Stem Cells, Vol. 16, No. 5, 329-333,
September 1998
© 1998 AlphaMed Press
Thrombopoietin in Patients with Hepatoblastoma
Emiko Komuraa,
Takafumi Matsumuraa,
Takashi Katob,
Tomoyuki Taharab,
Yuko Tsunodac,
Tadashi Sawadaa
a Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan;
b Kirin Brewery Co., Ltd., Takasaki, Japan;
c Department of Surgery, Showa University School of Medicine, Tokyo, Japan
Key Words. Thrombopoietin • c-mpl • Hepatoblastoma • Thrombocytosis
Dr. Takafumi Matsumura, Department of Pediatrics, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602-8566, Japan.
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Abstract
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Marked thrombocytosis (over 50 x 104/µl) is frequently seen in patients with hepatoblastoma. Thrombopoietin (TPO), c-mpl ligand, has recently been purified as the major physiological regulator of the thrombopoiesis and is mainly produced in the liver. Since it is possible that TPO participates in thrombocytosis and the tumor growth of this particular hepatic tumor, serum TPO levels in addition to interleukin 1ß (IL-1ß) and IL-6 levels were assessed in seven untreated patients by using a sandwich enzyme-linked immunosorbent assay. High serum TPO levels were observed in all of the examined patients. The level ranged from 3.15 to 11.02 (mean ± standard deviation; 6.08 ± 1.25) fmol/ml. IL-6 levels were also somewhat higher than normal. Platelet counts, however, appeared to correlate more with serum TPO levels (p = 0.1) than with IL-1ß (p = 0.5) and IL-6 (p = 0.2) levels. Furthermore, using the reverse transcriptase polymerase chain reaction method, the expression of c-mpl mRNA was found in five of eight hepatoblastoma tissues as well as TPO mRNA in all eight tissues. These observations suggest that thrombocytosis in hepatoblastoma patients results from the production of cytokine members, including TPO, within tumor tissues. Additionally, it is possible that TPO might act as a type of autocrine and/or paracrine system for cellular growth in this tumor.
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Introduction
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Hepatoblastoma is the most common malignant liver tumor in childhood [1]. In patients with hepatoblastoma, marked thrombocytosis (over 50 x 104/µl) is frequently seen at diagnosis [2-4]. The exact mechanism of this hematologic complication, however, remains unclear. Schweinitz et al. [5] have suggested interleukin 6 (IL-6) to be one of the mediators showing that hepatoblastoma cells induce IL-6 production in surrounding fibroblasts and endothelial cells by virtue of the endogenous secretion of IL-1ß. Recently, however, the major physiological regulator of thrombopoiesis has been purified as c-mpl ligand and termed thrombopoietin (TPO) [6-8]. TPO mRNA has been detected in several tissues and found most predominantly in the liver [6, 9]. Shimada et al. [10] demonstrated in a rat model that biologically active TPO was secreted from primary hepatocytes and hepatoma cell lines. These reports suggest that the liver could be the major source of TPO production. Therefore, an assumption can be made that thrombocytosis in hepatoblastoma patients results from TPO production in tumor tissue. In this report, the serum TPO levels as well as TPO and its receptor c-mpl mRNA expression are evaluated in patients with hepatoblastoma.
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Materials and Methods
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Patients
Eight children with untreated hepatoblastoma were referred to the Department of Pediatrics, Kyoto Prefectural University of Medicine, between 1985 and 1995. There were three boys and five girls, ranging in age from 3 to 29 months (mean, 12 months). They were all found to have thrombocytosis of more than 50 x 104/µl at diagnosis. The clinical data are summarized in Table 1.
Surgical Specimens
Surgical specimens were obtained from the primary lesions of patients who received a radical operation (cases 1-3) or a biopsy (cases 4-8). Case 3 received a transarterial embolization (TAE) before the initial operation. Fresh samples were quickly frozen in liquid nitrogen and cryopreserved at –80°C until use.
Xenograft
A human hepatoblastoma xenograft in nude mice, named USM [11], was subjected to reverse transcriptase polymerase chain reaction (RT-PCR) analysis for the mRNA expression of TPO and c-mpl. It was established from an 11-month-old girl and has serially passaged in nude mice.
RNA and cDNA Preparation
Total RNA was obtained from freshly frozen tumor samples using the acid-guanidium thiocyanate-phenol-chloroform method. Single-stranded cDNA synthesis from a 20 µg sample of template RNA with oligo-dT(12-18) was performed using reverse transcriptase isolated from AMV (>20 U) (Life Science; St. Petersburg, FL). After the extraction of cDNA with phenolchloroform, cDNA was resuspended in water.
PCR
RT-PCR amplification of cDNA with corresponding 2 µg RNA was performed in a 50 µl reaction mixture containing 10 mM Tris, 50 mM KCl, 200 µM each of 4 deoxyribonucleotide 5' triphosphates, 0.2 µM each of primers and 1.25 U Taq polymerase(TaKaRa Shuzo; Otsu, Japan). PCR protocols including the primer sequences are shown in Table 2 [12, 13]. The analysis of PCR products was performed by 2% agarose gel electrophoresis.
A Sandwich ELISA
Serum TPO, IL-6, and IL-1ß levels at diagnosis were assessed in all of the patients except case 3. Moreover, in case 8, TPO levels along with sequential chemotherapy were also evaluated. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method has been described in detail elsewhere [14], but briefly, it uses a mouse monoclonal antibody as the capture antibody and a biotinylated rabbit polyclonal antibody as the detector.
