age. Bone Marrow Transplant 1992; 9: 151–155. 3 Demuynck H, Delforge M, Zachee P et al. An update on peripheral blood progenitor cell transplantation. Ann Hematol 1995; 71: 29–33. 4 Gordon MY, Greaves MF. Physiological mechanisms of stem cell regulation in bone marrow transplantation and hematopoiesis. Bone Marrow Transplant 1989; 4: 335–338. 5 Cairo MS, Suen Y, Sender L. Circulating granulocyte colonystimulating factor (G-CSF) levels after allogeneic and autologous bone marrow transplantation: endogenous G-CSF production correlates with myeloid engraftment. Blood 1992; 79: 1869–1873. 6 Kawano Y, Takaue Y, Saito S et al. Granulocyte colonystimulating factor (CSF), macrophage-CSF, granulocyte– macrophage CSF, interleukin-3, and interleukin-6 levels in sera from children undergoing blood stem cell autografts. Blood 1993; 81: 856–860. 7 Miksitis K, Beyer J, Siegert W. Serum concentration of GCSF during high-dose chemotherapy with autologous stem cell rescue. Bone Marrow Transplant 1993; 11: 375–377. 8 Haas R, Gericke G, Witt B, Cayeux S, Hunstein W. Increased serum levels of granulocyte colony-stimulating factor after autologous bone marrow or blood stem cell transplantation. Exp Hematol 1993; 21: 109–113.

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