Leukemia Research Vol. 16, No. 5, pp. 529--535, 1992. Printed in Great Britain.

0145-2126/92 $5.00 + .00 © 1992 Pergamon Press Ltd

IN V I T R O SUSPENSION C U L T U R E REACTIONS TO 1,25 D I H Y D R O X Y V I T A M I N D3 IN R E L A T I O N TO BONE M A R R O W M O R P H O L O G Y A N D PROGNOSIS IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES EVA HELLSTROM-LINDBERG,* KARL-HENRIK ROBI~RT,* BIRGITTA /~HNSI~N,t ANNIKA ELMHORN-ROSENBORG,:~ YVONNE KOCK§ and/~KE OST§ tDepartment of Medicine and tDepartment of Immunology at Huddinge University Hospital, Department of Pathology at ,Huddinge University Hospital and §Karolinska Hospital, Stockholm, Sweden (Received 5 November 1991. Accepted 16 December 1991) Abstract--Thirty-four patients with MDS or AML following MDS were studied with regard to survival, peripheral blood values and bone marrow morphology. The effects of 1,25 dihydroxyvitamin D3 (D3) on differentiation (NBT positivity) and proliferation (3H-thymidine incorporation) were studied in suspension cultures of bone marrow cells. Twelve bone marrow donors served as controls. Normal cells showed spontaneous differentiation in vitro, but only 2/12 were induced to differentiation by D3. Myelodysplastic cells did not differentiate spontaneously, but cells from 18/34 patients differentiated after incubation with D3. Normal cells showed increased proliferation, myelodysplastic cells showed a heterogeneous response and leukemic cells reacted with decreased proliferation after D3 incubation. Poor survival was associated with low platelet counts, high percentage of bone marrow blasts (BM blast %), low spontaneous in vitro proliferation and absence of hypogranulation of myeloid cells. Platelet counts and hypogranulation retained their predictive value in a multi-variate analysis. Progression to AML was predicted by a high BM blast % and low scores for erythroid and total dysplasia. In conclusion, the pattern of in vitro proliferation showed prognostic value while the pattern of vitamin D3-induced differentiation failed to correlate to other parameters. An estimation of bone marrow dysplasia can be used to predict the development of AML. Our results add to the information about the biology of MDS and may be important for the evaluation of therapeutic trials. Key words: Myelodysplastic syndromes, proliferation, differentiation, 1,25 dihydroxyvitamin D3, morphology, prognosis.

INTRODUCTION

[5] and scoring systems such as the Bournemouth score [6] and the system presented by Sanz et al. [7] have been used to predict the prognosis of these patients. Other studies [8, 9] have shown that erythroid hyperplasia and ineffective erythropoiesis are less common in R A E B and RAEB-t than in R A and RAS and that patients with a low percentage of bone marrow erythropoiesis show poor survival. Proliferation and differentiation of myelodysplastic cells from bone marrow and peripheral blood have been studied both in suspension cultures [10-13] and in colony systems [8, 14-16]. Colony studies show that the spontaneous growth patterns of both myeloid and erythroid progenitors are correlated to the FAB classification and to prognosis. Undetectable myeloid colony growth in cultures from peripheral blood is for example a strong indicator of poor prognosis [16]. The clinical significance of suspension culture reactions have not been evaluated to the same extent.

PATIENTS with myelodysplastic syndromes (MDS) are heterogeneous with regard to clinical features, survival and response to different treatment alternatives. Several methods, such as the FAB classification [1, 2], cytogenetics [3, 4], bone marrow morphology Abbreviations: MDS, myelodysplastic syndromes; RA, refractory anemia; RAS, refractory anemia with sideroblasts; RAEB, refractory anemia with excess of blasts; RAEB-t, refractory anemia with excess of blasts in transformation; CMML, chronic myelomonocytic leukemia; AML, acute myelogenous leukemia; AML-m, acute myelogenous leukemia following MDS; FAB, the FrenchAmerican-British classification of MDS; 1,25 D3, 1,25 dihydroxyvitamin D3; NBT, nitro blue tetrazolium; 3HTdR, incorporation of tritiated thymidine. Correspondence to: Dr Eva Hellstrfm-Lindberg, Department of Medicine, Huddinge University Hospital, 14186 Huddinge, Sweden. 529

530

E. HELLSTROM-LINDBERGet al. TABLE 1. M D S SUBTYPES, MEDIAN VALUES FOR AGE (YS), HEMOGLOBIN (G/L), GRANULOCYTE AND PLATELET COUNTS (X 109/L), BONE MARROW BLASTS (%), SURVIVAL (MONTHS) AND DEVELOPMENT OF AML (NO AND %) FOR ALL 34 PATIENTS

FAB class

No

Age

Hb

Granutoc.

