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Blood Thrombopoietin Levels in Clonal Thrombocytosis and Reactive Thrombocytosis
Blood Thrombopoietin Levels in Clonal Thrombocytosis and Reactive Thrombocytosis Jen C. Wang, MD, Chi Chen, MD, PhD, Allan D. Novetsky, MD, Stephen M. Lichter, MD, Fakhiuddin Ahmed, MD, Neal M. Friedberg, MD BACKGROUND: Although the distinction between clonal and reactive thrombocytosis is clinically relevant because clonal thrombocytosis has more thrombohemorragic complications, the differential diagnosis of these two entities can be difficult. Methods such as the detection of unstimulated erythroid or megakaryocyte colony growth are not readily available. Therefore, we measured blood thrombopoietin levels to determine whether these levels can be used to distinguish the two conditions. PATIENTS AND METHODS: Thrombopoietin levels were measured in 73 patients with thrombocytosis (platelet count .500,000/mL), including 39 patients with clonal thrombocytosis (20 patients with essential thrombocythemia, 15 with agnogenic myeloid metaplasia, 1 patient with polycythemia vera, and 3 with undefined myeloproliferative disorders) and 34 patients with reactive thrombocytosis (17 with malignant tumors, 11 with inflammatory diseases, 4 with sickle cell disease, and 2 with iron deficiency anemia). Seventeen normal volunteers were used as controls.
RESULTS: Thrombopoietin levels were significantly higher (P ,0.05) in patients with clonal thrombocytosis (mean 6 SD of 555 6 585 pg/mL), including the subgroup with essential thrombocythemia (505 6 459 pg/mL), than in patients with reactive thrombocytosis (290 6 133 pg/mL) who had similar levels as controls (201 6 112 pg/mL). Thrombopoietin levels in patients with clonal thrombocytosis, including essential thrombocythemia, were not correlated with platelet counts. CONCLUSIONS: Thrombopoietin levels may be helpful in distinguishing between clonal thrombocytosis and reactive thrombocytosis. Thrombopoietin is probably responsible for the elevated platelet counts in clonal thrombocytosis including essential thrombocythemia, but not in reactive thrombocytosis. High thrombopoietin levels in patients with clonal thrombocytosis cannot be explained solely by platelet megakaryocyte mass. Am J Med. 1998;104:451– 455. q1998 by Excerpta Medica, Inc.
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(10,11), and the other laboratory tests require specialized techniques not commonly available. Recently thrombopoietin has been purified from the serum of thrombocytopenic animals (12–14) and its c-DNA has been cloned (12,14,15). Thrombopoietin is an important regulator of the proliferation and differentiation of hematopoietic progenitor cells into megakaryocytes (16,17). We measured blood thrombopoietin levels to see if such measurements might help in the differential diagnosis of thrombocytosis.
hrombocytosis in general can be divided into two categories. One group, termed clonal thrombocytosis, occurs in the context of the chronic myeloproliferative disorders, including essential thrombocythemia, primary myelofibrosis, and polycythemia vera. The other group, called reactive thrombocytosis, occurs in various inflammatory states (1,2), neoplastic diseases (3), iron deficiency anemia, and after splenectomy (2). The distinction between these two groups is clinically relevant because thrombohemorrhagic complications are more common in clonal thrombocytosis (4,5). Several clinical or laboratory parameters have been used to differentiate these two conditions, such as splenomegaly, bone marrow histology evaluations (6), cytogenetic studies (7), in vitro platelet function tests (8), platelet distribution width and platelet nucleotide ratio (9), and unstimulated burst forming and erythroid colony growth (9). But the frequency of palpable splenomegaly is less than 50% in patients with essential thrombocythemia
From the Division of Medical Oncology and Hematology, Brookdale University Hospital and Medical Center, Brooklyn, New York. Supported in part by the Lee Streich Memorial Fund for Cancer Research and Dworetz-Wolf-Hoffman Foundation. Requests for reprints should be addressed to: Jen C. Wang, MD, Division of Hematology/Oncology, Brookdale University Hospital Medical Center, Brooklyn, New York 11212. Manuscript submitted October 14, 1997 and accepted in revised form February 25, 1998. q1998 by Excerpta Medica, Inc. All rights reserved.
