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Keywords:

  • myelodysplastic syndromes;
  • CD34+CD38 bone marrow cells;
  • stem cells;
  • molecular targets;
  • flow cytometry

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited

Myelodysplastic syndrome (MDS) is a kind of clonal stem-cell disorder in which aberration within a hematopoietic stem cell (HSC) gives rise to the entire disease as in acute myeloid leukemia (AML). Studies have showed that contrasting normal stem cells, AML stem cells express CD96 and CD123, but lack of CD90, although both of them reside within the CD34+CD38 population. So far, little is known about expression of the markers on MDS HSC. In this study, we analyzed the immunophenotypic characteristics of CD34+CD38 bone marrow (BM) cells by multicolor flow cytometry in 38 patients with MDS and 10 control patients. We found that CD34+CD38 BM cells coexpressed CD13, CD33, CD117, CD133, and HLA-DR almost in all patients, but in MDS they expressed higher amounts of CD13 (79% ± 16% vs. 36% ± 13%, P < 0.05) and CD133 (66% ± 20% vs. 25% ± 13%, P < 0.05). CD90 was expressed in all control patients but just in 63% of patients with MDS. No control patients had an expression of CD2, CD5, CD7, CD44, CD96, and CD123, which expressed variable amounts in 17–53% of patients with MDS. The level of CD13 in RCMD (89% ± 7%), RAEB-1 (88% ± 11%), and RAEB-2 (81% ± 13%) were obviously higher than that of RA (63% ± 16%, P < 0.05). CD2, CD5, and CD7 were more frequently observed in RAEB or INT and HIGH-R cases. Taken together, we demonstrate MDS stem cells display deranged phenotypic abnormalities that may make them particularly difficult to eradicate using therapies targeted against surface antigens, and the percentage of cells expressing CD13 is notably higher in patients with high-grade MDS that may be a potential prognostic indicator of MDS in the future. © 2010 International Society for Advancement of Cytometry

Cancer stem cells (CSCs), defined as a small subset of cells within a cancer that have the exclusive ability to self-renew and to differentiate into the heterogeneous lineages of cancer cells that comprise the tumor (1), have attracted considerable attention in cancer research in recent years. So far, CSCs have already been confirmed in many kinds of cancers such as leukemia, multiple myeloma, and solid organ malignancies including brain, breast, prostate, colon, and pancreatic cancer (2). These rare cells may play an important role in tumor formation, metastasis, therapeutic resistance, and recurrence (2). So, it is crucial to functionally identify molecular targets in CSCs and to define “stem cell expression profiles” for various neoplasms, and this may provide insights into the development of predictive and prognostic markers and specific therapeutic interventions.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal malignant hematopoietic disorders, which is characterized by ineffective hematopoiesis and frequent progression to acute myeloid leukemia (AML) (3). Recent studies suggest that MDS is a stem-cell disorder in which aberration within a hematopoietic stem cell (HSCs) gives rise to the entire disease just as in AML (4, 5). Previous data have confirmed that similar to normal HSCs, human AML stem cells reside within the CD34+CD38 compartment of the leukaemic clone (6). However, contrasting normal stem cells, AML stem cells express substantial amounts of CD96 (7) and CD123 (8), which are thought to be leukemic stem cell-specific markers, but lack the expressions of CD90 (9). Other antigens that have been detected on the surface of AML stem cells include CD13, CD33, CD44, CD133, and CD117 (10–13). However, whether these markers including CD123 are specific for AML stem cells remains a matter of controversy (11–16). As for MDS stem cells, so far, only a few data about their characteristics have become available. It is now commonly viewed that the MDS stem cells have a similar phenotype of CD34+CD38 CD90+ as normal HSCs (17), whereas abnormal expression of CD7, CD13, CD33, HLA-DR, and CD117 have also been found on dysplastic CD34+ myeloid precursors (18–20) but they are not certain.

