Relation between S-phase fraction of myeloma cells and anemia in patients with multiple myeloma

Experimental Hematology 27 (1999) 1621–1626

Relation between S-phase fraction of myeloma cells and anemia in patients with multiple myeloma Alexander Fossåa,b, Dieter Brandhorsta, June Helen Myklebustb, Siegfried Seebera, and Mohammed Resa Nowrousiana a Department of Medical Oncology, West German Cancer Center, University of Essen, Essen, Germany; Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway

b

(Received 27 October 1998; revised 25 April 1999; accepted 20 July 1999)

In an attempt to define the relation among anemia, tumor mass, and proliferative activity of tumor cells in vivo, we measured the proportion and cell cycle distribution of erythropoietic cells and myeloma cells in the bone marrow of patients with multiple myeloma using four-parameter flow cytometry. Forty-three bone marrow samples from 33 patients with stage II or III disease and normal renal function at diagnosis (n 5 9), in partial remission (n 5 9), and in progression or relapse after chemotherapy (n 5 25) were evaluated. Early and late erythropoietic cells were discriminated based on published light scatter properties in combination with CD71 expression. Myeloma cells were detected by exploiting their strong CD38 positivity and light scatter characteristics. Cell cycle distribution of the three cell populations was determined by propidium iodine staining. In the whole group of patients, hemoglobin (Hb) concentration was inversely correlated with b2-microglobulin (p 5 0.03), percentage of marrow CD3811 cells (p 5 0.008), and percentage of CD3811 cells in S phase (S-CD3811; p , 0.001). Partial correlation analysis revealed S-CD3811 to be the only independent predictor of Hb concentration (p , 0.001). No correlation was found between Hb concentration and the S-phase fraction of erythropoietic cells. In the subgroup of patients with moderate to severe anemia, defined as Hb concentration ,11 g/dL, Hb level correlated negatively only with S-CD3811 (p , 0.001) but not with b2-microglobulin and percentage of marrow CD3811 cells. In addition, Hb and the S-phase proportion of early erythropoietic cells correlated positively (p 5 0.029). The strong inverse correlation between Hb concentration and percentage of myeloma cells in S phase suggests that in multiple myeloma, tumor proliferative activity may have a more important impact on the development of anemia than tumor mass. The S-phase fraction of tumor cells appears to be the most important pathogenic factor, especially in anemic patients. In these patients, the positive relation between Hb concentration and the S-phase fraction of erythro-

Offprint requests to: Mohammed Resa Nowrousian, Department of Medical Oncology, West German Cancer Center, University of Essen, Hufelandstraße 55; D-45147 Essen, Germany; E-mail: nowrousian@ uni-essen.de

poietic progenitors indicates that development of anemia is associated with inhibition of erythropoiesis. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Myeloma—Proliferation—Anemia—Erythropoiesis

Introduction Multiple myeloma is a B-lineage lymphoproliferative disease in which tumor cell proliferation is thought to be influenced by a large number of cytokines acting in autocrine or paracrine fashion on myeloma cells [1,2]. The proliferative activity of myeloma cells generally is assessed as the labeling index obtained after incubation of cells in the presence of tritium-labeled thymidine or bromodeoxyuridine [3,4]. These techniques are time consuming, relatively complex, and generally do not allow simultaneous analysis of malignant cells and subpopulations of residual normal bone marrow cells. Immunophenotypical studies by flow cytometry have revealed normal and malignant plasma cells to express high levels of the CD38 antigen, which allows clear distinction between these cells and other bone marrow cells [5,6]. Simultaneous staining for DNA with propidium iodide (PI) and the CD38 antigen has been used to calculate the cell cycle distribution of myeloma cells [6]. The technique has been reported to be relatively easy to perform and reproducible, and it offers the possibility of simultaneously studying the proliferative activity of different subpopulations of residual bone marrow cells [6,7]. Despite methodologic differences, both labeling index and flow cytometric assessment of the S-phase fraction of myeloma cells have been shown to be important markers of biologic behavior and prognosis in multiple myeloma [3,4,7,8]. Anemia is a frequent complication of multiple myeloma, causing morbidity and reduced quality of life [9,10]. The pathogenesis of anemia associated with multiple myeloma is not completely understood [11,12]. Possible mechanisms