MATERIALS AND METHODS Patients From 1994 through 1996, we enrolled 39 patients with clonal thrombocytosis (Table 1), (including 20 with essential thrombocythemia, 15 with agnogenic myeloid metaplasia (MF), 3 with undefined myeloproliferative disorders, and 1 with polycythemia vera), 34 patients with reactive thrombocytosis (Table 2), including 17 with malignant tumors (4 colon cancer, 3 malignant lymphoma, 2 uterine tumor, 2 hepatoma, and 1 each of carcinoma of lung, breast, bladder, pancreas, tongue, and larynx), 11 with chronic inflammatory disease or infectious process, 4 with sickle cell disease, and 2 with iron deficiency anemia. Seventeen normal volunteers were used as controls. Patients who were seen at the Hematol0002-9343/98/$19.00 451 PII S0002-9343(98)00090-4
Thrombopoietin Levels in Thrombocytosis/Wang et al
Table 1. Clinical Characteristics of Patients with Clonal Thrombocytosis Age (Years) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
83 34 48 63 66 80 70 70 73 47 83 90 31 83 58 91 78 65 39 49 81 72 70 70 76 77 70 73 55 84 62 75 63 90 75 80 90 77 84
Gender
Diagnosis
Platelet Counts (/mL)
Thrombopoietin (PG/ML)
Spleen Size (cm)*
ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MPD MPD MPD PV
521,000 535,000 573,000 633,000 635,000 640,000 663,000 864,000 881,000 900,000 949,000 1,166,000 1,292,000 1,300,000 1,400,000 1,720,000 1,800,000 2,020,000 2,900,000 1,000,000 533,000 575,000 590,000 629,000 629,000 640,000 673,000 751,000 790,000 882,000 889,000 1,000,000 1,050,000 1,060,000 1,200,000 585,000 606,000 1,050,000 614,000
256 575 321 240 882 1651 565 141 713 148 125 179 637 1545 886 137 92 655 50 298 68 1779 83 531 45 557 310 567 2116 1778 1901 404 101 232 102 285 307 69 332
0 0 2 0 0 2 0 0 0 0 2 2 2 0 2 0 0 2 0 2 16 20 6 16 20 12 6 10 10 12 8 6 4 4 4 4 2 4 2
F F F M F F F M F F F F M F F F F M F M F F F F M F M F F F F F F M F F F M F
* Denotes distance palpated below left costal margin. ET 5 essential thrombocythemia; MF 5 agnogenic myeloid metaplasia; MPD 5 undefined myeloproliferative disorders; PV 5 polycythemia vera.
ogy/Oncology Department at Brookdale Hospital with platelet counts greater than 500 3 103/mL were selected for study. Either serum or plasma was obtained and stored at 2708C. Spleen size was measured as length in centimeters by palpation below the left costal margin. Informed consent was obtained.
Definitions The term clonal thrombocytosis was applied to patients with elevated platelet counts and the diagnosis of myeloproliferative disease (essential thrombocythemia, primary myelofibrosis or polycythemia vera). Conventional 452
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criteria were used in the diagnosis, including Philadelphia chromosome negative primary myelofibrosis and the Polycythemia Vera Study Group criteria for essential thrombocythemia and polycythemia vera (18). The term reactive thrombocytosis was applied to patients without history of myeloproliferative disorders who had a disease known to be associated with thrombocytosis.
TPO Assay Thrombopoietin ELISA reagents were provided by Amgen (Amgen, Inc.; Thousand Oaks, California), and the assay was performed as previously described (19). Poly-
Thrombopoietin Levels in Thrombocytosis/Wang et al
Table 2. Characteristics of Patients with Reactive Thrombocytosis*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Age
Gender
Diagnosis
Platelet Counts (/mL)
TPO (pg/mL)
62 70 74 78 54 53 70 76 72 54 57 71 47 34 91 53 71 71 79 78 73 68 68 56 79 66 58 50 40 65 30 19 22 20
M M M F M M F M M M F M M M F F F F M F M F F M F M F F M F M M M F
Lung cancer Bladder cancer Breast cancer Colon cancer Colon cancer Larynx cancer Pancreas cancer Tongue cancer Colon cancer Colon cancer Hepatoma Hepatoma Lymphoma Lymphoma Lymphoma Uterine cancer Uterine cancer Arthritis Infection Arthritis Infection Infection Infection Infection Infection Infection Pericarditis Arthritis Iron deficiency anemia Iron deficiency anemia SS anemia SS anemia SS anemia SS anemia
580,000 778,000 717,000 524,000 756,000 1,200,000 703,000 580,000 692,000 789,000 642,000 673,000 528,000 703,000 789,000 692,000 692,000 540,000 560,000 565,000 582,000 600,000 600,000 640,000 756,000 948,000 808,000 1,200,000 552,000 734,000 603,000 620,000 847,000 610,000
219 599 260 166 480 351 181 295 179 298 310 437 337 597 279 216 476 208 158 232 397 372 240 296 244 186 155 149 508 156 194 142 547 103
* No patient with reactive thrombocytosis had palpable splenomegaly. SS anemia 5 sickle cell anemia.
