Exploratory Study On Pathogenesis For Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal malignant hematopoietic disorders, characterized by ineffective hematopoiesis in one or more of the lineages of the bone marrow and frequent progression to acute myeloid leukemia (AML). At present, there is no "gold standard" for diagnosis of MDS, which is diagnosed only by excluding other hematopoietic disorders with cytopenia. So it is very difficult to make diagnosis and prognosis for some untypical MDS. As MDS occurs more frequently in an aging population, allogeneic stem cell transplantation (allo-HSCT) is not considered as a normal option owing to the increased risk of treatment-related mortality. Although other treatments such as supportive therapy, immunomodulating agents and so on have improved the disease symptoms of several patients, no treatment is known to alter the natural history of the disease. Therefore, to explore the pathogenesis and elucidate the development and progression of MDS is helpful for us to develop new targets for MDS therapy and more simple diagnostic method and more effective therapeutic tool.The pathogenesis for MDS is unclear. Data suggests that cytogenetics, molecular defects and gene mutation are closely related to the progression and prognosis of MDS. Chromosomal abnormality and important gene mutation are commonly found in MDS patients. Clonal analytical technique shows that MDS is clonal disorders of hematopoietic stem cells (HSCs), and it progresses to AML since accumulated genetic and epigenetic alterations step by step. To study and explore specific molecular genetic alteration of MDS on its pathogenetic source is the key to recognize or cure MDS and is a focus in this field.The retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) is now considered to be a tumor suppressor. Decreased or lost expression of RIZ1 is found in various human solid tumors. The mechanism is probably through aberrant DNA methylation of the promoters CpG islands of RIZ1 gene. Suppression of RIZ1 expression is also found in a few of neoplastic hematologic disorders. However, it has not been reported in MDS to date.Recently, clonal analysis of MDS stem cells is mainly focus on primitive HSC populations such as CD34+CD38-subset. Various types of leukemic stem cells have been obtained successfully, from which the development of theory and practice on tumor stem cells has been extended. However, studies on CD34+CD38-population in MDS are rare because of their heterogeneity.In the present study, we analyzed the expression of RIZ1 in bone marrow cells and its correlation with risk stratification in MDS patients, and we also detected multiple molecular targets on the surface of CD34+CD38-cells in various MDS. Our aim is to find more specific surface marker, to provide new experimental and theoretical basis for elucidating the mechanism of development and progression of MDS, and to offer possible new direction and target for early diagnosis and targeted therapy in MDS. PartⅠStudy on expression of RIZ1 on bone marrow cells in MDS patientsObjective:To investigate the expression of RIZ1 which is considered as a tumor suppressor on bone marrow cells in MDS patients and health adults and analyze it in MDS patients of various subtypes and risk groups and the differences among these groups.Methods:A total number of 22 untreated patients with newly diagnosed MDS and 6 patients with nonneoplastic hematologic diseases as normal control were included in the present study. The mononuclearcells in bone marrow were collected and the expression of RIZ1 on them was detected by quantitative real-time reverse-transcription polymerase chain reaction assay (RT-PCR). According to the World Health Organization (WHO) criteria, patients were classified as follows:refractory anemia (RA),8 patients; refractory cytopenia with multilineage dysplasia (RCMD),2; refractory anemia with excess blasts-1 (RAEB-1),6; refractory anemia with excess blasts-2 (RAEB-2),5 and MDS associated with isolated del(5q) (5q-),1 case. Then patients were divided into two groups as follows: low risk group including RA, RCMD and 5q-subtype and high risk group including RAEB-1 and RAEB-2 subtypes. The expression of RIZ1 in various subtypes and risk groups and the differences among groups were analyzed. Relative quantitation was used for the expression of RIZ1 which was indicated by mean±standard deviation (x±s). Results:The expression of RIZ1 in MDS and control groups are 0.59±0.27 and 1.00±0.19 respectively, and the difference between two groups are statistically significant (P<0.05). As the only patient who suffered both from MDS and prostatic carcinoma was excluded, the expression of RIZ1 was significantly decreased in MDS group in 15 of 21 (71%) patients compared with the control group. Compared to control group, the expression of RIZ1 decreased significantly in RCMD (0.29±0.05), RAEB-1 (0.64±0.15) and RAEB-2 (0.31±0.09) subtype groups (P<0.05). No any significant differences in RIZ1 between RA group (0.84±0.36) and control group (P>0.05) were observed. Likewise, compared with control group the RIZl expression decreased obviously in high risk group (0.49±0.18, P<0.05), but had no statistical change in low risk group (0.70±0.35, P>0.05).Conclusion:Decreased gene expression of RIZ1 is very common in human MDS and it possibly refers to the pathogenesis of MDS. The expression levels of RIZ1 alter among WHO subtypes or risk groups, which may demonstrates its relationship with risk-stratification and important role in the progression in MDS. PartⅡStudy on expression of marker molecules on the surface of hematopoietic stem cells in MDS patientsObjective:To investigate the co-expression of multiple antigens on the surface of CD34+CD38-populations in MDS patients and the differences among various subtypes and risk groups, and to detect specific marker molecules on the surface of MDS stem cells as well as to find out the relationship between the abnormal antigen level and the progression and prognosis of MDS.Methods:A total number of 38 untreated patients with newly diagnosed MDS and 10 patients with nonneoplastic hematologic diseases as normal control were included in the present study. The mononuclearcells in bone marrow were collected and the expressions of a series of surface antigens on CD34+CD38-populations were detected by multi-color flow cytometry. According to the WHO criteria, patients were classified as follows:RA,11 patients; RCMD (including RCMD-RS),9; RAEB-1,7; RAEB-2,10 and 5q-,1 case. According to the International Prognostic Scoring System (IPSS),6 cases were classified as low-risk (LOW-R) MDS,20 as intermediate-1 (INT-1-R),8 as intermediate-2 (INT-2-R) and 4 cases as high-risk (HIGH-R) MDS. The differences of antigen expressions on CD34+CD38-populations among groups were analyzed.Results:In both groups, CD34+CD38-progenitors co-expressed CD13, CD33, CD117, CD 133 and HLA-DR almost in all patients. No significant differences between two groups were observed about the expression levels of CD33,CD117和HLA-DR, but MDS patients 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 MDS patients, with a significant difference between two groups about the expression levels (32±19% vs 12±3%, P<0.05). No control patients had an expression of CD2, CD5, CD7, CD44, CD96 and CD123, which expressed variable amounts in 17～53% of MDS patients: CD2 (47±22%), CD5 (44±28%), CD7 (20±9%), CD44 (35±15%), CD96 (43±19%)和CD123 (54±23%). Stem cells in both groups didn't express detectable levels of CD19. Myeloid markers CD13, CD33 and stem/progenitor cell markers CD117, CD133, HLA-DR expressed consistently in the vast majority of MDS samples 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, CD7 appeared to be expressed more frequently in RCMD, RAEB-1 and RAEB-2 subtypes than RA and 5q-subtype, although the positive rate of these markers were rather low. The positive case rate of CD90 decreased and that of CD96/CD123 increased gradually from RA to RAEB-2 subtype group, but these alterations didn't show statistical difference. The positive case rate of CD2, CD5, CD7, CD96 and CD 123 showed a rising tendency and that of CD90 presented a decline trend with risk increasing, but no statistical significance was obtainedConclusion:MDS stem cells display a complex phenotype different from both normal stem cells and AML stem cells which may make them particularly difficult to eradicate using therapies targeted against surface antigens, and the percentage of cells expressing CD 13 is notably higher in high-grade MDS patients which may be a potential prognostic indicator of MDS in the future.