Stem Cells, Vol. 18, No. 2, 112-119,
March 2000
© 2000 AlphaMed Press
Effect of Thrombopoietin on Proliferation of Blasts from Patients with Myelodysplastic Syndromes
Shan-Shun Luoa,
Kiyoyuki Ogataa,
Norio Yokosea,
Takashi Katob,
Kazuo Dana
a Third Department of Internal Medicine, Nippon Medical School, Tokyo, Japan;
b Pharmaceutical Research Laboratory, Kirin Brewery Co., Takasaki, Japan
Key Words. Thrombopoietin • Myelodysplastic syndromes • Blast proliferation
Correspondence:
Kiyoyuki Ogata, M.D., Ph.D., Third Department of Internal Medicine, Nippon Medical School, Sendagi 1-1-5, Bunkyo-ku, Tokyo 113-8603, Japan. Telephone: 81-3-3822-2131 (Ext. 6775); Fax: 81-3-5685-1793; e-mail: ogata{at}nms.ac.jp
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Abstract
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Thrombopoietin (TPO), a major cytokine involved in megakaryocytopoiesis/thrombopoiesis, may be effective for treatment of the thrombocytopenia associated with myelodysplastic syndromes (MDS). However, it has been unclear whether TPO stimulates proliferation of MDS blasts, as observed in de novo acute myeloid leukemia. This study examined this concern. When marrow cells from 37 MDS cases were cultured with or without recombinant human PEGylated TPO, TPO increased the blast number (stimulation index 
1.5) in 9 of 16 high-risk MDS cases (refractory anemia with excess blasts [RAEB] and RAEB in transformation) and 4 of 10 cases with MDS transformed to acute leukemia (MDS-AL), but none of 11 cases with low-risk MDS (RA and RA with ringed sideroblasts). When the cell cycle of cultured cells was determined by three-color flow cytometry, TPO activated the cell cycle of MDS cells (causing a decrease in G0-phase cells) in most of the cases whose blast number increased in response to TPO. Reverse transcriptase-polymerase chain reaction analysis detected TPO receptor messenger RNA in purified blasts from all six cases examined, irrespective of the response of their blasts to TPO in culture. Analysis of the patients' characteristics identified a high-serum lactate dehydrogenase (LDH) value as being associated with blast proliferation in high-risk MDS cases (p = 0.0036). We conclude that TPO stimulates in vitro proliferation of blasts from a fraction of MDS patients. High-risk MDS patients, especially those who have a high-serum LDH value, and MDS-AL patients should be monitored with particular care in clinical trials of TPO for MDS.
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Introduction
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Myelodysplastic syndromes (MDS), which are malignant disorders of hematopoietic progenitors, show various degrees of anemia, neutropenia and thrombocytopenia. Manifestations caused by the cytopenia, such as infections and bleeding, and transformation to acute leukemia are the major causes of death in MDS. No effective therapy for MDS has been established yet, except for allogeneic stem cell transplantations which are available for a limited number of patients [1]. Hematopoietic growth factors (HGFs), such as G-CSF, may be effective in preventing infections in neutropenic patients [2]. Thrombopoietin (TPO, mpl-ligand), a major HGF involved in the growth and development of megakaryocytes and platelet production [3-5], is a candidate for treatment of thrombocytopenia in MDS, and a clinical trial of TPO for MDS is now being conducted in Japan.
TPO receptor (TPO-R, c-mpl) is expressed mainly on platelets, megakaryocytic cells and hematopoietic progenitors [6, 7] in normal humans. Data from patients with acute myeloid leukemia (AML) indicate that blasts from a high proportion of AML patients have TPO-R messenger RNA (mRNA) [8, 9] and blasts from a fraction of these patients proliferate in response to TPO in vitro [10, 11]. Therefore, the clinical effect of TPO in AML patients should be carefully monitored. Regarding MDS, such analysis has been rare. By using Northern blotting, Bouscay et al. [12] detected TPO-R mRNA in mononuclear cells (MNCs) from the bone marrow or peripheral blood of 19 of 58 MDS patients. The same group also reported that TPO increased 3H-thymidine uptake by marrow MNCs, which consisted of various cell types responding to TPO, and enhanced cluster formation (probably derived from the MDS clone) induced by combined stimulus with interleukin 3 (IL-3), IL-6, erythropoietin, GM-CSF, and stem cell factor (SCF), in some MDS cases [13]. We have previously detected TPO-R protein, which was able to bind with and degrade TPO, on blasts from a patient with MDS transformed to acute leukemia (MDS-AL) [14].