clonal antibody raised against the receptor binding portion of thrombopoietin was selected as the captured antibody. Briefly, this antibody was coated onto 96 EIA plates, then samples and recombinant human thrombopoietin (rHUTPO), used as standard, were added. Signal antibody using rabbit anti-rHUTPO: horseradish peroxidase conjugate was added after the uncaptured proteins were washed. TMB-peroxidase substrate/peroxidase solution was then added for color development and read as optical density at 450 nm minus optical density 650 nm. The sensitivity and specificity of this assay was described previously (19). We have previously found that plasma and serum thrombopoietin levels are highly correlated: plasma level 5 0.87 (serum level) 2 20; r 5 0.96, P ,0.0001 (19). Therefore, all plasma levels are converted to serum levels.
Statistical Analysis
Continuous values are expressed as mean 6 SD. One-way analysis of variance was used to compare groups. When the F value was significant, the Bonferroni method was employed to determine the significance between groups. P ,0.05 was considered as significant. Correlations between variables were evaluated using the least squares method.
RESULTS Figure 1 shows that the serum thrombopoietin values in patients with clonal thrombocytosis (555 6 585 pg/mL, range 45 to 2116 pg/mL) were significantly higher than those in patients with reactive thrombocytosis (290 6 133 pg/mL, range 103 to 599 pg/mL; P ,0.05) or when May 1998
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Thrombopoietin Levels in Thrombocytosis/Wang et al
DISCUSSION
Figure 1. Mean serum levels of thrombopoietin (pg/mL) in 39 patients with clonal thrombocytosis [CT] including 20 patients with essential thrombocythemia, 15 with agnogenic myeloid metaplasia, 3 with undefined myeloproliferative disorders, and 1 with polycythemia vera), 34 patients with reactive thrombocytosis (RT), and 17 normal volunteer controls. The error bars represent the standard deviation of the mean. *P , 0.05 as compared with controls; **P , 0.05 as compared with patients with reactive thrombocytosis; and P , 0.01 as compared with controls.
compared with controls (P ,0.01). Serum thrombopoietin values in patients with similar to those in controls (201 6 112 pg/mL, range 46 to 375 pg/mL). Serum thrombopoietin values in patients with essential thrombocythemia (504 6 459 pg/mL, range 50.3 to 1651 pg/ mL, n 5 20) were significantly higher than in patients with reactive thrombocytosis (P ,0.05) or controls (P ,0.01). We also determined likelihood ratios (LR) by thrombopoietin levels (,250, 250 to 499, 500 to 749, .750 pg/mL). LR was defined as the proportion of those with clonal thrombocytosis at each level divided by proportion of those with reactive thrombocytosis at that level. Therefore, at thrombopoietin levels ,250 pg/mL, the LR was 0.76; at 250 to 499 pg/mL, the LR was 0.48; at 500 to 749 pg/mL, the LR was 2.3; at .750 pg/mL, the LR was infinite. Thus, thrombopoietin levels more than 500 pg/mL, and especially more than 750 pg/mL, are suggestive of clonal thrombocytosis, while thrombopoietin levels ,500 pg/mL do not distinguish between clonal and reactive thrombocytosis. We also evaluated likelihood ratios by platelet counts. At platelet counts between 500- and 800,000/mL, the LR was 0.52; between 800,000 and 1,000,000/mL, the LR was 30; while at platelet counts .1,000,000/mL, the LR was 70. This suggests platelet counts more than 800,000/mL, especially at more than 1,000,000/mL are more suggestive of clonal thrombocytosis. We found no significant correlation between serum thrombopoietin levels and either blood platelet counts (Figure 2) or degree of splenomegaly. 454
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The present study demonstrated that thrombopoietin levels were significantly greater in patients with clonal thrombocytosis, including essential thrombocythemia, than patients with reactive thrombocytosis. This finding varies from one previously reported (20), in which no apparent difference was found between essential thrombocythemia and reactive thrombocytosis. In that series, however, no statistical comparisons were made because of the small number of patients studied. In addition, since we have cared for these patients and observed their clinical courses closely, we believe we can distinguish clonal thrombocytosis from reactive thrombocytosis in some difficult cases. Furthermore, we studied more patients with reactive thrombocytosis than in the previous report. Other reports (21) also found no increase of serum thrombopoietin levels in 21 patients who developed thrombocytosis after major lower extremity trauma. If this finding is confirmed by further studies, thrombopoietin levels may be useful in the differential diagnosis of thrombocytosis, especially when levels are above 750 pg/mL. The cause of the elevated thrombopoietin levels in clonal thrombocytosis and essential thrombocythemia is not known. Thrombopoietin levels may be regulated by platelet and megakaryocyte mass. Both types of cells express the MPL receptor, in humans (22,23) and animals (24 –28). Clonal thrombocytosis, including essential thrombocythemia, is associated with both high platelet and megakaryocyte mass. However, since no correlation between thrombopoietin levels and platelet counts was found in our patients (Figure 2), the elevated thrombopoietin levels found in patients with clonal thrombocytosis cannot be explained solely by this mechanism. In a preliminary report, Li et al (29) found fewer thrombopoietin receptors on platelets from patients with essential thrombocythemia, leading to high thrombopoietin levels. These, in turn, could stimulate the production of platelets. Variant forms of endogenous thrombopoietin were found
Figure 2. Correlation between serum thrombopoietin levels and blood platelet counts in patients with clonal thrombocytosis.
