Analysis of hematopoietic progenitor cells in patients with myelodysplastic syndromes according to their cytogenetic abnormalities

Leukemia Research 28 (2004) 1181–1187

Analysis of hematopoietic progenitor cells in patients with myelodysplastic syndromes according to their cytogenetic abnormalities Natalia López-Holgado a , Jose Luis Arroyo a , Carmen Pata a , Eva Villarón a , Ferm´ın Sánchez Guijo a , Alejandro Mart´ın a , Jesús Mar´ıa Hernández Rivas a , Alberto Orfao b , Jesús Fernando San Miguel a , Ma Consuelo del Cañizo Fernández-Roldán a,∗ a

Department of Hematology, Hospital Cl´ınico Universitario de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain Servicie of Cytometry and Cancer Institute, Universidad de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain

b

Received 23 October 2003; accepted 23 February 2004 Available online 8 May 2004

Abstract The present work analyzes the hematopoietic progenitor cells (HPC) in myelodysplastic syndrome (MDS) patients using both an immunophenotypical and a functional approaches in order to know whether they are similar in patients with or without cytogenetic abnormalities. Among CD34+ HPC, the proportion of myeloid committed progenitors was higher in patients with an abnormal karyotype. Ninety MDS patients were studied. Patients with abnormal karyotype showed a similar platting efficiency than patients with normal cytogenetics. Trisomy 8 and 5q− showed a significant higher P.E. than patients with normal karyotype or monosomy 7. We observed that when the most immature HPC were studied, the total number of granulo-monocytic colonies produced by LTBMC was higher in the normal karyotype group. In summary, the present study shows that in MDS the HPC are impaired; this impairment is deeper in patients with abnormal karyotype. © 2004 Elsevier Ltd. All rights reserved. Keywords: MDS; Haematopoietic progenitor cells; Cytogenetic abnormalities; Immunophenotypical studies; LTBMC; Clonogenic assays; Abnormal karyotype

1. Introduction Myelodysplastic syndromes (MDS) are a group of clonal disorders of the hematopoietic stem cell (HSC) characterized by morphologic dysplasia, peripheral blood cytopenias and predisposition to progress to secondary acute myeloid leukaemia (sAML) [1,2]. In the great majority of MDS patients (excluding CMML) there is a decreased colony growth when bone marrow (BM) Abbreviations: HPC, haematopoietic progenitor cells; MDS, myelodysplastic syndromes; RA, refractory anemia; RARs, refractory anemia with ringed sideroblasts; RAEB, refractory anemia with excess blasts; RAEBt, refractory anemia with excess blasts in transformation; SSC, semi-solid culture; sAML, secondary acute myeloid leukaemia; CMML, chronic myelomonocytic leukaemia; LTBMC, long-term bone marrow cultures; HSC, haematopoietic stem cell; CFU-GM, granulo-monocytic unit-forming colonies ∗ Corresponding author. Tel.: +34-923-291384; fax: +34-923-294624. E-mail address: [email protected] (Ma . del Cañizo Fern´andez-Rold´an). 0145-2126/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2004.02.007

hematopoietic progenitor cells (HPC) are plated including multipotent and committed progenitors [3–5]. Other investigators have shown that proliferation in long-term bone marrow cultures (LTBMC) is also impaired [6]. Using a different approach in a previous study [7], we have shown that CD34+ haematopoietic progenitor cells from these patients can show phenotypic alterations when compared with HPC from healthy volunteers. It is well known that 40% of MDS patients show cytogenetical alterations at diagnosis [8], but it has been considered that the cytogenetic abnormalities are a secondary event occurring in an unstable haematopoietic stem cell. However, to the best of our knowledge there is no data available studying the relationship between cytogenetic alterations and ‘in vitro’ growth in MDS patients. The present study analyzed the HPC from a series of MDS patients in order to ascertain whether HPC could show either immunophenotypical or ‘in vitro’ growth abnormalities. We also analyzed whether there were any differ-