In this study, we performed in vitro studies to examine whether TPO increases the blast number and activates the cell cycle of blasts from MDS patients. We also sought a correlation between the proliferative response of blasts to TPO and various background characteristics of MDS patients and examined TPO mRNA expression by purified blasts from some patients.
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Materials and Methods
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Subjects
Twenty-seven MDS patients, who were diagnosed by the standard criteria [15], and 10 MDS-AL patients were the subjects in this study (Table 1
). For this study, we classified refractory anemia (RA) and RA with ringed sideroblasts (RARS) as a low-risk MDS, and RA with excess blasts (RAEB) and RAEB in transformation (RAEB-t) as a high-risk MDS.
Cell Preparation
Heparinized bone marrow cells were obtained from the patients after they gave informed consent. MNCs were separated by gradient centrifugation, using Histopaque-1077 (Sigma Chemical Co.; St. Louis, MO). To examine TPO-R mRNA expression, blasts were purified from six cases using immunomagnetic beads conjugated with anti-CD34 antibody (Dynal A.S.; Oslo, Norway) as described previously [16]. The purity of the blasts was 97% or more when determined for Wright-Giemsa-stained cytospin preparations.
Blast Proliferation Assay
The marrow MNCs were suspended in RPMI-1640 medium containing 10% fetal calf serum (5 x 105 cells/ml) and cultured at 37°C in air containing 5% CO2 in the presence of recombinant human PEGylated TPO (Kirin Brewery Co.; Tokyo, Japan) or a mixture of recombinant human HGFs, i.e., 100 ng/ml of G-CSF, IL-3 and SCF (Kirin Brewery Co.). This HGF mixture (HGFs/Mix) is a powerful stimulator of MDS cells, as described previously [17]. The cells were also cultured without any HGFs as a negative control in each case. After the culture, the nucleated cell counts were determined with a hemocytometer, and the percentage of blasts was determined for Wright-Giemsa-stained cytospin preparations. Then the blast number was calculated from these data. The stimulation index (SI) was calculated by dividing the number of blasts in TPO- or HGFs/Mix-stimulated cultures by the number in the negative control culture.
In preliminary experiments, the marrow MNCs were cultured with different concentrations of TPO for various lengths of time. In cases whose blast number increased in response to TPO, the maximum increase was observed at 100 ng/ml of TPO, and the SI values were comparable between 48-h and 96-h cultures. Although the viability of cells was maintained for up to 48 h of culture (97% by the trypan blue exclusion test), the viability decreased when the cells were cultured for 96 h in some cases. Thus, 100 ng/ml of TPO and a 48-h culture were used as the experimental conditions in this study. To examine the reproducibility of this assay, cell cultures not supplemented with any HGF were set in duplicate for 10 cases, and each duplicate culture was arbitrarily labeled A or B at the initiation of culture. When the blast number after the culture was determined, the coefficient of variation, calculated from the mean ± SD of I x II–1, was 14.1% (I = the blast number in culture A of each case, and II = the blast number in culture B of each case).
Cell Cycle Analysis
For this analysis, we selected high-risk MDS cases, whose blasts were positive for CD13. The CD13 positivity of the blasts was identified with antihuman CD13 monoclonal antibody (Becton Dickinson; San Jose, CA) using an immunomagnetic beads method, as described in our previous report [17]. Briefly, the marrow MNCs were reacted with antihuman CD13 monoclonal antibody, followed by incubation with immunomagnetic beads coated with antimouse IgG antibody. The incubated cells were subjected to cytospin preparation and Wright-Giemsa staining. Cases were selected if more than 50% of the blasts were bound to the beads. The reasons for using CD13 to identify the cells to be analyzed are described in detail in the Discussion. MDS-AL cases whose marrow MNCs contained more than 90% blasts were also subjected to the cell-cycle analysis.