Thrombopoietin Levels in Thrombocytosis/Wang et al
by Best et al (30). Further studies, including searches for mutations of MPL or thrombopoietin genes, definition of rates of production of thrombopoietin mRNA by the bone marrow or other organs, or enumeration of platelet thrombopoietin receptors, (cMPL), will be necessary to answer these questions. Comparable thrombopoietin levels in patients with reactive thrombocytosis and in normal controls (Figure 1) suggest that thrombopoietin is not a major cause of reactive thrombocytosis. Plasma or serum interleukin-6 levels are elevated in 60% to 80% of these patients (31–34). Taken together, these findings suggest that in reactive thrombocytosis cytokines such as interleukin-6, rather than thrombopoietin, are involved in the pathogenesis of thrombocytosis.
ACKNOWLEDGMENTS The authors thank Amgen Inc., Thousand Oaks, California, for providing the thrombopoietin ELISA reagents; and Drs. Janet Nichol and Stanley Lee for their critical review and editing of the manuscript.
REFERENCES 1. Morowitz DA, Allen LW, Kirsner JB. Thrombocytosis in chronic inflammatory bowel disease. Ann Intern Med. 1968;18:1013–1021. 2. Frenkel EP. Southwestern Internal Medicine Conference: The clinical spectrum of thrombocytosis and thrombocythemia. Am J Med Sci. 1991;301(1):69 – 80. 3. Levin J, Conley CL. Thrombocytosis associated with malignancy. Arch Intern Med. 1964;114:497–500. 4. Colombi M, Radaelli F, Zocchi L, Maiolo AT. Thrombotic and hemorrhagic complications in essential thrombocythemia: a retrospective study of 103 patients. Cancer. 1991;67:2926 –2930. 5. Cortelazzo S, Viero P, Finazzi G, et al. Incidence and risk factors for thrombotic complications in a historical cohort of 100 patients with essential thrombocythemia. J Clin Oncol. 1990;3:556 –562. 6. Burkhardt R, Bartl R, Jager K, et al. Working classification of chronic myeloproliferative disorders based on histological haematological, and clinical findings. J Clin Pathol. 1986;39:237–252. 7. Kopf I, Swolin B, Weinfeld A. Cytogenetic studies on primary thrombocythaemia. Hereditas. 1982;97:217–220. 8. Schafer AI. Bleeding and thrombosis in the myeloproliferative disorders. Blood. 1984;64:1–12. 9. Dudley JM, Messinezy M, Eridani S, et al. Primary thrombocythaemia: diagnostic criteria and a simple scoring system for positive diagnosis. Br J Haematol. 1989;71:331–335. 10. Fenaux P, Simon M, Caulier MT, et al. Clinical course of essential thrombocythemia in 147 cases. Cancer. 1990;66:549 –556. 11. Chistolini A, Mazzucconi MG, Ferrari A, et al. Essential thrombocythemia: a retrospective study on the clinical course of 100 patients. Haematologica. 1990;75:537–540. 12. deSauvage FJ, Hass PE, Spencer SD, et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-mpl ligand. Nature. 1994;369:533–538. 13. Kuter DJ, Beeler DL, Rosenburg RD. The purification of megapoietin: a physiological regulator of megakaryocyte growth and platelet production. Proc Natl Acad Sci USA. 1994;91:11104 –11108. 14. Bartley TD, Bogenerger J, Hunt P, et al. Identification and cloning of a megakaryocyte growth and develop-ment factor that is a ligand for the cytokine receptor mpl. Cell. 1994;77:1117–1124.