In this study, we analyzed the immunophenotypic characteristics of CD34+CD38 bone marrow (BM) cells from patients with MDS compared to normal/reactive BM. Our aim was to define the profile of immunoreactive cell surface target antigens expressed on CSCs in patients with MDS, which could contribute to distinguish MDS stem cells from normal/reactive HSCs, and to find out whether there was a relationship between the antigen level and the risk of MDS. Overall, our results showed that most patients with MDS displayed complex and aberrant phenotypic abnormalities in the immature CD34+CD38 BM cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited

Patients

A total number of 38 untreated patients (12 women, 26 men) with newly diagnosed MDS according to the World Health Organization (WHO) criteria (21) were included in this study. The median age was 55.5 years (ranging from 15 to 79 years). According to the WHO criteria (21) patients were classified as follows: refractory anemia (RA), 11 patients; refractory cytopenia with multilineage dysplasia (RCMD, including RCMD and ringed sideroblasts, RCMD-RS), 9 patients; RA with excess blasts-1 (RAEB-1), 7 patients; RAEB-2, 10 patients; and 5q-, 1 patient. According to the International Prognostic Scoring System (IPSS) (22), six cases were classified as low-risk (LOW-R) MDS, 20 as intermediate-1 (INT-1-R), eight as intermediate-2 (INT-2-R), and four cases as high-risk (HIGH-R) MDS. The patients' characteristics are shown in Table 1. For control purpose, we examined normal/reactive BM cells obtained from 10 patients (median age: 34.5 years; range: 20–47 years) with nonneoplastic hematologic diseases including iron deficiency anemia, other toxic or reactive cytopenias (for example, idiopathic trombopenia purpura, or secondary thrombocytopenia) and infection-associated leucopenias. All samples were collected at the Center for Stem Cell Research and Application and the Institute of Hematology of Union Hospital, Kindstar Molecular Diagnostic Center or High Trust Diagnostics after obtaining informed consent.

Table 1. Patients' characteristics
PATIENT NO.F/MAGE (Y)DIAGNOSIS AND SUBTYPEKARYOTYPEWBC × 109 L−1% CD34 on MNCs% CD34+CD38 on MNCs
  1. WBC, white blood cell; MNCs, mononuclear cells; f, female; m, male; y, years; NA, not available. MDS, myelodysplastic syndrome; ITP, idiopathic thrombocytopenia; LP, leucopenias; ST, secondary thrombocytopenia; NBM, normal bone marrow; IDA, iron deficiency anemia.

1#m72MDS-RA46,XY,add(11)(q23)5.801.8300.641
2#m47MDS-RA46,XY10.360.1300.027
3#m44MDS-RA47,XY,+82.831.5600.140
4#m63MDS-RA46,XY5.900.2600.036
5#m75MDS-RA46,XY2.810.5390.054
6#m69MDS-RA46,XY2.771.0900.327
7#m55MDS-RA46,XY2.401.7220.525
8#f36MDS-RA46,XX3.200.4740.221
9#f40MDS-RA46,XX3.700.4890.188
10#m63MDS-RA46,XY2.400.8000.040
11#f56MDS-RA46,XX6.400.6500.129
12#m35MDS-RCMD46,XY5.001.2680.535
13#f43MDS-RCMD46,XX2.401.1300.071
14#m47MDS-RCMD46,XY2.640.5000.085
15#f51MDS-RCMD46,XX6.101.9500.312
16#f52MDS-RCMD46,XX2.400.9130.455
17#f38MDS-RCMD46,XX2.100.1300.065
18#m79MDS-RCMD45,XY,-78.701.0890.147
19#m65MDS-RCMD-RS46,XY5.601.0130.344
20#m58MDS-RCMD-RS45,X,-Y2.110.6510.283
21#m44MDS-RAEB-146,XY,del(12)(p13)4.305.4823.330
22#f64MDS-RAEB-146,XX1.666.7301.720
23#m27MDS-RAEB-146,XY1.611.2600.753
24#m47MDS-RAEB-146,XY1.316.6102.181
25#m63MDS-RAEB-146,XY2.117.3401.426
26#f65MDS-RAEB-146,XX2.308.892.230
27#f54MDS-RAEB-146,XX1.904.850.817
28#m62MDS-RAEB-246,XY4.073.6400.728
29#m74MDS-RAEB-245,X,-Y,t(8;21)(q22;q22)2.4711.5381.516
30#m15MDS-RAEB-245,XY,-731.902.6601.014
31#m70MDS-RAEB-246,XY,del(20)(q11)0.636.0601.203
32#m44MDS-RAEB-246,XY6.005.4280.808
33#f67MDS-RAEB-246,XX0.907.3701.840
34#m55MDS-RAEB-246,XY21.4616.0002.84
35#m60MDS-RAEB-246,XY2.4412.3202.45
36#m69MDS-RAEB-245,XY,-70.8319.7003.12
37#m26MDS-RAEB-246,XY1.8010.5002.24
38#f72MDS-5q-46,XX,del(5q)2.371.0100.338
39#f44ITPNA10.450.7930.222
40#m43LPNA3.480.4960.074
41#f30LPNA2.750.6030.075
42#m20STNA5.600.5800.153
43#m26NBMNA9.900.4200.059
44#f30IDANA9.700.3520.028
45#f46LP46,XX2.500.6400.096
46#m36NBMNA8.700.6040.099
47#m47NBMNA8.600.5130.088
48#f33STNA9.200.5480.096

Sampling of Bone Marrow Cells

BM was obtained from the posterior iliac crest and collected in EDTA anticoagulated syringes. All patients gave written informed consent before BM puncture. BM samples were carried to the laboratory within 24 h after aspiration.