The cell cycles of the marrow MNCs cultured for 48 h were determined by three-color flow cytometry, as described previously in detail [17, 18]. Briefly, cells were labeled with antihuman CD13 monoclonal antibody conjugated with fluorescein (this step was omitted for the MDS-AL cases whose marrow MNCs contained more than 90% blasts). After washing, the cells were incubated in a buffer containing 0.004% saponin (Wako Chemical; Osaka, Japan). Then, the DNA and RNA in the cells were stained with 7-aminoactinomycin D (Sigma Chemical Co.) and pyronin Y (Polysciences; Warrington, PA), respectively. The cell cycle (G0-, G1-, S-, and G2/M-phases) of the fluorescein-stained cells (or nonstained cells in the MDS-AL cases) was analyzed with a FACScan (fluorescence-activated cell sorter) flow cytometer using CellFIT and Lysis II softwares (Becton Dickinson). In these analyses, lymphocytes in freshly isolated peripheral blood MNCs from normal volunteers were used as an internal standard for G0 cells, because a majority of unstimulated lymphocytes are, by definition, G0 cells [19, 20]. The coefficient of variation of this cell-cycle analysis method was determined by us and as previously reported [17].
The indices of change in the G0 cell percentage (G0IC) and the S cell percentage (SIC) were calculated with the following formulae: G0IC = G0 cell percentage in experimental culture x G0 cell percentage in control culture–1 x 102, SIC = S cell percentage in experimental culture x S cell percentage in control culture-1 x 102.
Detection of TPO-R mRNA by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
RNA was extracted from the purified blasts of three cell lines by the acid guanidinium thiocyanate-phenol-chloroform method [21]. The cell lines used were Dami cells (American Tissue Culture Collection; Rockville, MD) which express TPO-R mRNA, and K562 and Jurkat cells (RIKEN Cell Bank; Ibaraki, Japan) which do not express TPO-R mRNA [22].
RT-PCR for TPO-R mRNA was performed using an RNA PCR Kit (Ver. 2.1; Takara Shuzo; Otsu, Japan) as described previously [23]. In brief, one µg of total RNA was reverse-transcribed to cDNA in a final volume of 20 µl using 1 mM each of four deoxyribonucleotide 5' triphophates, 20 U RNase inhibitor, 2.5 µM of the random 9 mers, and 5 U AMV reverse transcriptase XL. The reaction was performed for 10 min at 30°C, 30 min at 42°C, 5 min at 99°C, and 5 min at 5°C, and the product was subjected to PCR. PCR was performed in a final volume of 100 µl containing 0.2 µM of the sense primer (nucleotide 843, 5'-TGGAGATGCAGTGGCACTTG-3'), 0.2 µM of the antisense primer (nucleotide 1029, 5'-TGATGTCTGGGGTGTCAAGA-3'), and 2.5 U of Taq polymerase. Amplification was performed for 35 cycles (denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min). Eight-µl aliquots of the products were analyzed by 2% agarose gel electrophoresis. The presence of intact mRNA was verified by concomitant amplification of ß-actin mRNA.
Measurement of Plasma TPO Concentration
Heparinized peripheral blood was drawn from the patients after obtaining informed consent, and the plasma was immediately separated and stored at –20°C until use. Plasma TPO concentrations were determined with a sensitive sandwich enzyme-linked immunosorbent assay, as described in our previous report [14].
Statistical Analysis
Differences between two groups of data for continuous variables were evaluated using the Mann-Whitney-U test. Three or more groups of data for continuous variables were compared by analysis of variance (ANOVA), and when the results were significant, these groups of data were further compared with one another by Scheffe's test. Differences in categorical variables were evaluated using the chi-square test. Definitions of cytogenetic subgroups and prognostic subgroups by the international prognostic scoring system (IPSS) were the same as the reported criteria [24]. Overall survival was calculated from the day of diagnosis until death. The progression-free survival of high-risk MDS was calculated from the day of diagnosis until disease progression (transformation to RAEB-t or MDS-AL in RAEB cases and to MDS-AL in RAEB-t cases). Kaplan-Meier product limit estimates were performed to determine these survivals. A p-value of less than 0.05 was considered to be statistically significant.
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Results
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Effects of TPO on Proliferation of MDS Blasts in Culture
First, we examined whether TPO increases the number of MDS blasts in the cultures. The change in the blast number in response to TPO or the HGFs/Mix, which is expressed as the SI of the blast number, is presented in Table 1
. When the data for low-risk MDS, high-risk MDS and MDS-AL were compared, the increase in the blast number induced by TPO differed significantly among the three groups (p = 0.0355 by ANOVA, p = 0.0119 for low-risk MDS versus high-risk MDS, p = 0.0646 for low-risk MDS versus MDS-AML). The increase in the blast number induced by the HGFs/Mix did not differ significantly among the three patient groups (p = 0.7262 by ANOVA).