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ences between patients with and those without cytogenetic abnormalities. 2. Material and methods 2.1. Patients A total of 90 patients were studied at diagnosis and classified according FAB criteria as follows: RA 47, RARs 22, RAEB 10, and RAEBt 11. Patients with CMML were not included because their ‘in vitro’ behavior is different [9–11]. Median age was 74 years (13–90) and sex ratio male/female was 55/35. Bone marrow from 17 healthy donors for haematopoietic transplantation was used as controls after informed consent had been obtained. 2.2. Methods 2.2.1. Marrow processing In the patient group the bone marrow samples were obtained from the posterior iliac crest or sternal puncture. For clonogenic CFU-GM assay, mononuclear cells (MNC) were separated by centrifugation on a Ficoll-Hypaque gradient (Lymphoprep TM, Niergaard, Oslo, Norway) (d = 1.077 g/ml). Interface cells were washed and resuspended on IMDM. For LTBMC, cells were obtained by gravity sedimentation using a solution of 0.1% methylcellulose. The cells remaining in suspension were washed with IMDM-FCS [12]. 2.2.2. Immunophenotypic studies Whole BM samples (approximately 2 × 106 cells in 100 ␮l/test) were stained using a stain-and-then-lyse direct immunofluorescence technique in which the following monoclonal antibodies were used in triple-stainings-fluorescein isothiocyanate (FITC), phycoerythrin (PE), and PE cyanine5 (Cy5): CD34/CD33/CD38 and CD15/CD34/HLADR. Briefly, BM samples were incubated (15 min at room temperature), in the presence of 5–20 ␮l of each MoAb, according to the recommendations of the manufacturers. After lysing the non-nucleated red cells with 2 ml/tube of FACS lysing solution (Becton/Dickinson Biosciences, San José, CA, USA), cells were centrifuged and resuspended in 0.5 ml of PBS/tube until analysis in the flow cytometer [13]. All MoAb reagents were purchased from Becton/Dickinson Biosciences (BDB) except CD38−PE Cy5 which was obtained from Caltag Laboratories (San Francisco, CA, USA). Data acquisition was performed in two consecutive steps on a FACScalibur (BDB) flow cytometer using the CellQUEST software (BDB). In the first step, a total of 20,000 events/tube were acquired, corresponding to the total nucleated BM cells. In the second step, acquisition through electronic ‘live-gates’ drawn on CD34+ cells was performed according to their SSC and antigenic expression.

In this latter step, 3 × 105 events were measured with information only obtained for those events that fulfilled the live-gate criteria. For data analysis the Paint-A-Gate PRO software program (BDB) was used. CD34+ hematopoietic stem and progenitor cells (HPC) compartment was analyzed for immunophenotyping as well as the following cell subsets: CD34+/CD33−/CD38−, CD34+/CD33+/CD38−, CD33+/CD38+, CD33−/CD38 + [14]. 2.2.3. Cytogenetic studies Cytogenetic analysis was performed according to standard procedures [15]. Abnormal clones were identified according to criteria defined by the International System for Human Cytogenetic Nomenclature, 1995 (ISCN, 1995). 2.2.4. Clonogenic assays In all cases semi-solid cultures were performed as previously reported [16] in order to analyze committed granulo-monocytic progenitor cells. Briefly, 2 × 105 mononuclear cells/ml in Iscove’s modified Dulbecco’s medium (IMDM) (Gibco, Grand Island, NY, USA) were plated on 35-mm petridishes in 0.9% methylcellulose containing 10% PHA-leucocyte conditioned medium, 10% bovine serum albumin and 10% human AB serum. Cultures were incubated at 37 ◦ C in a fully humidified atmosphere with 5% CO2 and scored at day 14 under an inverted microscope. Colonies were scored as aggregates with more than 40 cells; aggregates of between 4 and 40 cells were scored as clusters. 2.2.5. Long-term bone marrow cultures (LTBMC) In 26 cases in whom a sufficient number of cells were available, LTBMC were established according to the methods of Gartner and Kaplan with slight modifications [12]. Cells (2 × 106 cells/ml; 10 × 106 total cells) were inoculated in tissue culture flasks (Falcon) in LTBMC medium: IMDM 350 mOsm/kg supplemented with 10% pre-selected FCS, 10% horse serum (BioWittaker) and 5 × 10−7 M hydrocortisone sodium succinate (Sigma). The cultures were incubated in a humidified atmosphere with 5% CO2 in air at 33 ◦ C for 6 weeks. At weekly intervals before re-feeding, the stromal layer formation was analyzed under an inverted microscope. For re-feeding, half of the supernatant was removed and replaced with fresh LTBMC medium. The non-adherent cells harvested were counted and assayed for their CFU-GM content. After 6 weeks of culture, the total supernatant was removed and the adherent layer was detached by exposure to trypsin. Cells were recovered, washed, counted and assayed for their CFU-GM content. 2.2.6. Statistical analysis Statistical analysis was carried out with the SPSS 10.0 program. The following non-parametric tests were used: Mann–Whitney’s U test for unpaired data and Pearson or Spearman test for quantitative correlation.