15. Lok S, Kaushansky K, Holly RD, et al. Cloning and expression of murine thrombopoietin cDNA and /timulation of platelet production in vivo. Nature. 1994;369:565–568. 16. Kaushansky K, Lok S, Holly RD, et al. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl Ligand thrombopoietin. Nature. 1994;369:568 –571. 17. Wendling F, Maraskovsky E, Debili N, et al. cMpl Ligand is a humoral regulator of megakaryocytopoiesis. Nature. 1994;369:571– 578. 18. Tefferi A, Silverstein MN, Hoagland HC. Primary thrombocythemia. Semin Oncol. 1995;22:334 –340. 19. Wang JC, Chen C, L-H Lou, Mora M. Blood thrombopoietin, IL-6, IL-11 levels in patients with agnogenic myeloid metaplasia. Leukemia. 1997;11:1827–1832. 20. Nichol JL. Serum levels of thrombopoietin in health and disease. In: Kuter DJ, Hunt P, Sheridan W, Zucker-Franklin D, eds. Thrombopoiesis and Thrombopoietins. Totowa, New Jersey: Human Press; 1997;359 –375. 21. Choe EI, Kasabian AK, Kolker AR, et al. Thrombocytosis after major lower extremity trauma: mechanism and possible role in free flap failure. Ann Plastic Surg. 1996;36:489 – 494. 22. Chang M, Yu S, Meng G, et al. Differential mechanisms in the regulation of endogenous levels of thrombopoietin and Interleukin-11 during thrombocytopenia: insight into the regulation of platelet production. Blood. 1996;88:3354 –3362. 23. Kosugi S, Kirata Y, Tomiyama Y, et al. Circulating thrombopoietin level in chronic immune thrombocytopenic purpura. Br J Haematol. 1996;93:704 –706. 24. Kuter DJ, Rosenberg RD. The reciprocal relationship of thrombopoietin (c-MPL ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood. 1995;85: 2720 –2730. 25. McCarty JM, Sprugel KH, Fox NE, et al. Murine thrombopoietin mRNA levels are modulated by platelet count. Blood. 1995;86: 3668 –3675. 26. Alexander WS, Roberts AW, Nicola NA, et al. Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietin receptor c-Mpl. Blood. 1996;87:2162–2170. 27. Cohen-Solal K, Villeval J-L, Titeux M, et al. Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis. Blood. 1996;88:2578 –2584. 28. Stoffel R, Wiestner S, Skoda RC. Thrombopoietin in thrombocytopenic mice: evidence against regulation at the nRNA level and for a direct regulatory role of platelets. Blood. 1996;87:567–573. 29. Li J, Xia Y, Kuter DJ. Analysis of the thrombopoietin receptor (MPL) on platelets from normal and essential thrombocythemia (ET) patients. Blood. 1996;88:545a. Abstract. 30. Best D, Hornkohl A, Selesi D, et al. Evaluation of biological activity, molecular weight and circulating levels of TO in plasma from normal donors and patients with abnormal platelet numbers. Exp Hematol. 1996;24:1069. Abstract. 31. Hollen CW, Henthorn J, Koziol JA, Burnstein SA. Elevated serum interleukin-6 levels in patients with reactive thrombocytosis. Br J Haematol. 1991;79:286 –290. 32. Hollen CW, Henthorn J, Koziol JA, Burnstein SA. Serum interleukin-6 levels in patients with thrombocytosis. Leukem Lymphoma. 1992;8:235–241. 33. Gastl G, Plante M, Finstad CL, et al. High IL-6 levels in ascitic fluid correlate with reactive thrombocytosis in patients with epithelial ovarian cancer. Br J Haematol. 1993;83:433– 441. 34. Tefferi A, Ho TC, Ahmann GJ, et al. Plasma interleukin-6 and CReactive protein levels in reactive versus clonal thrombocytosis. Am J Med. 1994;97:374.
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RESULTS: Thrombopoietin levels were significantly higher (P ,0.05) in patients with clonal thrombocytosis (mean 6 SD of 555 6 585 pg/mL), including the subgroup with essential thrombocythemia (505 6 459 pg/mL), than in patients with reactive thrombocytosis (290 6 133 pg/mL) who had similar levels as controls (201 6 112 pg/mL). Thrombopoietin levels in patients with clonal thrombocytosis, including essential thrombocythemia, were not correlated with platelet counts. CONCLUSIONS: Thrombopoietin levels may be helpful in distinguishing between clonal thrombocytosis and reactive thrombocytosis. Thrombopoietin is probably responsible for the elevated platelet counts in clonal thrombocytosis including essential thrombocythemia, but not in reactive thrombocytosis. High thrombopoietin levels in patients with clonal thrombocytosis cannot be explained solely by platelet megakaryocyte mass. Am J Med. 1998;104:451– 455. q1998 by Excerpta Medica, Inc.