When a significant increase in the number of MDS blasts was arbitrarily defined as SI 1.5, the TPO and HGFs/Mix induced a significant increase in 13 cases (35.1%) and 33 cases (89.2%), respectively, among the total of 37 cases. Regarding the MDS subtype, a positive response to TPO (SI 1.5) was observed only in the high-risk MDS and MDS-AL cases (7 of the 11 RAEB cases, two of the five RAEB-t cases, and 4 of the 10 MDS-AL cases), but not in any of the 11 low-risk MDS cases.
To explore whether the increase in the number of MDS blasts is accompanied by cycle activation of quiescent blasts (G0-phase blasts), we performed cell-cycle analysis of the cultured cells. To obtain reliable results, only cases whose bone marrow samples contained a substantial proportion of blasts (high-risk MDS and MDS-AL) were included in this cell-cycle analysis. The cell cycle data of the examined cases are summarized in Table 2, while the indices of the cell cycle (G0IC and SIC) of each case are presented in Table 1.
Figure 1 illustrates the indices of the cell cycle in patients whose blast number increased in response to TPO (SI 1.5) and in patients whose blast number did not increase (SI < 1.5). TPO decreased the G0IC and increased the SIC in most cases belonging to the former patient group. Further, cell-cycle activation was more marked in the former patient group compared with the latter patient group (SIC 200 was observed in 7 of the 12 cases with SI 1.5 but not in any of the six cases with SI < 1.5, p = 0.0167; G0IC 
50 was observed in 5 of the 12 cases with SI 1.5 but not in any of the six cases with SI < 1.5, p = 0.0628).
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Figure 1. TPO-induced cell cycle change (G0IC and SIC) in the cases whose SI of the blast number was 1.5 and in the cases whose SI was <1.5. Circles indicate high-risk MDS and squares indicate MDS-AL.
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Correlation between Increase in Blast Number in Response to TPO and Clinical and Biological Variables
Because TPO induced blast proliferation in a fraction of the high-risk MDS and MDS-AL cases, we examined whether there were any patient background characteristics that correlated with the TPO-induced blast proliferation (Table 3). Regarding the high-risk MDS cases, only the lactic dehydrogenase value differed significantly between the cases whose SI was 1.5 or more and the cases whose SI was less than 1.5 (p = 0.0036, Fig. 2). Regarding the MDS-AL cases, there was no characteristic that differed between the two groups divided according to the SI value.
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Figure 2. Serum LDH values of the high-risk MDS cases whose SI of the blast number was 1.5 and the cases whose SI was <1.5. Serum LDH values were significantly different between the two groups (p = 0.0036).
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The overall survival and progression-free survival did not differ between these two groups in the high-risk MDS cases or the MDS-AL cases (p > 0.2 for all analyses).
Expression of TPO-R Transcripts in MDS Blasts
We were able to purify blasts from six cases (Cases 16, 18, 21, 26, 27 and 36) and subject them to RT-PCR analysis for TPO-R mRNA. Although the in vitro proliferation response of the blasts to TPO differed among these cases (SI of blast number: 1.5 in three cases and <1.5 in the other three cases), TPO-R mRNA was detected in blasts from all six cases (Fig. 3).
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Figure 3. RT-PCR analysis of TPO-R mRNA (t) and ß-actin mRNA (a) for MDS blasts and cell lines. M, D, K and J indicate markers, Dami cells, K562 cells and Jurkat cells, respectively. The numbers indicate the case numbers. The SI of the blasts was 1.5 in Cases 21, 18, and 16 and <1.5 in the other three cases.