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Results
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Serum TPO, IL-1ß, and IL-6 Levels
The serum TPO level at diagnosis significantly increased in all patients examined, ranging from 3.15 to 11.02 (mean ± standard deviation [SD]; 6.08 ± 1.25; the normal range is 1.97-2.23 in children from two months to four years of age [15]) fmol/ml ( Table 3). Serum IL-1ß and IL-6 levels (mean ± SD) were 0.32 ± 0.08 and 22.9 ± 7.9 pg/ml, respectively. Platelet counts correlated more with serum TPO levels (p = 0.1, Fig. 1) than with IL-1ß (p = 0.5), and IL-6 (p = 0.2) levels. Additionally, a serial measurement in case 8 indicated that TPO levels decreased along with the normalization of platelet counts following sequential chemotherapy ( Table 4).
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Figure 1. RT-PCR analysis for TPO, c-mpl, IL-1ß, and IL-6 mRNAs in tumor samples from patients with hepatoblastoma; M =  X174Hae III cut size marker, P = positive control, N = negative control, 1~8 = cases, 9 = USM (human hepatoblastoma cell line transplantable into nude mice [11]).
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Expression of TPO, c-mpl, IL-1ß, and IL-6 mRNA in Tumor Tissues
TPO mRNA was expressed in all eight tumor samples examined, while its receptor c-mpl mRNA was expressed in five samples. IL-1ß and IL-6 expression were also observed in all and four patients, respectively ( Fig. 2).
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Figure 2. Relationship between serum TPO level and platelet counts at diagnosis in patients with hepatoblastoma (p = 0.1).
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Discussion
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In this study, the focus was on TPO and the thrombocytosis associated with hepatoblastoma. TPO has been cloned as the cognate ligand for the c-mpl encoded receptor [6-8]. The proto-oncogene c-mpl is a member of the hematopoietin receptor superfamily [16], and TPO acting through c-mpl is essential for normal thrombopoiesis. Experiments using c-mpl antisense oligonucleotide demonstrated the inhibitory effect on megakaryocyte colony formation, whereas erythroid and granulocyte-macrophage colony formations were not impaired [12]. In addition, c-mpl knock-out mice develop severe but nonlethal thrombocytopenia with a reduction in megakaryocytes [17]. TPO induces the proliferation of megakaryocyte progenitors and their differentiation into large polyploid, platelet-producing megakaryocytes. Normally, serum TPO levels are inversely related to the numbers of circulating platelets and of bone marrow megakaryocytes [14]. When platelet levels are high, an increased amount of TPO is taken up by platelets and megakaryocytes, resulting in a decrease in the circulating levels of the cytokine, thereby limiting megakaryocyte production. Increased serum TPO levels were seen in patients with aplastic anemia and acute leukemia whose platelet counts were low because of decreased platelet production. In our study, however, all eight patients with hepatoblastoma demonstrated a high serum TPO level despite their marked thrombocytosis. Additionally, a serial measurement in a patient indicated that TPO levels decreased along with the normalization of platelet counts during sequential chemotherapies. These observations suggest that thrombocytosis in hepatoblastoma resulted from the overproduction of TPO. In a normal physiological state, the major source of TPO production has been identified with the kidney, liver, spleen, and bone marrow [6, 9]. Especially when using an in situ hybridization technique, the strong expression of TPO mRNA was evident in the hepatocytes of the liver from the fetus stage to adulthood [18, 19]. Therefore, it is likely that hepatoblastoma tissues produce an excessive amount of TPO. Meanwhile, other hematopoietic regulators, such as IL-3 [20], IL-6 [21, 22], leukemia inhibitory factor [23], and IL-11 [24], proved to have the potential to increase platelets. In our series, serum levels of IL-1ß and IL-6 were also higher than normal, but TPO appeared to correlate to the number of peripheral platelets. Thus, there is a possibility that multiple cytokines cooperate to induce thrombocytosis in hepatoblastoma. Furthermore, cytokine members including TPO might act as a type of autocrine and/or paracrine system for cellular proliferation in this tumor. Further investigations are of great interest for clarifying the biological significance of these cytokines in the cellular growth of hepatoblastoma.
Histopathologically, hepatoblastoma exhibits a wide range of epithelial and mesenchymal lines of differentiation [25, 26]. The histological classification distinguishes between pure epithelial and mixed hepatoblastoma, the latter containing both epithelial and mesenchymal components. The epithelial hepatoblastomas include the fetal and embryonal subtypes, so named on the basis of histological similarities with the developmental stages of the normal liver. Such a multidirectional spectrum of differentiation has led to the hypothesis that hepatoblastoma derives from a pluripotent stem cell [27, 28]. In this respect, it is considered that our finding of the expression of TPO mRNA in all eight cases and c-mpl mRNA in five tumors is derived not only from hematologic cells comprised in tumor tissues but also from such hepatic stem cells, i.e., tumor cells. Notably, we have confirmed both TPO and c-mpl mRNA expression in human hepatoblastoma xenografts in nude mice ( Fig. 1). Since our primers for human TPO, c-mpl and other cytokines do not crossreact with those of murine, the expression in xenograft must have derived exclusively from human hepatoblastoma cells—but not from murine hematopoietic cells.
Further study needs to be done to clarify the biological significance of TPO in this particular tumor, i.e., how it cooperates with other cytokines on the platelet production and whether it participates in tumor growth.
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accepted for publication July 1, 1998.
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Abstract
Introduction