N. L´opez-Holgado et al. / Leukemia Research 28 (2004) 1181–1187 Table 1 Chromosomal abnormalities (n = 25) in MDS series (n = 90) Patients

FAB classification

Karyotype

6 7 8 13 14 17 22 23 33 34 36 40

RA RA RA RA RA RA RA RA RA RA RA RA

48 49 51 52

RARS RARS RARS RARS

55 60 67 68 69 72 79 84 86

RARS RARS RARS RARS RARS RAEB RAEB RAEB-t RAEB-t

46, XX, del(5)(q13q31) [8]/46, XX [12] 46, XX, del(5)(q13q31) [10]/46, XX [10] 46, XX, del(5)(q13q31) [6]/46, XX [14] 45, XX, −7 [10]/46, XX [10] 46, XX, del(5)(q13q31) [18]/46, XX [2] 45, XY, -mar(C) [8]/46, XY [12] 46, XX, del(5)(q13q31) [14]/46, XX [6] 47, XX, +8 [7]/46, XX [13] 47, XX, +8 [7]/46, XX [13] 47, XY, +8 [3]/46, XY [5] 47, XX, del(5)+8 [2] 46, XX [13] 47, XX, del(1)(p11),+ del(1)(p11)-22[8]/46, XX [2] 46, XY, del(5)(q13q31) [4]/46, XY [16] 47, XX, +8 [8]/46, XX [14] 47, XX, +8 [16]/46, XX [4] 45, XY, del(3)(q13q27),del(5),−7,−12,+18[14]/46, XY [6] 47, XX, +8 [5]/46, XX [15] 45, X, -Y[16]/46, XY [4] 47, XX, del(3)(q12), +18[2]/46, XY [17] 45, X, -Y[22] 46, XX, del(1)(q14q32) [2]/46, XX [20] 47, XY, +8 [14]/46, XY [11] 45, X, -X, t(3;3)[6] 47, XY, +11[10]/46, XY [12] 47, XY, +13[4]/46, XY [18]

3. Results 3.1. Cytogenetic analysis Twenty-five out of 90 MDS patients showed cytogenetic abnormalities, 48 showed a normal karyotype and in 17 patients analyzable mytosis was not achieved. The abnormal karyotypes are shown in Table 1. The most frequent anomalies were 5q− and +8. 3.2. In vitro cultures When the whole group was analyzed we observed that median CFU-GM was similar to control when both, clusters and colonies were considered (188 versus 180; P = NS). However, when only the number of colonies was analyzed, patients showed a significantly lower number of colonies