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(10,11), and the other laboratory tests require specialized techniques not commonly available. Recently thrombopoietin has been purified from the serum of thrombocytopenic animals (12–14) and its c-DNA has been cloned (12,14,15). Thrombopoietin is an important regulator of the proliferation and differentiation of hematopoietic progenitor cells into megakaryocytes (16,17). We measured blood thrombopoietin levels to see if such measurements might help in the differential diagnosis of thrombocytosis.
hrombocytosis in general can be divided into two categories. One group, termed clonal thrombocytosis, occurs in the context of the chronic myeloproliferative disorders, including essential thrombocythemia, primary myelofibrosis, and polycythemia vera. The other group, called reactive thrombocytosis, occurs in various inflammatory states (1,2), neoplastic diseases (3), iron deficiency anemia, and after splenectomy (2). The distinction between these two groups is clinically relevant because thrombohemorrhagic complications are more common in clonal thrombocytosis (4,5). Several clinical or laboratory parameters have been used to differentiate these two conditions, such as splenomegaly, bone marrow histology evaluations (6), cytogenetic studies (7), in vitro platelet function tests (8), platelet distribution width and platelet nucleotide ratio (9), and unstimulated burst forming and erythroid colony growth (9). But the frequency of palpable splenomegaly is less than 50% in patients with essential thrombocythemia
From the Division of Medical Oncology and Hematology, Brookdale University Hospital and Medical Center, Brooklyn, New York. Supported in part by the Lee Streich Memorial Fund for Cancer Research and Dworetz-Wolf-Hoffman Foundation. Requests for reprints should be addressed to: Jen C. Wang, MD, Division of Hematology/Oncology, Brookdale University Hospital Medical Center, Brooklyn, New York 11212. Manuscript submitted October 14, 1997 and accepted in revised form February 25, 1998. q1998 by Excerpta Medica, Inc. All rights reserved.
MATERIALS AND METHODS Patients From 1994 through 1996, we enrolled 39 patients with clonal thrombocytosis (Table 1), (including 20 with essential thrombocythemia, 15 with agnogenic myeloid metaplasia (MF), 3 with undefined myeloproliferative disorders, and 1 with polycythemia vera), 34 patients with reactive thrombocytosis (Table 2), including 17 with malignant tumors (4 colon cancer, 3 malignant lymphoma, 2 uterine tumor, 2 hepatoma, and 1 each of carcinoma of lung, breast, bladder, pancreas, tongue, and larynx), 11 with chronic inflammatory disease or infectious process, 4 with sickle cell disease, and 2 with iron deficiency anemia. Seventeen normal volunteers were used as controls. Patients who were seen at the Hematol0002-9343/98/$19.00 451 PII S0002-9343(98)00090-4
Thrombopoietin Levels in Thrombocytosis/Wang et al
Table 1. Clinical Characteristics of Patients with Clonal Thrombocytosis Age (Years) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
83 34 48 63 66 80 70 70 73 47 83 90 31 83 58 91 78 65 39 49 81 72 70 70 76 77 70 73 55 84 62 75 63 90 75 80 90 77 84
Gender
Diagnosis
Platelet Counts (/mL)
Thrombopoietin (PG/ML)
Spleen Size (cm)*
ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET ET MF MF MF MF MF MF MF MF MF MF MF MF MF MF MF MPD MPD MPD PV
521,000 535,000 573,000 633,000 635,000 640,000 663,000 864,000 881,000 900,000 949,000 1,166,000 1,292,000 1,300,000 1,400,000 1,720,000 1,800,000 2,020,000 2,900,000 1,000,000 533,000 575,000 590,000 629,000 629,000 640,000 673,000 751,000 790,000 882,000 889,000 1,000,000 1,050,000 1,060,000 1,200,000 585,000 606,000 1,050,000 614,000
256 575 321 240 882 1651 565 141 713 148 125 179 637 1545 886 137 92 655 50 298 68 1779 83 531 45 557 310 567 2116 1778 1901 404 101 232 102 285 307 69 332
0 0 2 0 0 2 0 0 0 0 2 2 2 0 2 0 0 2 0 2 16 20 6 16 20 12 6 10 10 12 8 6 4 4 4 4 2 4 2
F F F M F F F M F F F F M F F F F M F M F F F F M F M F F F F F F M F F F M F
* Denotes distance palpated below left costal margin. ET 5 essential thrombocythemia; MF 5 agnogenic myeloid metaplasia; MPD 5 undefined myeloproliferative disorders; PV 5 polycythemia vera.
ogy/Oncology Department at Brookdale Hospital with platelet counts greater than 500 3 103/mL were selected for study. Either serum or plasma was obtained and stored at 2708C. Spleen size was measured as length in centimeters by palpation below the left costal margin. Informed consent was obtained.