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include displacement of normal bone marrow by myeloma cells, inadequate erythropoietin production, and suppression of erythropoiesis by cytokines, renal failure, hemodilution, and effects of treatment. Great emphasis has been put on the contribution of cytokines capable of directly or indirectly inhibiting erythropoiesis, such as interleukin 6 (IL-6), interleukin 1 (IL-1), tumor necrosis factor (TNF), and interferon gamma (IFN-g) [12]. Furthermore, a reduced erythropoietin production has been observed both in patients with reduced and normal renal function [13]. IL-1, TNF, and IFN-g have been shown in vitro to inhibit proliferation of erythropoietic precursor cells, either at the level of burst-forming unit erythroid (BFU-E) or colony-forming unit erythroid (CFU-E) or both, and clinical studies have linked the serum levels of IL-1 and TNF to the development of anemia [14]. However, the effect of the complex and aberrant cytokine network associated with multiple myeloma on erythropoietic progenitor cells in vivo is largely unknown. In an attempt to study the influence of cell cycle distribution of myeloma cells on the development of anemia, we studied bone marrow cells from patients with multiple myeloma using four parameter flow cytometry and CD38/PI double staining. Simultaneous CD71/PI double staining was used to study the in vivo cell cycle distribution of erythropoietic progenitor cells.

Patients and methods Patients After obtaining informed consent, a total of 43 bone marrow samples were obtained from 33 anemic and nonanemic patients with multiple myeloma (Table 1). Diagnostic criteria included detectable monoclonal serum or urine myeloma protein and marrow plasmocytosis .20% with or without lytic bone lesions [15]. Staging was performed according to Durie and Salmon [16]. Further inclusion criteria were a normal serum creatinine level and readily detectable iron stores in bone marrow reticulum cells as judged by iron staining of the bone marrow smears. Patients with bone marrow infiltration by myeloma cells with low or absent CD38 expres-

Table 1. Patient characteristics No. of patients Sex (male/female) Paraprotein IgG/IgA Bence Jones proteinuria Nonsecretory Age of patients [years; median (range)]* Stage (IIA/IIIA)* b2 microglobulin [g/L; median (range)]* Treatment status* No chemotherapy In partial remission At relapse or progression *At time of sampling.

33 18/15 22/8 2 1 58 (41–72) 11/32 2.4 (0.7–12.1) 9 9 25

sion were excluded. Sampling was performed twice in 4 patients, three times in 2 patients, and four times in 1 patient. Repeated sampling from each patient occurred at different time points of the disease, i.e., at diagnosis, in remission, or at later relapse or progression. Nine samples originated from patients at diagnosis, 9 from patients in partial remission [15], and 25 from patients in progression or relapse after previous chemotherapy. Chemotherapy consisted of standard melphalan and steroids or conventional dose combination chemotherapy in all but two patients who had a relapse after high-dose melphalan with autologous peripheral stem cell transplantation. No patient had received blood transfusions within the prior 4 months, and no erythropoietin treatment had been given. On the day of bone marrow sampling, the following routine laboratory tests were performed in blood or serum: complete blood count, total serum protein, serum protein electrophoresis, creatinine, urea, calcium and b2-microglobulin. The latter was determined by means of the AUTODELFIA™ b2-micro kit (WallacADL-GmbH, Germany). Normal bone marrow was obtained from volunteer bone marrow donors at the Department of Bone Marrow Transplantation, West German Cancer Center, University of Essen. Cell preparation and flow cytometry Bone marrow aspirates were obtained by puncture of the iliac crest and sodium citrate 3.13% (w/v) added to prevent clotting. The samples were diluted 1:1 in phosphate-buffered saline (PBS) at pH 7.2. Aliquots of 100 mL containing approximately 106 nucleated cells were incubated for 30 minutes at room temperature in the dark with FITC-labeled anti-CD38 (Dianova-Immunotec GmbH, Hamburg, Germany) or FITC-labeled anti-CD71 (Dianova-Immunotec GmbH) antibodies, respectively. After washing in PBS, the cells were resuspended in 75 mL of fetal calf serum and processed with the DNA-Prep system (Coulter Electronics GmbH, Krefeld, Germany) containing RNase and PI according to the manufacturer’s instructions. Quantitative fluorescence analysis and cell sorting were performed within 30 minutes on an Coulter Epics CS flow cytometer (Coulter-Electronics GmbH, Krefeld, Germany) that was equipped with an Innova 90 laser at 488 nm (Coherent, Rödermark, Germany). Fluorescence compensation between FITC and PI was established using DNA-Check FITC-beads (Coulter Electronics GmbH) and PI-stained chicken erythrocyte nuclei included in the DNA-Prep kit. A minimum of 105 cells were counted per sample. The percentage of CD38 strong positive myeloma cells (CD3811 cells) was determined as described by Orfao et al. [6]. Within the group of CD71 strong positive cells, two different populations were differentiated based on forward and side scatter properties (Fig. 1). CD71 strong positive cells with light scatter properties similar to lymphocytes have been reported to represent mainly mature erythropoietic cells, i.e., normoblasts and polychromatic erythroblasts (herein referred to as M-CD7111 cells) [17]. CD71 strong positive cells with physical properties characteristic of the blast window of normal bone marrow (referred to as I-CD7111 cells) have been shown to consist mostly of immature erythropoietic cells, i.e., proerythroblasts and basophilic erythroblasts [17]. Cell cycle analysis Cell cycle analysis was performed separately for CD3811, M-CD7111, and I-CD7111 cell gates as described earlier, counting