Antibodies and Other Reagents

A number of conjugated monoclonal antibodies (mAbs) included fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll-protein (PerCP), or allophycocyanin (APC) labeled CD2, CD5, CD7, CD10, CD13, CD19, CD33, CD34, CD38, CD44, CD90, CD96, CD117, CD123, CD133, HLA-DR, and isotype control IgGs were used to define the phenotype of MDS CSCs. The reagents and 12 mm × 75 mm Falcon™ capped polystyrene test tubes were provided by Becton-Dickinson Bioscience (BD Bio), Santa Cruz Biotechnology or Miltenyi Biotechnology. The combinations of mAbs in four color stainings (FITC/PE/PerCP/APC) were systematically used as follow: CD38/CD96/CD34/CD44; CD38/ CD33/CD34/CD2; CD7/CD13/CD34/CD38; CD10/CD19/CD34/CD38; CD5/CD133/CD34/CD38; CD90/CD123/CD34/CD38; HLA-DR/CD117/CD34/CD38.

Multicolor Flow Cytometry and Characterization of MDS Stem Cells

EDTA anticoagulated BM cells (106 leukocytes per tube) were first incubated with four-color direct fluorescent-labeled Abs (see earlier) at room temperature for 20 min and then added with 2 ml Flow Cytometer (FCM) Lysing Solution (Becton Dickinson, San Jose, CA), and incubated for 10 min. After that, tubes were centrifuged at 1500 rpm for 5 min, supernatant aspirated, and 2 ml of PBS was added to wash the cells two times. Now cells were resuspended with PBS and analyzed on a FCM Calibur™ (BD Bio) using CellQuest software (BD Bio). Data acquisition was performed in two consecutive steps. In the first step, information about a total of 30,000 events per tube corresponding to the whole BM cellularity was acquired; in the second step, information on a minimum of 3 × 103 CD34+ cells was specifically acquired through an electronic gate. MDS stem cells were defined by their typical low forward/side scatter characteristics and their unique phenotype (CD34+CD38). Expression of additional antigens on MDS stem cells (CD34+CD38) was examined subsequently by multicolor flow cytometry. The gating strategy is shown in Figure 1. Antibody reactions were controlled using isotype-matched control antibodies. Positivity was defined as no less than 10% of cell expression on CD34+CD38 cells, unless CD90 (≥5%) (9).

thumbnail image

Figure 1. Gating strategy of immature CD34+CD38 cells. FACS data analysis of a file from patient 15 (see Table 1). For each antibody combination, an ungated file (A–C) as well as a gated file (D–L) using a CD34+ live gate were acquired. R1, live gate; R2, CD34+ live gate; R3 & R4, CD34+CD38 live gate. A, all events of the file acquired; B & C, all events of R1 gate; D & J, all events of R2 gate; E–I, all events of R3 gate; K–L, all events of R4 gate.

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Cytogenetic Analysis

BM cells were cultured for 24 h to prepare for conventional chromosome detection, and karyotype was analyzed with G-banding technique. Each karyotype was named according to International Human Chromosomes Nomendature (ISCN 1995).

Statistical Analysis

The data were analyzed by using the SPSS software package (version 13.0 for Windows, SPSS, Chicago, IL) in this study. All variables were presented as mean values and SD or median and interquartile range. Comparisons between two or more groups for quantitative variables were made using the Student's t-test or ANOVA (for parametric data) and either the Mann–Whitney U or the Kruskal–Wallis tests (for nonparametric data). Differences between groups for qualitative variables were analyzed by Fisher's exact probability tests. Correlation coefficients including Pearson, Spearman's rho, or Kendall's tau-b were used for correlation analysis. P-values < 0.05 was considered to be associated with statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited

Identification of Molecular Targets on CD34+CD38 BM Cells in MDS

In this study, the cell surface target profile of CD34+CD38 BM cells was analyzed in patients with MDS and compared with the profile of molecular targets identified on normal stem cells (Table 2). Similar to normal stem cells, the CD34+CD38 progenitors in MDS were found to coexpress CD13, CD33, CD117, CD133, and HLA-DR almost in all patients, but the CD34+CD38 cells in MDS group expressed obviously higher amounts of CD13 (79% ± 16% vs. 36% ± 13%, P < 0.05) and CD133 (66% ± 20% vs. 25% ± 13%, P < 0.05). CD90 was found to be expressed on these stem cells in all the patients in control group but just in 63% of the patients in MDS group, and there was significant difference in expression amounts between the two groups (32% ± 19% vs. 12% ± 3%, P < 0.05). By contrast, no patients in control group had an expression of CD2, CD5, CD7, CD44, CD96, and CD123 on CD34+CD38 cells, which expressed variable amounts in a few part (17–53%) of patients with MDS: CD2 (47% ± 22%), CD5 (44% ± 28%), CD7 (20% ± 9%), CD44 (35% ± 15%), CD96 (43% ± 19%), and CD123 (54% ± 23%). 20% of patients express CD10 (25% ± 13%) in control group but 11% of patients express CD10 (36% ± 19%) in MDS group. Stem cells in both groups did not express detectable levels of CD19.

Table 2. Expression of cell surface antigens on CD34+CD38 cells of the patients with MDS and nonneoplastic hematologic diseases
ANTIGENCONTROLMDS
  • All values of positive expression were presented as positive/studied cases (positive rate) and percentage of reactive cells.

  • a

    P < 0.05 versus control group.

CD20/10 (0%)0%6/35 (17%)47 ± 22%
CD50/10 (0%)0%7/35 (20%)44 ± 28%
CD70/10 (0%)0%7/35 (20%)20 ± 9%
CD102/10 (20%)25 ± 13%4/38 (11%)36 ± 19%
CD1310/10 (100%)36 ± 13%35/35 (100%)79 ± 16%a
CD190/10 (0%)0%0/38 (0%)0%
CD3310/10 (100%)52 ± 7%34/35 (97%)55 ± 21%
CD440/10 (0%)0%9/35 (26%)35 ± 15%
CD9010/10 (100%)12 ± 3%22/35 (63%)32 ± 19%a
CD960/10 (0%)0%13/38 (34%)43 ± 19%
CD11710/10 (100%)55 ± 10%37/38 (97%)62 ± 20%
CD1230/10 (0%)0%19/36 (53%)54 ± 23%
CD1339/10 (90%)25 ± 13%34/35 (97%)66 ± 20%a
HLA-DR10/10 (100%)73 ± 10%38/38 (100%)68 ± 21%

Comparison of Marker Profiles on CD34+CD38 BM Cells in Various MDS Subtypes

The expression of various cell surface antigens on CD34+CD38 cells of the MDS patients with different WHO subtypes are given in Table 3. We found that myeloid markers CD13, CD33 and stem/progenitor cell markers CD117, CD133, HLA-DR expressed consistently in the vast majority of MDS samples (>80% cases) in every subtype and the differences of the percentage of positive reactive cells between subtypes were not significant except CD13. The levels of CD13 on CD34+CD38 cells in RCMD (89% ± 7%), RAEB-1 (88% ± 11%), and RAEB-2 (81% ± 13%) groups were obviously higher than that of RA group (63% ± 16%, P < 0.05). Lymphoid markers CD2, CD5, and CD7 appeared to be expressed more frequently in RCMD, RAEB-1, and RAEB-2 subtypes than RA subtype, although the positive rate of these markers were rather low (Table 3, Fig. 2A). A remarkable observation was that the positive rate of CD90 and CD96/CD123 gradually decreased or increased from low- to high-grade MDS subtypes (Table 3, Fig. 2A), but these alterations did not show statistical difference. Likewise, the levels of other markers such as CD10 and CD44 were of no statistical difference among subtype groups.

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Figure 2. CD2, CD5, CD7, CD90, CD96, CD123 expression in different WHO subtypes and risk groups. A, Positive rate of CD2, CD5, CD7, CD90, CD96, CD123 expression in different WHO subtypes; B, Positive rate of CD2, CD5, CD7, CD90, CD96, CD123 expression in control and risk groups.

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Table 3. Expression of cell surface antigens on CD34+CD38- cells of the patients with MDS according to different WHO subtypes
ANTIGENRARCMDRAEB-1RAEB-25Q-
  • All values of positive expression were presented as positive/studied cases and percentage of reactive cells (values in brackets).

