Bmi-1 is useful as a novel molecular marker for predicting progression of myelodysplastic syndrome and patient prognosis

Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic disorders characterized by aberrant hematopoiesis and dysplasia.^1,2  Because the disease entity includes quite heterogeneous pathogeneses, the clinical course and prognosis are highly variable. The International Prognostic Scoring System (IPSS) has achieved international acceptance to estimate prognosis in patients with MDS.^3,4  However, IPSS score is not necessarily enough to evaluate the patient for the purpose of choosing intervention therapies. It would be helpful for the clinical treatment of patients with MDS to establish molecular markers that clearly reflect the disease progression.

Because the most critical event for the progression of MDS and prognosis of patients with MDS is susceptibility to acute leukemia, it is necessary to monitor the appearance and increase of leukemic stem cells (LSCs) in patients with MDS. Thus, we focused on molecular mechanisms supporting LSCs. Bmi1 is a member of the Polycomb group of transcriptional repressor genes, which may be expressed restrictedly in stem cells and progenitors.^5,6  Some studies have demonstrated that Bmi-1 is required to regulate the adult self-renewing hematopoietic and LSCs.^7-12  Others have shown that overexpression of the Bmi-1 gene could cause neoplastic proliferation of cells.^13-16  A number of reports on Bmi-1 provide perspectives on the close association of its expression with the progression of hematopoietic malignancies.^17-21 

In this study, we therefore examined the positivity of Bmi-1 expression in CD34⁺ cells from patients with MDS by flow cytometry to test whether Bmi-1 would be a novel biomarker that is well correlated with the disease progression and prognosis of the patients.

Four acute myeloid leukemia (AML) cell lines (KG1, HL60, HEL, and U937) were obtained from American Type Culture Collection (ATCC; Manassas, VA). Mono7 originated from MDS was available in our laboratory. Normal T cells were obtained from healthy donors and cultured in RPMI-1640 complete medium in the presence of interleukin-2 and/or PHA.

We studied bone marrow samples obtained from 50 patients with newly diagnosed MDS or MDS-AML (case 19 occurred in the same patient as case 27, which developed into refractory anemia with excess blasts [RAEB]). We used 10 bone marrow samples from healthy donors and from patients with renal anemia, iron deficiency anemia, and lymphoma without bone marrow involvement as controls. The patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS) received supportive care such as blood-cell transfusion and low-intensity therapy, including granulocyte colony-stimulating factor (G-CSF), erythropoietin, and cyclosporine. The patients with RAEB, RAEB in transformation (RAEB-T), and MDS-AML received high-intensity therapy, including chemotherapy and bone marrow transplantation. Informed consent was obtained from these patients and donors. An independent pathologist confirmed the diagnosis.

Immunoblot analysis using anti–Bmi-1 monoclonal antibody (Upstate Cell Signaling Solutions, Lake Placid, NY) was performed. The immunoblot results demonstrated that Bmi-1 was highly expressed in 5 AML cell lines. For flow cytometric analysis, cells were stained with anti-CD34 antibody-PE (BD Biosciences, San Jose, CA). After fixation with 3% paraformaldehyde in PBS supplemented with 5% bovine serum albumin, the cells were incubated with anti–Bmi-1 monoclonal antibody. They were incubated with goat anti–mouse IgG antibody-FITC (BD Biosciences). We confirmed that the Bmi-1 expression of all cell lines was detected by flow cytometric analysis.

The correlation between the positivity of Bmi-1 expression of CD34⁺ cells and IPSS was evaluated by the Spearman correlation coefficient. We used the Fisher protected least significant difference (Fisher PLSD) to evaluate differences in Bmi-1 expression ratio of CD34⁺ cells according to French-American-British (FAB) classification. We then used the Cox proportional hazards model to evaluate the correlation of prognosis with the percentage of Bmi-1 expression. P values less than .05 were considered statistically significant. These studies were approved by the Hiroshima University institutional review board for these studies.