Platelets

Blasts

Surv.

AML

(% AML)

RA RAS RAEB CMML RAEB-t MDS-AML All patients

2 4 17 4 2 5 34

66 72 72 73 74 75 72

96 78 78 112 97 85 87

3.3 1.6 1.7 8.8 0.25 6.6 1.75

155 292 110 168 75 56 112

4 2.5 9 8.5 21 37 9

32.5 16.5 31 42 28 5 28

0 0 5 2 2

(0) (0) (29.4) (50) (100)

9/29

(31)

A low s p o n t a n e o u s D N A synthesis [17] has b e e n shown to be correlated to a p o o r prognosis but the role of differentiation capacity in these cultures has not been clarified. Vitamin D3 has b e e n r e p o r t e d to alter colony growth and to induce differentiation in suspension [11, 18-20]. It has also b e e n suggested that studies of in vitro reactions to various substances could be a way of predicting response to various drugs. H o w e v e r , only a few reports have s u p p o r t e d this hypothesis [21, 22]. In a previous study [13] we s h o w e d that myelodysplastic patients could be divided into those with b o n e m a r r o w cells showing in vitro differentiation in response to several drugs and those showing no differentiation at all. Vitamin D3, in this study, was a m o r e p o t e n t differentiation inducer than retinoic acid, a-interferon and cytosine arabinoside. The aim of the present study was to assess the clinical significance of vitamin D 3 - i n d u c e d proliferation and differentiation of myelodysplastic b o n e m a r r o w cells in suspension cultures. T h e results of the in vitro assays were therefore related to peripheral b l o o d values, b o n e m a r r o w m o r p h o l o g y , patient survival and d e v e l o p m e n t of A M L .

MATERIALS AND METHODS Patients The patients were classified according to the FAB system. Twenty-nine had MDS and 5 had AML following MDS (MDS-AML). The patients with MDS-AML all had a history of MDS of I>4 months prior to AML development. Two of the patients with AML had 30% blasts in the peripheral blood. Seventeen of the patients were females. All patients had clinical symptoms related to cytopenia. The patients were previously untreated. The median time from diagnosis of MDS to inclusion into the study was 5 months. Hemoglobin level, platelet count, granulocyte count and bone marrow morphology were analyzed on the same day as bone marrow was taken for the in vitro assay. Survival was estimated from the date of diagnosis. Table 1 shows MDS subtypes, median values and ranges for age, per-

ipheral blood counts, survival and development of AML. A Bournemouth score was calculated for each patient with MDS. All patients were observed until death or as a minimum 18 months after the in vitro analysis. Bone marrow morphology Bone marrow smears and clot preparations were made on bone marrow from all patients. All bone marrow samples were analyzed by a morphologist who did not know about the outcome of the in vitro tests or the clinical course of the patients. Bone marrow samples were assessed for percentage of blasts, cellularity, percentage of erythropoiesis, dysplastic scores, sideroblasts and fibrosis. Dysplastic scores were calculated for two variables in each poiesis as described in Table 2. For each variable, the score could vary between 0 and 2 and the total dysplasia score could reach a maximum of 12. The presence or absence of fibrosis was estimated on sections of the clot preparation, stained with silver impregnation. Suspension culture Bone marrow obtained by aspiration was anticoagulated with heparin and layered on a single step Ficoll-Isopaque gradient (1077g/ml) to remove mature erythroid and myeloid cells. The mononuclear cells obtained from the interface of the gradient were washed and resuspended in Eagle's essential medium. 1,25 dihydroxyvitamin D3 (kindly provided by Roche Co.) was suspended and stored in absolute alcohol at an initial concentration of 10 -3 M. For each assay, the drugs were diluted in medium to provide a concentration of 10 -7 M. The final ethanol concentration did not exceed 0.1%. Manipulations with vitamin D3 were performed in subdued light. Mononuclear bone marrow cells were plated at a concentration of 5 x 105 ml Eagle's medium supplemented with 10% human AB serum. The cell suspensions were plated in 3 ml portions on 35 mm Nunc's multidish 6, containing either medium supplemented with 10% human AB serum or 1,25 dihydroxyvitamin D3 10 -7 M. The culture plates were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air for 4 days. After incubation and removal of the cells, the plates were checked under light microscope and the amount of remaining adherent cells was found to be negligible. Cell viability was defined as the percentage of viable cells in the cultures on day 4 as compared to day 0. Nonviable cells were excluded by the trypan blue method. Bone marrow samples from 12 healthy bone marrow donors served as controls.