  • a

    P < 0.05 versus the percentage of reactive cells in RA group.

CD20/9 (0%)2/8 (56 ± 28%)2/7 (41 ± 5%)2/10 (45 ± 33%)0/1 (0%)
CD50/9 (0%)2/8 (56 ± 43%)2/7 (45 ± 21%)3/10 (35 ± 20%)0/1 (0%)
CD71/9 (25 ± 0%)1/8 (14 ± 0%)2/7 (19 ± 6%)3/10 (22 ± 14%)0/1 (0%)
CD101/11 (72 ± 0%)2/9 (30 ± 9%)1/7 (14 ± 0%)0/10 (0%)0/1 (0%)
CD139/9 (63 ± 16%)8/8 (89 ± 7%)a7/7 (88 ± 11%)a10/10 (81 ± 13%)a1/1 (68 ± 0%)
CD190/11 (0%)0/9 (0%)0/7 (0%)0/10 (0%)0/1 (0%)
CD339/9 (47 ± 18%)8/8 (49 ± 27%)7/7 (71 ± 16%)9/10 (56 ± 16%)1/1 (66 ± 0%)
CD441/9 (33 ± 0%)4/8 (39 ± 26%)2/7 (32 ± 11%)2/10 (34 ± 4%)0/1 (0%)
CD907/9 (26 ± 11%)7/8 (54 ± 22%)4/7 (19 ± 1%)3/10 (13 ± 10%)1/1 (40 ± 0%)
CD963/11 (22 ± 7%)3/9 (41 ± 21%)3/7 (55 ± 7%)4/10 (51 ± 17%)0/1 (0%)
CD11711/11 (54 ± 18%)8/9 (77 ± 16%)7/7 (69 ± 19%)10/10 (52 ± 18%)1/1 (68 ± 0%)
CD1233/10 (62 ± 19%)4/8 (66 ± 22%)4/7 (46 ± 30%)8/10 (50 ± 17%)0/1 (0%)
CD1339/9 (51 ± 20%)8/8 (85 ± 17%)7/7 (70 ± 20%)9/10 (63 ± 18%)1/1 (65 ± 0%)
HLA-DR11/11 (60 ± 22%)9/9(67 ± 26%)7/7 (75 ± 19%)10/10 (69 ± 19%)1/1 (81 ± 0%)

Correlation of Immunophenotype on CD34+CD38 BM Cells to Risk-based Classifications

According to the percentage of BM blasts, karyotype and cytopenias, we evaluated IPSS scores of all MDS patients and classified them as LOW-R (0), INT-1-R (0.5–1.0), INT-2-R (1.5–2.0), and HIGH-R (≥2.5) MDS groups (22). The expression of various cell surface antigens on CD34+CD38 cells of the MDS patients with different risk groups are showed in Table 4. Just as in subtype groups, the positive rate of CD2, CD5, CD7, CD96, and CD123 showed a rising tendency and that of CD90 presented a decline trend with risk increasing, but no statistical significance was obtained (Table 4, Fig. 2B). In addition, no significant differences in amounts of the various CD antigens among the risk groups of patients analyzed could be substantiated.

Table 4. Expression of cell surface antigens on CD34+CD38 cells of the patients with MDS according to different risk groups
ANTIGENLOW-RINT-1-RINT-2-RHIGH-R
  1. All values of positive expression were presented as positive/studied cases and percentage of reactive cells (values in brackets).

CD20/5 (0%)3/18 (55 ± 19%)3/8 (39 ± 26%)0/4 (0%)
CD51/5 (99 ± 0%)3/18 (35 ± 21%)1/8 (12 ± 0%)2/4 (47 ± 18%)
CD70/5 (0%)4/18 (19 ± 5%)2/8 (11 ± 0%)1/4 (43 ± 0%)
CD100/6 (0%)2/20 (55 ± 16%)2/8 (17 ± 3%)0/4 (0%)
CD135/5 (76 ± 13%)18/18 (77 ± 18%)8/8 (82 ± 13%)4/4 (85 ± 12%)
CD190/6 (0%)0/20 (0%)0/8 (0%)0/4 (0%)
CD335/5 (49 ± 15%)18/18 (58 ± 25%)8/8 (51 ± 14%)3/4 (61 ± 23%)
CD442/5 (24 ± 8%)4/18 (41 ± 25%)2/8 (33 ± 4%)1/4 (38 ± 0%)
CD904/5 (48 ± 24%)14/18 (31 ± 17%)3/8 (21 ± 20%)1/4 (29 ± 0%)
CD961/6 (32 ± 0%)9/20 (37 ± 23%)2/8 (45 ± 6%)3/4 (52 ± 23%)
CD1176/6 (56 ± 23%)19/20 (66 ± 18%)8/8 (72 ± 15%)4/4 (31 ± 7%)
CD1233/6 (70 ± 25%)6/18 (59 ± 23%)7/8 (45 ± 19%)3/4 (52 ± 18%)
CD1335/5 (65 ± 26%)18/18 (69 ± 19%)8/8 (65 ± 14%)3/4 (60 ± 23%)
HLA-DR6/6 (62 ± 22%)20/20 (70 ± 22%)8/8 (66 ± 17%)4/4 (70 ± 20%)