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when compared with normal BM (10 versus 50; P = 0.000), (Table 2). This data conditioned a very high cluster/colony ratio in the majority of patients (57 ± 108) (normal value in our laboratory: 1.22 ± 0.18). The production of CFU-GM during the period of 6 weeks of LT-BMC is shown in Fig. 1. The weekly production was lower than that obtained with normal BM with a very low production during the last 3 weeks. The total number of granulo-monocytic colonies and total MNC produced by LTBMC was higher in the control group (P = 0.000 and 0.001), Table 2. 3.3. Phenotypical analysis The median number of CD34+ cells in the whole group of MDS patients was 0.9% ranging from 0 to 31%. The results of the different CD34+ cell subpopulations are shown in Table 3. The value of CD34+ cells in normal BM is 0.7±0.2 in our hands. 3.4. Comparison between MDS patients with normal or abnormal karyotype In order to know whether the progenitor cell compartment was similar in patients with karyotypic abnormalities when compared with those with a normal cytogenetic analysis, the proportion of different CD34+ cell subpopulations and the ‘in vitro’ behavior was analyzed. 3.4.1. Phenotypical data The results are shown in Table 3. The proportion of CD34+ cells was similar in both groups of patients. However, when CD34+ subpopulations were studied some differences could be observed: the proportion of the myeloid committed progenitor cells was significantly higher in patients with an abnormal karyotype (P = 0.02). By contrast, the non-committed progenitor cells were higher in patients without chromosomal abnormalities (Table 3). These features conditioned a CD34+ CD33/CD34+ 33− ratio being significantly higher in the AK group (2.8 versus 6.6; P = 0.02). When this analysis was performed in patients with specific cytogenetic abnormalities we could observe that patients carrying a 5q− deletion showed the most striking differences when compared with patients with normal karyotype. By contrast, patients with trisomy 8 did not show any significant difference (Table 4).

Table 2 Comparison between controls and the whole group of MDS patients

Donors (n = 17) MDS (n = 90)

CFU-GM (cl + col)

CFU-GM (col)

CFU-GM tot/LTBMC

Total cells/LTBMC

180 (68–548) 188 (2–3,348)

50 (20–160)∗ 10 (0–1,205)

366 (140–1,161)∗ 110 (18–1,064)

870,000 (272,000–1,591,000)∗∗ 474,000 (188,000–968,000)

CFU-GM: progenitors produced/105 cells plated; cl: cluster; col: colonies. CFU-GM/LTBMC: progenitors produced by LTBMC during 6 weeks of culture. Total cells: cells produced by LTBMC during 6 weeks of culture. ∗ P < 0.005. ∗∗ P < 0.01.

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p=0,003 100 p=0,720

CFU-GM

80

p=0,000 60

p=0,000

40

p=0,004

20

MDS

p=0,020

Healthy Donors

0 2

1

4

3

5

6

Weeks Fig. 1. Production of CFU-GM in LTC in donors and MDS patients. CFU-GM was significantly lower in MDS patients when compared with normal BM during the 6 weeks of culture.

Table 3 Phenotypical analysis of haematopoietic progenitor cells CD34+ and their subpopulations

All

NK (n = 39)

AK (n = 23)

P

CD34+ CD34+ 38− 33− CD34+ 38+ 33− CD34+ 33+ CD34+ 33− CD34+ 33+/CD34+ 33−

0.9 (0–31.5) 3.3 (0–83) 11.1 (0–79) 79.2 (4.4–100) 20.8 (0–90) 3.8 (0–247)

0.9 (0–31.5) 4.8 (0–63.8) 19.0 (0–78) 74.2 (10–100) 26.0 (0–90) 2.8 (0.1–247)

1.5 (0.1–19) 2.3 (0–70.2) 4.4 (0–38.6) 86.2 (10.5–100) 13.0 (0–90) 6.6 (0.1–248)

0.18 0.49 0.01 0.02 0.02 0.02

Percentage of CD34+ cells among MNC. Results expressed as % of positive cells among CD34+ cells. CD 34+ cells in normal donors: 0.7 ± 0.2. NK: normal karyotype; AK: abnormal karyotype. P: when NK and AK were compared.