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for CD3811, M-CD7111, and I-CD7111 cells were below 3% in all cases analyzed. An illustration of DNA measurement in an aneuploid case of multiple myeloma is shown in Figure 1. For six samples the number of CD3811 cells in the bone marrow sample was too low for cell cycle analysis that could not be overcome by counting a larger number of cells. Four of these patients were in remission after chemotherapy, and two had multifocal osteolytic bone lesions but no detectable bone marrow infiltration in a biopsy taken at the same time as the FACS analysis. Morphologic studies For morphologic analysis, bone marrow aspirates with 3.13% (w/ v) sodium citrate from normal volunteers or patients were diluted 1:20 in PBS and mononuclear cells prepared by density centrifugation in lymphodex (Fresenius, Bad Homburg, Germany). Cells were resuspended in PBS and labeled with anti-CD38 or antiCD71 antibodies as described earlier. After washing, cells were sorted on the Coulter Epics CS flow cytometer according to the gates for CD3811, M-CD7111, and I-CD7111 cells given earlier. Duplicate cytocentrifuge slides were prepared for each sorted fraction of cells. The slides were air dried and stained with WrightGiemsa solution. Differential counts were made for each sample. Statistics To estimate the significance of differences between groups of patients, the Mann-Whitney U -test was used. To test for the relationship between variables, Spearman’s rank correlation coefficient was calculated either as bivariate or partial correlation coefficients. All statistical analyses were performed using SPSS 7.0 for Windows (SPSS Inc., Chicago, IL).

Results

Figure 1. Flow cytometric detection of mature (M-CD7111) and immature (I-CD7111) erythropoietic cells and CD3811 myeloma cells and distribution of DNA content in bone marrow of a patient with multiple myeloma. (A) Whole bone marrow with an aneuploid G0/G1 peak detectable along with euploid residual bone marrow cells. The definition of M-CD7111 cells and I-CD7111 cells within the lymphocyte and blast region of normal bone marrow and DNA distribution are shown in (B) and (C), respectively. (D) Definition of CD3811 myeloma cells and their distribution of DNA content. The cell cycle distribution of each cell population was as follows. M-CD7111: G0/G1: 96.6%; S: 2.0%; G2/M: 1.4%. I-CD7111: G0/G1: 33.0%; S: 31.4%; G2/M: 35.6%. CD3811: G0/G1: 93.8%; S: 3.4%; G2/M: 2.8%.