Percentages of CD34+ or CD34+CD38 cells on mononuclear cells (MNCs) of the patients in different groups and its correlation to clinical features and cytogenetics.

At last, the percentages of CD34+ or CD34+CD38 cells on MNCs, ages and WBC counts of the patients were analyzed among different groups (Table 5). We found that compared with control group, the percentages of CD34+ or CD34+CD38 cells on MNCs in MDS, RAEB-1, RAEB-2, INT-1-R, INT-2-R, and HIGH-R groups were significantly higher (P < 0.05), and the patients in MDS were obviously older (P < 0.05). The percentages of CD34+ cells on MNCs were significantly different among subtypes except that between RA and RCMD; percentages of CD34+CD38 cells on MNCs were significantly different among these groups except that between RA and RCMD or RAEB-1 and RAEB-2. Moreover, percentages of CD34+ or CD34+CD38 cells on MNCs were significantly different among risk groups except that between INT-2-R and HIGH-R (P < 0.05). The ages and WBC counts of patients among subtypes or risks were statistically the same. The results also showed that both percentages of CD34+ and CD34+CD38 cells on MNCs were related to subtype (R = 0.643, P < 0.01 or R = 0.630, P < 0.01), risk (R = 0.594, P < 0.01 or R = 0.518, P < 0.01), and WBC counts (R = −0.328, P < 0.05 or R = −0.342, P < 0.05), while poorly associated with age. In addition, karyotype was related to risk (R = 0.506, P < 0.01) but poorly associated with age, WBC counts, subtype or percentages of CD34+ and CD34+CD38 cells on MNCs.

Table 5. Percentages of CD34+ or CD34+CD38 cells on MNCs of the patients in different groups and its correlation to clinical features and karyotype
GROUP% CD34 ON MNCs (MEAN ± SD)% CD34+CD38 ON MNCs (MEAN ± SD)AGE (YEARS) (MEAN ± SD)WBC (×109 L−1) (MEDIAN ± IQR)KARYOTYPE (CASES)
GOODINTERMEDIANPOOR
  • Karyotype of all patients was classified as Good, Intermedian and Poor according to IPSS score system [22].

  • a

    P < 0.05 versus control group. Multiple comparison among groups of RA, RCMD, RAEB-1, and RAEB-2 showed that percentages of CD34+ cells on MNCs were significantly different among subtype groups except that between RA and RCMD; percentages of CD34+CD38 cells on MNCs were significantly different among subtype groups except that between RA and RCMD or RAEB-1 and RAEB-2. Multiple comparison among groups of LOW-R, INT-1-R, INT-2-R, and HIGH-R showed that percentages of CD34+ or CD34+CD38 cells on MNCs were significantly different among risk groups except that between INT-2-R and HIGH-R. The ages and WBC counts of patients among subtype or risk groups were statistically the same. Both percentages of CD34+ and CD34+CD38 cells on MNCs were related to WBC counts (R = −0.328, P < 0.05, or R = −0.342, P < 0.05), subtype (R = 0.643, P < 0.01 or R = 0.630, P < 0.01), and risk (R = 0.594, P < 0.01 or R = 0.518, P < 0.01), while poorly associated with age. In addition, karyotype was related to risk (R = 0.506, P < 0.01) but poorly associated with age, WBC counts, subtype and percentages of CD34+ and CD34+CD38 cells on MNCs.