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Discussion
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Although a clinical trial of TPO for thrombocytopenia in MDS is being conducted, there are no data which clarify whether TPO induces blast proliferation in vitro and/or disease progression in vivo in MDS patients. A prior report showed that TPO increased 3H-thymidine uptake by bone marrow MNCs in some high-risk MDS cases [13]. However, because the marrow MNCs consist of various types of cells and TPO may stimulate myeloid and erythroid cells as well as megakaryocytic cells [25-27], it is unknown whether TPO induced 3H-thymidine uptake by MDS blasts in that prior report. In this study, we showed that TPO increased the blast number (SI 1.5) in in vitro liquid culture in nearly half of the cases with high-risk MDS and MDS-AL, but not in any of 11 cases with low-risk MDS. In contrast, a combination of HGFs (HGFs/Mix) increased the blast number in most cases, including the low-risk MDS cases. In these experiments, we used a short-term culture (48 h), because viable MDS blasts were not able to be maintained well in longer culture. Therefore, it remains unknown whether long-term stimulus by TPO induces blast proliferation in low-risk MDS cases. Nevertheless, our data suggest that the risk of blast proliferation by TPO is higher in high-risk MDS and MDS-AL than in low-risk MDS.
The increase in blast number by TPO indicates that TPO induces proliferation of blasts in the active cell cycle and/or TPO induces G0-phase blasts to enter the cell cycle and proliferate. Thus, we also examined whether TPO modulates the cell cycle of MDS cells by using the recently developed flow cytometric analysis method [28]. In applying this method, we employed an anti-CD13 antibody, which reacts not only with MDS blasts but also with more mature myeloid cells and monocytic cells, to identify the cells to be analyzed instead of other relatively blast cell-specific antibodies, i.e., anti-CD34 and anti-c-kit antibodies. Our main reason for using anti-CD13 antibody was that the expressions of CD34 and c-kit are restricted to a low percentage of blasts in a limited number of MDS patients [29]. On the contrary, a majority of blasts in most MDS patients expresses CD13 [29]. Moreover, we previously showed that the cell cycle did not differ significantly between CD13+ cells and purified blasts in an MDS case [17]. Our present results also showed that the cell cycle of examined cells from both MDS-AL (whose MNCs consisted of more than 90% blasts in this study) and high-risk MDS cases was stimulated by TPO in most cases whose blast number increased in response to TPO. Taken together, the present data indicate that TPO induces G0-phase blasts to enter the cell cycle and proliferate, at least in some high-risk MDS and MDS-AL cases. In Cases 14 and 36, although their blast number increased in response to TPO, this was not accompanied by a decrease in G0-phase cells. It is probable that in such cases only cycling blasts were stimulated by TPO. In the meantime, it is known that AML blasts produce HGFs which stimulate blast proliferation in some cases [30]. Therefore, another point of interest is whether MDS blasts also produce HGFs which cooperate with TPO in stimulating blast proliferation in our cases. However, we currently have no data elucidating this point.
Identification of patient characteristics that correlate with blast proliferation in response to TPO is essential for safe clinical application of TPO. Our analysis identified the serum LDH value as a predictive factor for the blast response to TPO in high-risk MDS. Other serum enzymes, such as GOT, GPT, and ALP, were not associated with the blast response to TPO in the present subjects (data not shown). Although the mechanism responsible for this association remains unknown, we conclude that patients with high-risk MDS who have a high-serum LDH value should be monitored with particular care in clinical trials of TPO for MDS.
Data regarding the expression of TPO-R by MDS blasts are sparse. By using Northern blot analysis for marrow MNCs, Bouscay et al. [12] detected an elevated TPO-R mRNA level in 11 of 26 patients with RAEB or RAEB-t, but a normal TPO-R mRNA level (faint band by blotting, as observed in the normal marrow samples) in all 14 low-risk MDS patients. In the present study, we obtained purified MDS blasts to minimize contamination by mRNA from other cells. Because the number of purified blasts was small, we used RT-PCR instead of a ligand-binding assay or Northern blotting to examine TPO-R expression. TPO-R mRNA was detected in blasts from all six patients but not in control cells which had been reported to have no TPO-R mRNA. It is noted that the presence of TPO-R mRNA was not related with an in vitro blast response to TPO in our subjects. This finding is consistent with the data for de novo AML, in which an in vitro response of blasts to TPO is not significantly associated with the TPO-R mRNA expression by the blasts [10, 11].
The data generated in this study will be useful for planning clinical trials of TPO for MDS and monitoring patients in such trials. We hope that, in the near future, appropriate use of TPO will contribute to the treatment of this intractable disease.
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accepted for publication on February 14, 2000.
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Abstract
Introduction