a minimum of 104 cells per gate using the Epics CS Flow cytometer and the Para 1 software program (Coulter Electronics GmbH). In brief, the Para 1 software estimates the parameters for gaussian distribution of the Go/G1, G2/M, and, if detectable, a third peak from a least square analysis of the actual DNA histogram. Any skewness resulting from overlap of Go/G1 and G2/M peaks with S-phase cells is corrected for by using the left-hand side of the Go/G1 distribution or the right-hand side of the G2/M distribution for the least square analysis. The range of 2 SD was used for fitting. The need for doublet correction, indicated by the presence of triplet peaks in the DNA histogram, was not encountered. The coefficients of variation

Influence of treatment on cell kinetic parameters To exclude an effect of treatment on the laboratory and cell kinetic parameters tested in this study, samples derived from patients at diagnosis (n 5 9) and patients with prior chemotherapy (n 5 34) were compared. There was no significant difference between the two groups with regard to hemoglobin concentration, b2-microglobulin level, percentage of CD3811 myeloma cells, S-phase fraction of mature or immature erythropoietic, cells or any of the routine laboratory parameters. S-phase fraction of CD3811 cells was significantly higher in pretreated patients; however, this difference was only evident for patients not responding to chemotherapy, whereas patients in remission or at relapse after a previous response had similar values as untreated patients. Therefore, a treatment effect was not evident, and all parameters could be analyzed for untreated and previously treated patients together. Morphologic studies CD3811 cell fractions were sorted in 12 patients and routinely consisted of .98% myeloma cells, which is consistent with results reported by others [6]. M-CD7111 and I-CD7111 cell fractions were sorted from four patients and three normal bone marrow samples. Differential counts of a

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Table 2. Distribution of morphologically identifiable cells in normal bone marrow sorted according to light scatter properties and CD71 positivity Gate Proportion in gate* Cell type† Undifferentiated blasts Proerythroblasts Basophilic erythroblasts Polychromatophilic erythroblasts Eosinophilic erythroblasts Lymphocytes Myeloblasts Promyelocytes Myelocytes Metamyelocytes Rods Neutrophils Basophils Eosinophils Monocytes/monoblasts Plasma cells

M-CD7111

I-CD7111

3.4

1.9

2 2 8 15 55 16 ,1

12 38 30 12 ,1 ,1 3 ,1 ,1 ,1 1 ,1 2

Gates M-CD7111 and I-CD7111 are described in the legend to Figure 1. *Numbers are given in percent of all cells and represent the average of two separate sample preparations. † Numbers are given as percentage of cells within the corresponding gate and represent the average cell counts of two independent experiments. Numbers are rounded to the nearest whole figure; therefore, sums for each gate may exeed 100. No value is given when no cell of the corresponding type was detected.

representative example of normal bone marrow is shown in Table 2. CD7111 cell sorting in myeloma patients yielded similar results, with ,3% myeloma cells in the M-CD7111 and I-CD7111 gates (data not shown). Factors influencing anemia As a first step, the influence of routine clinical parameters and the cell kinetic parameters on hemoglobin level in all

patients was examined (Table 3A). A significant negative correlation with hemoglobin was found for b2-microglobulin, the percentage of CD3811 cells in the bone marrow, and the S-phase fraction of CD3811 cells (S-CD3811). The other laboratory parameters and the S-phase fractions of mature and immature erythroid progenitor cells were not significantly correlated with hemoglobin level. A partial correlation analysis incorporating the b2-microglobulin level, and the percentage of CD3811 cells and S-CD3811 revealed that the latter was the only parameter independently related to hemoglobin. A separate analysis was performed in patients with moderate to severe anemia, which was defined as a hemoglobin value ,11 g/dL (Table 3B). Fourteen patients had a hemoglobin value ,11 g/dL at the time of sampling. In this group of patients, S-CD3811 again was significantly negatively correlated with hemoglobin, whereas the S-phase fraction of immature erythroid progenitors (S-I-CD7111) was positively correlated with the degree of anemia. The percentage of marrow CD3811 cells, the fraction of mature erythroid precursor cells in S-phase, b2-microglobulin, and the clinical parameters tested were not significantly correlated with anemia. In this group of patients, there was a weak negative correlation between S-CD3811 and S-I-CD7111 (r 5 20.62; p 5 0.051). The mutual relations among hemoglobin, S-CD3811, and S-I-CD7111 for anemic patients are depicted graphically in Figure 2.