In vitro reactions to vitamin D3 in relation to morphology and prognosis in MDS

531

TABLE 2. BONE MARROW MORPHOLOGY. MEDIAN VALUES AND RANGES FOR THE PERCENTAGES OF BLASTS, ERYTHROPOIESIS AND CELLULARITY, NUMBER OF POSITIVE/TOTAL PATIENTS FOR FIBROSIS AND RING SIDEROBLASTS. SCORES FOR DYSPLASIA

Assay

Median value (range)

Percentage of Blasts Cellularity Erythropoiesis Presence of Fibrosis* Ringed sideroblasts* Lineage dysplasia Monolinear Bilinear Trilinear Erythroid dysplasia Total Megaloblastic Abnormal nuclei Myeloid dysplasia Total Hypogranulation Pseudopelger Megacaryocytic dysplasia Total Small megacaryocytes Hypolobation

Pos/total

9 (2-38) 80 (60-100) 23.5 (1-90) 16/34 13/34 1/34 15/34 18/34 21/34 15/34 17/34 30/34 23/34 23/34 32/34 32/34 32/34

* Presence or absence of ring sideroblasts and fibrosis, irrespective of percentage. Differentiation assays Nitro blue tetrazolium (NBT) reduction was performed using the standard method [23]. The total number/ml of positive cells was estimated from 200 cells on day 0 and day 4. An increase of NBT-positive cells was defined as an increase of >50 x 103 cells/ml. In order to estimate the percentage of erythropoietic cells in the suspension cultures, differentiation counts on day 0 and on day 4 were made on cytospin preparations from 5 cases. 3H- TdR incorporation Aliquots containing 1.0 x 105 cells in 0.2 ml medium were incubated in triplicates on microplates (Nunc's microdishes), with 1 IxCi (20 Ixl of 50 ~tCi/ml) 3H-labeled thymidine for 24 h at 37°C. Incubation was initiated at day 0, 2 and 4. 3H-labeled DNA on day 1, 3 and 5 was measured by harvesting the aliquots with a Titertek Cell Harvest (Skatron, Norway), absorbing the cells on pieces of filter which were put in a scintillation fluid (2 ml toluene Merch D-6100 with Permablend from a stem solution that was made from 2.51 of toluene and 13 g of Permablend). The radioactivity was measured with a liquid scintillation spectrometer (Beta-counter). Statistical methods The results from the in vitro assays were compared using Wilcoxon's signed rank test and the Mann-Whitney U-test. Correlations between different parameters was measured using Pearson's method. Bi-variate and multi-variate Cox regression analysis was used to predict survival. Factors

predicting AML evolution from MDS were estimated using Fisher's exact test. RESULTS Clinical outcome

Patient data are shown in Table 1. Twenty-four patients died during the time of observation and the median survival of all patients was 28 months. Nine of the 29 patients with MDS progressed into acute myelogenous leukemia. Obtained variables were analyzed both for the MDS patients separately and for all patients. Exclusion of the A M L patients did not change the outcome of the statistical analyses. The results are therefore shown for all 34 patients. Morphological analysis

Results are shown in Table 2. Two patients had monolinear, 14 had bilinear and 18 had trilinear dysplasia. Dysplastic features in the megacaryocytic lineage were observed in 32/34 patients. Myeloid dysplasia was observed in 30/34 patients and erythroid dysplasia in 22/34 patients. Three of the patients who lacked erythroid dysplasia had less than 5% erythropoiesis in the bone marrow, the other 9 patients had between 13 and 42%. The median value

532

E. HELLSTROM-LINDBERG et al. TABLE 3. RESULTS FROM THE I N VITRO ASSAYS. MEDIAN VALUES AND RANGES FOR VIABILITY, POSITIVITY AND 3H-TdR INCORPORATION IN 34 PATIENTS AND 12 HEALTY CONTROLS

Assay Viability NBT medium* NBT D3* Diff NBT$ 3H-TdR mediumt 3H-TdR D3t Diff 3H-TdR§