Table 4 Phenotypical analysis of haematopoietic progenitor cells according to specific karyotypic alterations CD34+ and their subpopulations

NK (n = 39)

5q− (n = 5)

P

+8 (n = 6)

P

CD34+ CD34+ 38− 33− CD34+ 38+ 33− CD34+ 33+ CD34+ 33− CD34+ 33+/CD34+ 33−

0.9 (0–31.5) 4.8 (0–63.8) 19 (0–78) 74.2 (10–100) 26 (0–90) 2.8 (0.1–247)

3 (0.1–3.7) 0 (0–3) 0.5 (0–39) 99.3 (58–100) 0.6 (0–41.5) 6.6 (0.1–248)

0.49 0.00 0.04 0.00 0.00 0.00

1.5 (0.3–3.4) 4.8 (0–25) 6 (0–25) 89 (50–100) 11 (0–50) 8.6 (1–247)

0.63 0.95 0.12 0.13 0.14 0.15

Percentage of CD34+ cells among MNC. Results expressed as % of positive cells among CD34+ cells. NK: normal karyotype.

3.4.2. Cell culture analysis Patients with abnormal karyotype showed a similar P.E. (cluster + colony numbers) and cluster/colony ratio than patients with normal cytogenetics (Table 5). However when specific cytogenetic abnormalities were analyzed we observed that patients with trisomy 8 and 5q− showed a significant higher P.E. when compared with those showing a normal karyotype. By contrast patients with monosomy 7 had lower P.E. and cluster/colony ratio (Table 6). The weekly production of CFU-GM in LTBMC was lower in the group of patients with an abnormal karyotype when compared with the group of patients without cytogenetic alterations throughout the period of culture. Furthermore, CFU-GM stopped being produced earlier in abnormal kary-

Table 5 Cell cultures in MDS patients

Colonies Clusters + colonies Clusters/colonies CFU-GM tot/LTBMC Total MNC/LTBMC

NK (n = 48)

AK (n = 25)

P

9 (0–408) 180 (2–3348) 0.3 (0–5.4) 140 (8–1064) 4.3 (1.9–9.7)

11 (0–1205) 254 (14–3104) 15 (2–710) 66 (44–99) 4.9 (2.5–8.0)

NS NS NS 0.005 NS

Values represent median (range) of colony-forming cells per 1 × 105 plated MNC. NS: non statistically significant. CFU-GM/LTBMC: progenitors produced by LTBMC during 6 weeks of culture. Total MNC: cells produced by LTBMC during 6 weeks of culture.

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Table 6 Semi-solid cultures according to specific cytogenetic alterations

Colonies Clusters + colonies Clusters/colonies

Normal karyotype (n = 48)

No mytosis (n = 16)

+8 (n = 8)

5q− (n = 5)

−7 (n = 3)

9 (0–408) 180 (2–3348) 18 (0–380)

9 (0–250) 160 (8–1850) 39 (3–398)

22 (0–502) 415∗ (40–1506) 16 (2–40)

12 (0–58) 467∗∗ (280–710) 59 (7–710)

0 (0–12) 38∗∗ (14–124) 10∗∗∗ (2–14)

∗

P > 0.01 and <0.1 when compared with no mytosis and or normal karyotype patients. P < 0.05 when compared with no mytosis patients. ∗∗∗ P < 0.05 when compared with normal karyotype patients. ∗∗

120

100

CFU-GM (Median)

Normal Karyotype Abnormal Karyotype

p=0,02

80

60

40 p=0,17 20 p=0,06 p=0,55

p=0,74 p=0,67

0 1

2

3

4

5

6

weeks Fig. 2. Production of CFU-GM in LTC according to the results of cytogenetic analysis during the 6 weeks of culture.

otype patients (Fig. 2). These features ensured that the total number of granulo-monocytic colonies produced by LTBMC was higher in the normal karyotype group (P = 0.005; Table 5).