Discussion Our results show that, among the parameters tested, the proportion of myeloma cells in S-phase is closely and independently correlated with the degree of anemia in patients with multiple myeloma. Interestingly, factors believed to reflect the total tumor burden in patients with normal renal function, such as b2-microglobulin or myeloma cell infiltration

Table 3. Results of correlation analysis for biochemical and cell kinetic parameters with hemoglobin concentration Partial correlation coefficient

Spearman’s correlation coefficient A: All patients (n 5 43) CD3811 cells in bone marrow (%) CD3811 cells in S phase (%) b2 microglobulin (g/L) Immature CD7111 cells in S phase (%)

20.40 20.57 20.47 0.19

p 5 0.008 p , 0.001 p 5 0.003 NS

20.32 20.60 20.28

p 5 0.082 p , 0.001 p 5 0.128

Spearman’s correlation coefficient B: Patients with anemia (n 5 14) CD3811 cells in bone marrow (%) CD3811 cells in S phase (%) b2 microglobulin (g/L) Immature CD7111 cells in S phase (%)

20.38 20.92 20.36 0.69

NS p , 0.001 NS 0.029

Only factors with significant correlation are given. A: All patients. B: Patients with anemia defined as a hemoglobin concentration ,11 g/dL. In this group, no partial correlation coefficient was calculated due to the low numbers of samples. NS 5 nonsignificant.

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Figure 2. Scatter plots for mutual correlation of hemoglobin concentration, S-phase fraction of CD3811 myeloma cells, and S-phase fraction of immature erythropoietic (I-CD7111) cells. (A) S-phase fraction of CD3811 myeloma cells vs hemoglobin concentration (r 5 20.92; p , 0.001). (B) S-phase fraction of I-CD7111 cells vs hemoglobin concentration (r 5 0.69; p 5 0.029). (C) S-phase fraction of I-CD7111 cells vs S-phase fraction of CD3811 myeloma cells (r 5 20.62; p 5 0.051).

of the bone marrow, did not appear to be as closely associated with the hemoglobin concentration. It is well recognized that anemia may occur in myeloma patients, even in the absence of massive bone marrow infiltration or low neutrophil or platelet counts. Similarly, patients with extensive infiltration of the bone marrow need not be anemic [11].

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The finding that the S-phase fraction of myeloma cells correlates with the degree of anemia suggests that pathogenic mechanisms determining myeloma cell proliferation may be linked to the development of anemia. Myeloma cell proliferation is thought to be regulated by a complex and aberrant cytokine network. The role of cytokines in the pathogenesis of monoclonal gammopathies, such as multiple myeloma, has been reviewed [1,2]. IL-6 of autocrine or paracrine origin appears to be the major growth factor for malignant plasma cells. A number of other cytokines are known to be frequently overexpressed in tumor samples from patients with multiple myeloma, such as IL-1, transforming growth factor b, granuloctye colony-stimulating factor, and TNF [18–20]. Cytokine loops have been proposed in which IL-1 and TNF, produced by myeloma cells, induce IL-6 production by the tumor cells themselves or the bone marrow microenvironment [20,21]. Erythropoietic progenitor cells in the bone marrow are exposed to the same cytokine network, and, as outlined earlier, IL-1 and TNF have been implicated in the development of anemia through suppression of erythropoiesis. Although shown to support the growth and differentiation of BFU-E in vitro [22], IL-6 may contribute to anemia through other hitherto unknown mechanisms. In earlier studies on prognostic factors in multiple myeloma, anemia was consistently found to be an independent indicator of poor prognosis [10,16]. More recent studies incorporating cell kinetic parameters show tumor proliferative activity assessed in the form of labeling index or proportion of cells in S-phase to be one of the most important prognostic factors [23,24]. Cell kinetic parameters have been included in simple and powerful prognostic models for this disease [7,23,24]. The results of the present study explain why anemia may lose prognostic significance when cell kinetic parameters are included in the analysis. Interestingly, in low tumor mass asymptomatic myeloma patients, mild anemia was found to independently predict the time to disease progression [25]. This clinical observation also may be due to the close relationship between anemia and myeloma cell proliferation. Anemia in patients with multiple myeloma can be corrected in up to 85% of cases by treatment with erythropoietin [26–28]. Because the treatment is costly, factors predicting the likelihood of response are important so as to tailor treatment and reduce costs. Some pretreatment parameters predicting a response have been identified, including low relative erythropoietin concentrations and serum levels of TNF and IL-1 [14,27,28]. The strong correlation of S-phase fraction of myeloma cells and anemia suggests that cell kinetic parameters may be valuable predictors of response to erythropoietin. To our knowledge this has not yet been formally tested. Based on the limited number of patients studied, the present results show a decreased proliferation in vivo of early erythroid progenitor cells in patients with anemia as-