Control0.555 ± 0.0890.099 ± 0.03535.50 ± 9.248.65 ± 3.30NANANA
MDS4.094 ± 3.763a0.926 ± 0.793a54.37 ± 15.11a2.56 ± 2.113053
RA0.868 ± 0.4970.212 ± 0.15856.36 ± 13.28a3.20 ± 2.77920
RCMD0.960 ± 0.3660.255 ± 0.14552.00 ± 13.81a2.64 ± 2.26801
RAEB-15.880 ± 1.728a1.780 ± 0.686a52.00 ± 13.86a1.90 ± 1.62610
RAEB-29.522 ± 4.490a1.776 ± 0.722a54.20 ± 19.88a2.46 ± 0.88622
LOW-R0.503 ± 0.2190.158 ± 0.09451.17 ± 12.06a5.75 ± 3.58600
INT-1-R2.539 ± 2.173a0.650 ± 0.523a54.70 ± 13.34a2.40 ± 2.101820
INT-2-R7.729 ± 3.909a1.798 ± 0.928a54.63 ± 16.37a4.19 ± 1.96611
HIGH-R9.990 ± 5.630a1.713 ± 0.703a57.00 ± 28.08a1.65 ± 0.58112

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited

Previous studies have indicated that both human normal and AML stem cells reside within the CD34+CD38 subpopulation, and these cells are capable of initiating long-term growth of normal myelopoiesis (normal stem cells) or leukemias (leukemic stem cells) in NOD/SCID mice (6, 23, 24). We infer that MDS stem cells may reside within the CD34+CD38 population, although we have not proven this yet by transplantation experiments. In this study, we have analyzed expression of various molecular antigens on the surface of CD34+CD38 BM cells in patients with MDS in an effort to define the phenotype of CSCs in patients with MDS and find potential therapeutic targets.

CD34 is a marker of HSC. In AML, the positivity of leukemic cells for CD34 is obviously different in various FAB subtypes (25, 26), and in MDS CD34+ cell clusters were found in 23% of patients and were associated with WHO categories with excess of blasts and poor-risk cytogenetics (27, 28). Consistent with these reports, our study showed that both percentages of CD34+ and CD34+CD38 cells on MNCs by definition increased with more advanced stage of MDS (and greater than in normal); and they were related to subtype, risk and WBC counts in MDS.

CD90, which is expressed on 5–25% of normal CD34+ cells but lack on AML stem cells (9), has now been found to be expressed on CD34+CD38 MDS stem cells (17). In line with these studies, our results showed that CD90 was expressed on normal stem cells in all control patients but only in 63% of patients with MDS. Furthermore, the expression amounts between two groups was significant different (P < 0.05). More recently, several leukemic stem cell-specific markers have been proved to express on CD34+CD38 cells, such as CD123 (8) and CD96 (7). CD123 has been reported to be expressed on stem cells in AML but not in normal BM (8). However, since important control experiments have not yet been reported, and high CD123 expression on non-AML-regenerating BM CD34+CD38 cells in five such cases have been found, some researchers think this issue needs serious attention (14). In our research, we detected no expression of CD123 on CD34+CD38 cells in any control patient, but we found CD123 was expressed in 53% of patients with MDS (54% ± 23% in 19 of 36 cases), which confirmed Jordan CT's report (8). CD96 has been demonstrated to be expressed on the majority of CD34+CD38 AML cells in many cases (74.0% ± 25.3% in 19 of 29 cases), whereas weakly expressed on only a few (4.9% ± 1.6%) cells in the normal HSC-enriched population (Lin-CD34+CD38CD90+) (7). We detected CD96 on a few part of CD34+CD38 MDS stem cells in about 34% of patients (43% ± 19% in 13 of 38 cases), and negative on normal stem cells. According to the expression of CD90, CD96, and CD123 on MDS CD34+CD38 cells, we might speculate it is probable that normal HSC (with a phenotype of CD90+CD96CD123) and leukemic stem cells (CD90CD96+CD123+) coexist in MDS stem cell pool.

CD33 is highly expressed on normal CD34+CD38 stem cells (14), whereas Hauswirth et al find CD33 is specially expressed on AML stem cells but not on normal ones (12). Abnormal expression of CD33 on dysplastic CD34+ myeloid precursors is also detected in MDS (18). We found both normal and MDS CD34+CD38 BM cells coexpressed CD33. The majority of studies have shown that the CD34+CD38 AML stem cells coexpress CD13, CD117, and CD133 (11, 29), but usually lack of HLA-DR (16). Nevertheless, abnormal expression of CD13, CD117, CD133, and HLA-DR on dysplastic CD34+ myeloid precursors or BM myeloblasts have been reported in MDS (18, 30, 31). CD34+CD38 subset in MDS had a phenotype of HLA-DR+CD13+CD33+ (32). Likewise, we detected CD13, CD117, CD133, and HLA-DR on CD34+CD38 cells with variable amounts both in MDS and in control patients. It is noteworthy that the CD34+CD38 cells in MDS expressed obviously higher amounts of CD13 and CD133 compared with controls. The difference in the percentage of cells expressing CD13 may be due to increased abnormal myeloid progenitors that ultimately lead to progression of the dysplasia. CD133 is expressed primarily on a small subset of stem cells (CD34+) (33), and increased numbers of CD133 positive cells are also found to be present in the majority of patients with MDS by other groups (34). The level of these two molecules might be a potential measure standard to discriminate normal and MDS stem cells.