4. Discussion Severe haematopoietic alterations have been observed, both in vivo and in vitro, in MDS patients. Examples of the latest have been reported by several authors [17,18]: reduced levels of primitive and mature progenitors, deficient proliferation in LTBMC, impaired responses to stimulatory cytokines, etc. Phenotypical abnormalities in the CD34+ HPC in these patients have been reported [7,19]. Also, it has been well established that about half of patients with primary MDS have cytogenetic abnormalities, the most common of which are trisomy 8, monosomy 7 and 5q− [20,21]. Chromosomal changes may be observed at the onset of symptoms and/or evolve over time as disease progresses. Specific cytogenetic abnormalities strongly correlate with prognosis in MDS. In general, patients with multiple cytogenetic abnormalities do poorly, and abnormalities involving chromosome 7 are especially hazardous [22], while patients with 5q− as an isolated anomaly have a better prognosis

[23] suggesting that the haematopoietic process could be differently impaired according to these cytogenetic abnormalities. However, there is no available data analyzing the HPC behavior in these patients and their relationship with the cytogenetic features. In the present study we have analyzed whether the immunophenotype of HPC and their proliferative capacity in clonogenic and LTBMC of BM progenitor cells from MDS could be related to cytogenetic data. For this purpose we have previously analyzed the plating efficiency of a series of patients and compared them with normal donors. We have observed that the median number of CFU-GM was lower in MDS patients than it was in controls (Table 2), with a very high cluster/colony ratio, which confirms previously published data [24]. Also, more immature progenitors were analyzed using an LTBMC approach and according to previous reports [6,7,17] we have also observed a significantly decreased production of CFU-GM and total MNC in LTBMC from MDS patients; moreover, the progenitor cell production finishes earlier in these patients with very low levels since the third week of culture (Fig. 1). It has been argued by several authors that this in vitro growth defect is caused by the presence of inhibitory factors [25], a defect in accessory cells [5,26,27] or indeed a defect in the proliferative rate [6].

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We have shown in a previous paper [7] that, when the immunophenotypical approach was used, the proportion of myeloid committed progenitors increased in MDS patients. This feature is also shown in the present analysis. However, using the functional approach, the number of CFU-GM was reduced in the majority of patients, showing a deep functional impairment in these progenitors. This impairment was even more striking in patients with an abnormal karyotype, with higher proportion of CD34+ CD33+ progenitor cells. On the best of our knowledge these features have not been previously reported. As well observed with committed progenitors, noncommitted myeloid progenitors are more severely impaired in patients with an abnormal karyotype, according to both immunophenotype and functional approaches. The explanation for this behavior could be as follows: there is an increased of committed myeloid progenitors seeking to ameliorate PB cytopenias but the functional impairment prevent the number of blood cells from being increased and this problem is more profound in patients with abnormal karyotype, whose disease is more evolved. In order to ascertain whether the specific cytogenetic abnormalities could condition a different in vitro behavior we have analyzed separately patients with the following alterations: trisomy 8, the 5q− and monosomy seven and we have verified that patients with monosomy 7 showed the lowest number of CFU-GM. It is well known that this cytogenetic abnormality conditions a very poor prognosis, perhaps because the HPC are more profoundly impaired in their behavior. In summary, the present study shows that in MDS the HPC are all altered. When examined by immunophenotypical and in vitro approaches we observed that this impairment is deeper in patients with an abnormal karyotype showing a more evolved disease. So, patients with 5q− or +8 alterations show a higher number of CD34+ CD33+ cells and higher production of clusters in semi-solid cultures. By contrast, in the three cases with deletion in chromosome 7 only analyzed by its behavior in vitro, we observed a lower growth than in the other patients. This data could compensate the higher growth obtained in the group of abnormal karyotype patients as a whole and make the difference less significant. In conclusion, the present study shows that patients with MDS show abnormalities in the hematopoietic progenitor cells, when they are studied using both immunophenotypical and functional approaches. It is striking that the impaired behavior of progenitors in LTBMC is more marked in patients with karyotypic abnormalities.

Acknowledgements This work has been supported by a grant from FIS, no.: 98/1184.

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