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sociated with myeloma. Anemic myeloma patients have been shown to have a reduced number of circulating BFU-E [29]. However, these early erythroid progenitor cells display highly variable responses to erythropoietin in vitro, ranging from markedly reduced to normal [30]. These experiments may not be representative because the progenitor cells are isolated from their bone marrow microenvironment, which may suppress their responsiveness to growth signals in vivo. In a larger study on untreated myeloma patients, SanMiguel et al. [7] demonstrated decreased proliferation of normal residual bone marrow cells in patients with anemia. Using a modification of the technique described by SanMiguel et al., we analyzed the proliferative activity of CD71 strong positive blast cells, which morphologically contained undifferentiated blast cells, proerythroblasts, and basophilic erythroblasts. According to Loken et al. [17], this cell population contains the majority of bone marrow BFU-E and CFU-E. However, the results of the present study were obtained from a patient population that includes patients who underwent prior chemotherapy. Although no effect of treatment on the cell kinetic parameters could be demonstrated, the results need to be confirmed in a larger number of untreated myeloma patients. References 1. Lust JA (1994) Role of cytokines in the pathogenesis of monoclonal gammopathies. Mayo Clin Proc 69:691 2. Klein B (1995) Cytokine and cytokine receptors in human multiple myeloma. In: Malpas JS, Bergsagel DE, Kyle RA (eds.) Myeloma biology and management. Oxford: Oxford University Press, pp. 63–81 3. Durie BGM, Salmon SE, Moon TE (1980) Pretreatment tumour mass, cell kinetics and prognosis in multiple myeloma. Blood 55:364 4. Greipp PR, Kyle RA (1983) Clinical, morphological, and cell kinetic differences among multiple myeloma, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma. Blood 62:166 5. Harada H, Kawano MM, Huang N, Harada Y, Iwato K, Tanabe O, Tanaka H, Sakai A, Asaoku H, Kuramoto A (1993) Phenotypic difference of normal plasma cells from mature myeloma cells. Blood 81:2658 6. Orfao A, Garcia-Sanz R, Lopez-Berges MC, Vidriales MB, Gonzalez M, Caballero MD, SanMiguel JF (1994) A new method for the analysis of plasma cell DNA content in multiple myeloma samples using a CD38/propidium iodide double staining technique. Cytometry 17:332 7. SanMiguel JF, Garcia-Sanz R, Gonzalez M, Moro MJ, Hernandez JM, Ortega F, Borrego D, Carnero M, Casanova F, Jimenez R, Portero JA, Orfao A (1995) A new staging system for multiple myeloma based on the number of S-phase plasma cells. Blood 85:448 8. Greipp PR (1994) Prognosis in multiple myeloma. Mayo Clin Proc 69:895 9. Riccardi A, Gobbi PG, Ucci G, Bertoloni D, Luoni R, Rutigliano L, Ascari E (1991) Changing clinical presentation of multiple myeloma. Eur J Cancer 27:1401 10. Medical Research Council’s Working Party on Leukemia in Adults (1980) Prognostic features in the third MRC myelomatosis trial. Br J Cancer 42:831 11. Meharchand JM (1995) Management of haematological complications of myeloma. In: Malpas JS, Bergsagel DE, Kyle RA (eds.) Myeloma biology and management. Oxford: Oxford University Press, pp. 353–374 12. Beguin Y (1995) Erythropoiesis and erythropoietin in multiple myeloma. Leuk Lymphoma 18:413

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