Median 0.8

95 175 53 23 20.5 9

Patients Range

p-value

Median

Controls Range

0.2-2.2

1.0

0.3-1.8

4-649 0-532 -337-+396 1-67 0-68 -42-+26

91 88 0 23 26.5 7

29--288 14-360 -63-+72 6-49 8-58 -6-16

0.061 0.44

NBT

p-value

0.84 0.015

* Number x 1000/ml. t Counts/minute x 1000. The difference between NBT positivity in the control medium and after vitamin D3 induction. § The difference between 3H-TdR incorporation in the control medium and after vitamin D3 induction. for the total dysplasia score was 4 (range 2-8). Ring sideroblasts were found in 13 patients (i>15% in 8 cases). Fibrosis was observed in 16 patients. In vitro results Results are shown in Table 3. There was no difference in NBT-positive cells between patients and controls on day 0 (median values of 65 and 75 × 103 positive cells/ml respectively). Normal cells showed more spontaneous differentiation in the medium control than the myelodysplastic cells. The median increase was 40 × 103 in the control group compared to 3.5 × 103 in the patient group. Vitamin D3 incubation resulted in increased NBT positivity in cells from 18/34 patients and 2/12 control cases. In the patient group, the median increase in NBT positivity after D3 incubation was 53 x 103 NBT-positive cells/ ml (range -337 × 103 to 396 x 103). Thep-value for this increase was 0.061. In the control group, the median increase was 0 (range -63 x 103 to +72 x 103). The difference in response to D3 between patients and controls did not reach statistical significance (p = 0.19). Vitamin D3 induction in the control group resulted in a median increase in 3HTdR incorporation of 7 x 103 counts/minute (range - - 6 × 103 t o + 16 x 103). Thep-value for this increase was 0.015. In the patient group the median change in 3H-TdR incorporation was 0 (range -42 × 103 to +26 x 103). The difference between patients and controls was significant (p = 0.034). Within the patient group, all AML cases showed decreased 3HTdR incorporation after D3 incubation, while the MDS cases showed a heterogeneous response. In neither the controls nor the patient group could a correlation between changes in NBT positivity and spontaneous or induced 3H-TdR be found. After Ficoll separation, the percentage of erythropoiesis was 14-100% of the percentage found in the

morphological analysis of the bone marrow smear. During the time in the suspension culture, the percentage of erythropoiesis continued to decrease and was at the fourth day 25-53% of the value on day 0. The contribution of proliferating erythropoietic cells in the suspension culture seems thus to have been negligible.

Correlations between in vitro results and hematological parameters p-values for the relevant correlations are shown in Table 4. Cells from patients with high bone marrow blast percentage and low platelet levels had lower spontaneous NBT positivity and lower viability in oitro. The percentage of bone marrow blasts was inversely correlated to both platelet counts and increase in 3H-TdR after vitamin D3 incubation. Hemoglobin values were correlated to spontaneous 3H-TdR. The ability to increase the percentage of NBT-positive cells after D3 induction showed no correlations with any hematological parameter analyzed. Correlations between in vitro results and morphological data Hypogranulation of the myeloid bone marrow cells was more pronounced in patients with high spontaneous 3H-TdR. Patients with high scores for total dysplasia and megaloblastic morphology showed more increase in 3H-TdR after induction with vitamin D3 in the in oitro assays (Table 4). Correlations between hematological parameters and morphological data Patients with low hemoglobin levels more often had trilinear dysplasia and erythroid dysplasia. Erythroid dysplasia, total dysplasia, and the percentage of bone marrow erythropoiesis were cor-

In vitro reactions to vitamin D3 in relation to morphology and prognosis in MDS

533

TABLE 4. SIGNIFICANT RELATIONSHIPS BETWEEN CLINICAL AND MORPHOLOGICAL DATA AND IN VITRO RESULTS

Variable

State

Hemoglobin

Erythroid dysplasia Trilineage dysplasia 3H-TdR in medium BM blast % Viability in vitro Ring sideroblasts+ BM cellularity % Erythropoiesis Total dysplasia NBT in medium Viability in vitro Diff 3H-TdR % Erythropoiesis 3H-TdR in medium Erythroid dysplasia Diff 3H-TdR

Platelets Granulocytes BM blast %

BM cellularity Hypogranulation Total dysplasia

R-factor -0.5184 -0.3654 0.3795 -0.3657 0.3653 0.4141 0.4691 -0.3535 -0.3721 -0.4373 -0.3584 -0.3522 -0.3924 0.3563 0.7031 0.3938

p-value 0.002 0.034 0.027 0.033 0.034 0.015 0.005 0.040 0.030 0.010 0.037 0.041 0.022 0.039