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Objective: The present study aimed to investigate the origins and differentiation potencies of 4 common malignant clonal cell types (+8, 5q-/-5, 20q-/-20, 7q-/-7) in myelodysplastic syndrome (MDS) and to investigate whether the trisomy of chromosome 8 occurs subsequently to other chromosomal abnormalities., Methods: The present study analyzed a total of 46 cases of chromosomal abnormalities in MDS patients. The magnetic activated cell sorting technique (MACS) was used to sort the CD34(+)CD38(-) (pluripotent hematopoietic stem cells) and CD34(+)CD38(+) cells (committed progenitor cells) from the bone marrow mononuclear cells (BMNCs) of these patients; the sorted cells were then individually smeared. Meanwhile, cytospins were prepared from the remaining CD34(-) BMNCs after cell sorting. The clonal cell proportions in these three types of smears were detected by fluorescence in situ hybridization (FISH). Cases in which +8 was associated with another abnormality (2 cases each in combination with abnormalities in chromosomes 7, 5, and 20) were dually hybridized with the cep8 probe and another corresponding probe., Results: (1) for abnormalities of +8, 5q-/-5, 20q-/-20 or chromosome 7 involvements, clonal cells above the baseline level were detected in the pluripotent stem cell level. (2) The average clonal cell proportion in the committed progenitor cells of the 46 cases increased to 75.3% from 57.3% at the level of stem cell (p < 0.001). The groups with +8 and chromosome 5 abnormalities showed a statistically significant increase in clonal cells at the progenitor cell stage. At the individual level, 33 of 46 cases showed significant increases in clonal cells at the progenitor cell stage relative to the stem cell stage, whereas the clonal cell proportion in the CD34(-) BMNCs generally did not increase relative to the committed progenitor cell population. (3) The dual hybridization analysis showed that if +8 and another abnormality were present in the same abnormal clone according to G-banding, +8 always coexisted with the other chromosomal abnormality at the single cell level; there were no situations in which +8 occurred later than the other chromosomal abnormality., Conclusion: It seems that the all malignant MDS clones originated at the pluripotent hematopoietic stem cell stage and that the proliferation and differentiation potencies were retained partly in these clonal cells. The present study failed to confirm that the trisomy 8 occurred subsequently to the other abnormalities, but some in vitro or transplant experiments maybe prove the succession of clonal origination.

To determine the treatment response and disease progression in strictly selected patients with myelodysplastic syndrome undergoing immunosuppressive therapy (IST), patients were required to have an international prognostic scoring system [corrected] (IPSS) score ≤ 1.0 and at least one of the following conditions: (1) expression of the HLA-DR15 allele, (2) bone marrow (BM) cellularity of less than 30%, and (3) abnormal immune index of BM T-lymphocytes.The exclusion criteria were as follows: (1) ≥ 5% marrow myeloblasts, (2) poor karyotype, and (3) diagnosis of concurrent nonhematological malignancy. Patients received antithymocyte globulin followed by cyclosporine A (CsA) or CsA alone for at least 3 months. Seventy-one cases were analyzed. The total response rate was 77.5% (55/71 cases) with 11 complete responses. The response rate was positively correlated with the number of recruitment criteria met. Patients with an abnormal CD8, an abnormal CD4, or both had similar response rates. Patients who responded to treatment had significantly lower Th1 and Tc1 levels after treatment (P < 0.01 and P < 0.001, respectively), and six of eight patients with abnormal chromosomes did not show obviously abnormal clonal expansion when reassessed after IST. During the median observation period of 24 months, only two cases exhibited disease progression. At the median observation of 24 months, 35 of 55 responders (63.6%) maintained a hematological response, and 60 of 71 patients (84.5%) were still alive. The strictly selective use of IST may yield high response rates and can avoid treatment-related acute myeloid leukemia transformation. IST significantly reduces Th1 and Tc1 levels without causing malignant clonal expansion., (Copyright © 2011 Wiley Periodicals, Inc.)

We performed flow cytometric analysis of CD34(+) cell apoptosis in 22 patients with myelodysplastic syndrome (MDS) and 7 patients with de novo acute myeloid leukemia (AML) using annexin V-FITC, which binds to exposed phosphatidylserine on apoptotic cells. Apoptosis was significantly increased in low-grade MDS group (IPSS score ≤ 1.0) compared with high-grade MDS group (21.33 vs. 7.27%, P < 0.001) and patients with de novo AML (21.33 vs. 7.53%, P < 0.001). There was no correlation between apoptosis and patient's age or gender. Our results confirmed that CD34(+) cell apoptosis was significantly increased in low-grade MDS, which was featured as bone marrow failure. CXCR4 protein expression on CD34(+) cell surface in the low-grade MDS was lower than high-grade MDS (10.42 vs. 16.97, P = 0.014) and AML group (10.42 vs. 20.26, P < 0.001). But there was no statistical significance between low-grade MDS and the control group (P = 0.496). Furthermore, we measured SDF-1 levels in BM plasma from patients by enzyme-linked immunosorbent assay (ELISA), and no difference was found. We found a negative correlation between apoptosis and CXCR4 expression. Our data indicate that CXCR4 might be the prognostic marker of MDS.