Definitions The term clonal thrombocytosis was applied to patients with elevated platelet counts and the diagnosis of myeloproliferative disease (essential thrombocythemia, primary myelofibrosis or polycythemia vera). Conventional 452
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criteria were used in the diagnosis, including Philadelphia chromosome negative primary myelofibrosis and the Polycythemia Vera Study Group criteria for essential thrombocythemia and polycythemia vera (18). The term reactive thrombocytosis was applied to patients without history of myeloproliferative disorders who had a disease known to be associated with thrombocytosis.
TPO Assay Thrombopoietin ELISA reagents were provided by Amgen (Amgen, Inc.; Thousand Oaks, California), and the assay was performed as previously described (19). Poly-
Thrombopoietin Levels in Thrombocytosis/Wang et al
Table 2. Characteristics of Patients with Reactive Thrombocytosis*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Age
Gender
Diagnosis
Platelet Counts (/mL)
TPO (pg/mL)
62 70 74 78 54 53 70 76 72 54 57 71 47 34 91 53 71 71 79 78 73 68 68 56 79 66 58 50 40 65 30 19 22 20
M M M F M M F M M M F M M M F F F F M F M F F M F M F F M F M M M F
Lung cancer Bladder cancer Breast cancer Colon cancer Colon cancer Larynx cancer Pancreas cancer Tongue cancer Colon cancer Colon cancer Hepatoma Hepatoma Lymphoma Lymphoma Lymphoma Uterine cancer Uterine cancer Arthritis Infection Arthritis Infection Infection Infection Infection Infection Infection Pericarditis Arthritis Iron deficiency anemia Iron deficiency anemia SS anemia SS anemia SS anemia SS anemia
580,000 778,000 717,000 524,000 756,000 1,200,000 703,000 580,000 692,000 789,000 642,000 673,000 528,000 703,000 789,000 692,000 692,000 540,000 560,000 565,000 582,000 600,000 600,000 640,000 756,000 948,000 808,000 1,200,000 552,000 734,000 603,000 620,000 847,000 610,000
219 599 260 166 480 351 181 295 179 298 310 437 337 597 279 216 476 208 158 232 397 372 240 296 244 186 155 149 508 156 194 142 547 103
* No patient with reactive thrombocytosis had palpable splenomegaly. SS anemia 5 sickle cell anemia.
clonal antibody raised against the receptor binding portion of thrombopoietin was selected as the captured antibody. Briefly, this antibody was coated onto 96 EIA plates, then samples and recombinant human thrombopoietin (rHUTPO), used as standard, were added. Signal antibody using rabbit anti-rHUTPO: horseradish peroxidase conjugate was added after the uncaptured proteins were washed. TMB-peroxidase substrate/peroxidase solution was then added for color development and read as optical density at 450 nm minus optical density 650 nm. The sensitivity and specificity of this assay was described previously (19). We have previously found that plasma and serum thrombopoietin levels are highly correlated: plasma level 5 0.87 (serum level) 2 20; r 5 0.96, P ,0.0001 (19). Therefore, all plasma levels are converted to serum levels.
Statistical Analysis
Continuous values are expressed as mean 6 SD. One-way analysis of variance was used to compare groups. When the F value was significant, the Bonferroni method was employed to determine the significance between groups. P ,0.05 was considered as significant. Correlations between variables were evaluated using the least squares method.
RESULTS Figure 1 shows that the serum thrombopoietin values in patients with clonal thrombocytosis (555 6 585 pg/mL, range 45 to 2116 pg/mL) were significantly higher than those in patients with reactive thrombocytosis (290 6 133 pg/mL, range 103 to 599 pg/mL; P ,0.05) or when May 1998
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Thrombopoietin Levels in Thrombocytosis/Wang et al
DISCUSSION
Figure 1. Mean serum levels of thrombopoietin (pg/mL) in 39 patients with clonal thrombocytosis [CT] including 20 patients with essential thrombocythemia, 15 with agnogenic myeloid metaplasia, 3 with undefined myeloproliferative disorders, and 1 with polycythemia vera), 34 patients with reactive thrombocytosis (RT), and 17 normal volunteer controls. The error bars represent the standard deviation of the mean. *P , 0.05 as compared with controls; **P , 0.05 as compared with patients with reactive thrombocytosis; and P , 0.01 as compared with controls.