Bmi-1 is known to play a role for sustaining self-renewing cell activity by repressing the INK4A locus encoding p16^INK4A and p19^ARF, which are capable of inducing growth arrest, cellular senescence, and apoptosis.^22-24  In this context, to determine whether cells expressing Bmi-1 proliferate, a BrdU-labeling assay was performed. Normal proliferating T cells, which incorporated BrdU, were predominantly stained with anti–Bmi-1 antibody. Furthermore, Bmi-1 positivity was also higher in CD34⁺ cells mobilized with G-CSF than for controls (data not shown). Thus, it appears that Bmi-1 protein not only sustains self-renewing stem cells, but it also plays an important role in providing cells the potential for proliferation.

Because CD34⁺ cells, in which LSCs are supposed to be enriched, have an important role in leukemogenesis, by applying flow cytometric analysis, we examined the percentage of Bmi-1 expression in CD34⁺ bone marrow cells from 51 patients with cases of MDS and MDS-AML, whose profiles are summarized in Table 1. Representative results of flow cytometric patterns are shown in Figure 1A. Higher positivity rate of Bmi-1 was preferentially seen in RAEB, RAEB-T, and MDS-AML compared with RA and RARS. The percentages of Bmi-1⁺ cells in CD34⁺ cells in RA and RARS were almost the same as that in controls (Figure 1B). Interestingly, the positivity of Bmi-1 expression at diagnosis was significantly higher in the patients with progression to RAEB (cases 6, 10, 11, 18, and 19) (28.29% ± 20.30%) compared with those without progression (4.03% ± 3.53%) (P < .001). Especially, even though the patient with case 19, whose percentage of Bmi-1⁺ cells was as high as 63.44% at diagnosis, had IPSS score of 1.0, the disease developed into RAEB in 3 months. However, the patient with case 32 with RAEB-T, had IPSS score of 2.0 and Bmi-1 positivity of about 7.58%, and has remained alive for more than 18 months after diagnosis without chemotherapy. Individuals with cases 20 to 31 with RAEB have died, except for the patient with case 25, who received successful allogeneic bone marrow transplantation, and the patient with case 20, who has been observed for just 7 months. Furthermore, Bmi-1 positivity in CD34⁺ cells was positively correlated with IPSS score (Figure 1C) (P < .001). Within a given IPSS group, cases of RA and RARS that progressed to RAEB showed the highest percentage of Bmi-1. Bmi-1 positivity in CD34⁺ cells independently seemed to predict the risk of mortality (hazard ratio, 1.025; P < .001). We then analyzed whether Bmi-1 is expressed more strongly in the stem-cell subpopulations among CD34⁺ cells, which are supposed to be heterogeneous. However, there was no significant difference in the positivity of Bmi-1 expression between hematopoietic stem cells (CD34⁺CD38^- cells)²⁵  and progenitors (CD34⁺CD38⁺ cells) in MDS and MDS-AML. Further phenotypic analyses did not reveal any correlation of Bmi-1 positivity in CD34⁺ cells in MDS and MDS-AML with other maturation markers, including CD13, CD33, or CD7 (data not shown). These results suggest that increased Bmi-1 expression could reflect qualitative difference of CD34⁺ cells. Although heterogeneity in study population of patients with MDS has prevented us from predicting the disease progression and prognosis, it might be possible to stratify patients to estimate them according to Bmi-1 expression in CD34⁺ cells. The detection and follow-up of this marker might also tell us when to initiate chemotherapy and/or stem-cell transplantation. Because our study suggested that Bmi-1 expression is useful for a molecular marker to predict progression and prognosis of MDS, the prospective study with a large group of patients is required to further evaluate Bmi-1 as a molecular maker for MDS.

We thank M. Kuroda and R. Matsumoto for their technical support. We also thank Dr H Harada for his help in collecting patient samples.