Autologous activated CD4(+) T lymphocytes (CD4(+) /CCR5(+) double-positive cells) that derived from BMNCs of patients with low and intermediate-1 risk myelodysplastic syndrome were depleted or added to in vitro cultures. The BMNCs depleted of CD4(+) /CCR5(+) T cells exhibited significantly increased numbers of colony-forming units (CFUs). Conversely, the bone marrow mononuclear cells cultures with a fourfold augmentation of CD4(+) /CCR5(+) T lymphocytes exhibited no colonies in cultures in vitro. The apoptotic index (AI) of colony cells was decreased compared with that of preculture counterparts. After depletion of CD4(+) /CCR5(+) in vitro cultures, the clonal cells increased in patients with chromosome 5q- or 20q- abnormalities but remained unchanged in patients with trisomy 8. In addition, after removal of CD4(+) /CCR5(+) T cells, the number of CFUs was increased in those patients with a higher number of BM Th1 (CD4(+) / IFN-γ(+) ) cells, hypocellularity, or bearing the DR15 allele. We concluded that the selective removal of autologous activated CD4(+) T cells can increase the generation of CFUs. However, whether the increased CFUs consisted of cells derived from residual normal hemopoiesis or clonal hemopoiesis remains unknown., (© 2010 John Wiley & Sons A/S.)

Purpose: To investigate how unselected bone marrow (BM) biopsy examinations could help in the diagnosis of clinically unknown nonhematological tumors., Methods: 10,112 plastic-embedded BM biopsy sections were retrospectively analyzed. In the analysis we focused on the following aspects: (1) the frequency of BM involvement arising from clinically unknown nonhematological malignancies, (2) the clinical indication for BM biopsy examination in cases with tumor BM metastasis, (3) the primary sites of the metastatic tumors, and (4) the advantage of plastic-embedded biopsy sections over paraffin-embedded samples and the complementarity of biopsy with aspiration smears., Results: Of the 10,112 BM samples analyzed, 101 (1.0%) were interpreted as being nonhematological tumor metastases. In cases with metastatic tumors, the nonhemocyte-related changes, such as skeletal pain (25%) and bone destruction (5%), were considerably higher than in those without metastasis (3.7 and 0.32%, respectively; P < 0.001). Primary lesions were documented antemortem in 50 of the 101 biopsy-positive cases (49.5%); the most frequent being in the lung, gastric tract, and breast. Using this assay, a higher incidence of metastatic tumors was detected when compared with previously reported paraffin-embedded samples. The frequency of metastatic tumors based on aspiration smears when the positive results for biopsy sections were used as a reference was 74.3%. All the 101 sections with metastatic tumors showed various degrees of myelofibrosis., Conclusions: We concluded that the routine BM biopsy examination is helpful in detecting insidious metastatic nonhematological malignancies. Skeletal pain and bone destruction are critical indications for susceptible patients to undergo BM examination. Plastic embedding of biopsy sections appears to be more sensitive than the paraffin embedding of samples and is an excellent complementation to isolated aspiration smears.

To verify the expression of type 1 insulin-like growth factor receptor (IGF-IR) and its impact on hematopoietic cells apoptosis in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), marrow samples from 16 patients with MDS and 16 patients with AML were examined along with 16 healthy donors as controls. Immunocytochemical methods (alkaline phosphatase anti-alkaline phosphatase) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (fluorescence) were used simultaneously on nucleated cell cytospins. The ratio of IGF-IR positive cells and apoptotic cells as well as the relationship between them were then analyzed separately. A quantitative real-time reverse transcriptase-polymerase chain reaction (PCR) was administrated for six MDS cases and two normal controls to validate IGF-IR expression detected by immunochemistry. In our assay, IGF-IR appeared to have higher to lower expression rate in turn from AML (86.8+/-13.8%) to MDS (56.8+/-14.3%) and then to normal controls (40.4+/-9.6%) (P<0.01 between each group). In MDS nucleated cells, IGF-IR showed stronger expression in refractory anemia with excess blasts (RAEB)/RAEB in transformation/chronic myelomonocytic leukemia subgroup when compared to RA/RA with ringed sideroblasts cases (64.1+/-3.2 vs 53.5+/-16.2%) (P>0.05). Nucleated cells from MDS marrow underwent more apoptosis (5.4+/-3.0%) than that in normal marrow (1.2+/-0.9%) (P<0.01) and AML marrow (0.3+/-0.4%) (also, P<0.01 between each compared group). For both AML and MDS cases, apoptotic signals presented mainly in individual IGF-IR negative cells (9.0+/-4.8%) and less so in IGF-IR positive cells (1.4+/-2.4%) (P<0.01). When analyzed by groups, cell number with IGF-IR expression showed a negative correlation to apoptotic cells amount (r=-0.852; P<0.01) but positive correlation to their blast count (r=0.677; P<0.01). Outcome from real-time quantitative PCR appeared to have a trend of enhanced IGF-IR expression in advanced MDS subtypes. In conclusion, overexpression of IGF-IR existed in hematopoietic cells in MDS and AML marrows, which appeared to be contributed to disease progress.