compared with controls (P ,0.01). Serum thrombopoietin values in patients with similar to those in controls (201 6 112 pg/mL, range 46 to 375 pg/mL). Serum thrombopoietin values in patients with essential thrombocythemia (504 6 459 pg/mL, range 50.3 to 1651 pg/ mL, n 5 20) were significantly higher than in patients with reactive thrombocytosis (P ,0.05) or controls (P ,0.01). We also determined likelihood ratios (LR) by thrombopoietin levels (,250, 250 to 499, 500 to 749, .750 pg/mL). LR was defined as the proportion of those with clonal thrombocytosis at each level divided by proportion of those with reactive thrombocytosis at that level. Therefore, at thrombopoietin levels ,250 pg/mL, the LR was 0.76; at 250 to 499 pg/mL, the LR was 0.48; at 500 to 749 pg/mL, the LR was 2.3; at .750 pg/mL, the LR was infinite. Thus, thrombopoietin levels more than 500 pg/mL, and especially more than 750 pg/mL, are suggestive of clonal thrombocytosis, while thrombopoietin levels ,500 pg/mL do not distinguish between clonal and reactive thrombocytosis. We also evaluated likelihood ratios by platelet counts. At platelet counts between 500- and 800,000/mL, the LR was 0.52; between 800,000 and 1,000,000/mL, the LR was 30; while at platelet counts .1,000,000/mL, the LR was 70. This suggests platelet counts more than 800,000/mL, especially at more than 1,000,000/mL are more suggestive of clonal thrombocytosis. We found no significant correlation between serum thrombopoietin levels and either blood platelet counts (Figure 2) or degree of splenomegaly. 454
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The present study demonstrated that thrombopoietin levels were significantly greater in patients with clonal thrombocytosis, including essential thrombocythemia, than patients with reactive thrombocytosis. This finding varies from one previously reported (20), in which no apparent difference was found between essential thrombocythemia and reactive thrombocytosis. In that series, however, no statistical comparisons were made because of the small number of patients studied. In addition, since we have cared for these patients and observed their clinical courses closely, we believe we can distinguish clonal thrombocytosis from reactive thrombocytosis in some difficult cases. Furthermore, we studied more patients with reactive thrombocytosis than in the previous report. Other reports (21) also found no increase of serum thrombopoietin levels in 21 patients who developed thrombocytosis after major lower extremity trauma. If this finding is confirmed by further studies, thrombopoietin levels may be useful in the differential diagnosis of thrombocytosis, especially when levels are above 750 pg/mL. The cause of the elevated thrombopoietin levels in clonal thrombocytosis and essential thrombocythemia is not known. Thrombopoietin levels may be regulated by platelet and megakaryocyte mass. Both types of cells express the MPL receptor, in humans (22,23) and animals (24 –28). Clonal thrombocytosis, including essential thrombocythemia, is associated with both high platelet and megakaryocyte mass. However, since no correlation between thrombopoietin levels and platelet counts was found in our patients (Figure 2), the elevated thrombopoietin levels found in patients with clonal thrombocytosis cannot be explained solely by this mechanism. In a preliminary report, Li et al (29) found fewer thrombopoietin receptors on platelets from patients with essential thrombocythemia, leading to high thrombopoietin levels. These, in turn, could stimulate the production of platelets. Variant forms of endogenous thrombopoietin were found
Figure 2. Correlation between serum thrombopoietin levels and blood platelet counts in patients with clonal thrombocytosis.
Thrombopoietin Levels in Thrombocytosis/Wang et al
by Best et al (30). Further studies, including searches for mutations of MPL or thrombopoietin genes, definition of rates of production of thrombopoietin mRNA by the bone marrow or other organs, or enumeration of platelet thrombopoietin receptors, (cMPL), will be necessary to answer these questions. Comparable thrombopoietin levels in patients with reactive thrombocytosis and in normal controls (Figure 1) suggest that thrombopoietin is not a major cause of reactive thrombocytosis. Plasma or serum interleukin-6 levels are elevated in 60% to 80% of these patients (31–34). Taken together, these findings suggest that in reactive thrombocytosis cytokines such as interleukin-6, rather than thrombopoietin, are involved in the pathogenesis of thrombocytosis.
ACKNOWLEDGMENTS The authors thank Amgen Inc., Thousand Oaks, California, for providing the thrombopoietin ELISA reagents; and Drs. Janet Nichol and Stanley Lee for their critical review and editing of the manuscript.
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