CD44 has recently been described as a target on leukemic CD34+CD38 stem cells; however, it is also weakly expressed on normal CD34+CD38 cells, more differentiated hematopoietic cells and many different tissues (12). Our data showed CD44 had minor expression on the CD34+CD38 cell compartment in MDS (9/30 cases, 35% ± 15%) and no expression in normal ones.

So far, the reports about expression of lymphoid markers on CD34+CD38 cell compartment in AML or MDS patients are rare. CD7 expression was found to be altered on immature precursors in almost half (41%) of the patients with MDS (19), and CD7 positivity of enriched blast cells was an independent variable for a poor prognosis in MDS (32). Our findings indicated that not only CD7 but also CD5 and CD2, which were found no expression on normal stem cells, were expressed on CD34+CD38 MDS BM cells in a relatively small percentage of cases (20%, 20%, and 17%, respectively). Besides, CD10 was detected on CD34+CD38 subset in both groups.

As it is known, WHO subtype and risk stratification of MDS are in close relation with prognosis of individuals suspected of MDS and the evolution to AML. So we analyzed the phenotypic abnormalities present in different WHO subtypes or risk groups of CD34+CD38 cells from MDS versus normal/reactive BM. Consistent with Matarraz's study that CD13 expression was found to be decreased in the very early phases of the disease (RA) (19), we found the levels of CD13 on CD34+CD38 cells in RCMD, RAEB-1 and RAEB-2 groups were notably higher than that of RA group (P < 0.05). Matarraz also found altered levels of CD33 and CD117 among CD34+ precursors, consisting of an abnormally low expression of CD33 in low-grade MDS (RA, RCMD, INT-1, and LOW-R cases) and significantly increased amounts in RAEB-2 cases, and increased expression of the CD117 being more frequently observed among patients with high- versus low-grade MDS (19). However, we did not see any markedly differences of these myeloid-associated markers except CD13 among subtypes or risk groups. One reason for this discrepancy may be that our phenotypic data were referred to overall population of BM CD34+CD38cells, and what they analyzed were CD34+ neutrophil precursors. An alternative explanation would be that the numbers of patients included in every subtype or risk group were too small.

In AML, patients with positive CD7 on blasts have poor prognosis. In MDS, HIGH-R cases more frequently show increased percentages of CD7+ cells on immature precursors (19). Our data displayed that lymphoid markers such as CD2, CD5, and CD7 were more frequently observed in RAEB subtypes or INT and HIGH-R cases, although the level of these markers was rather low. We may infer that the lymphoid markers presented on MDS stem cells probably imply a high-grade MDS. Another remarkable observation was a progressively higher positive rate of CD96 and CD123 and lower positive rate of CD90 were observed from low- to high-grade MDS, but these alterations could not show statistical difference since there were not enough cases included in every subtype or risk group. Whatever, this tendency appeared to show a relationship between positive rate of CD90, CD96, and CD123 and MDS subtype or risk.

All in all, our study may demonstrate that MDS stem cells display a deranged phenotype different from both normal stem cells and AML stem cells, and this may make them particularly difficult to eradicate using therapies targeted against surface antigens. The percentage of cells expressing CD13 is notably higher in patients with high-grade MDS may be a potential prognostic indicator of MDS in the future. However, this indicator should be used together with a flow cytometric scoring system (35) or gene expression profiles (36) and data on the sensitivies and specificities is required. Since very recently Bonnet's group showed that some AML stem cells (defined by in vivo assay) are CD34 (37), to define the phenotype of the entire stem cells population in MDS may require further research not only on CD34+CD38 subset but also on CD34 subset.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited

We thank the staff in Center for Stem Cell Research and Application and the Institute of Hematology in Wuhan Union Hospital, Kindstar Molecular Diagnostic Center in Wuhan and High Trust Diagnostics in Peking for providing MDS and normal BM samples. We also thank the patients for their consent in participating in this study.

Literature Cited

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
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