Intramedullary apoptosis of hematopoietic cells in myelodysplastic syndrome patients can be massive: apoptotic cells recovered from high-density fraction of bone marrow aspirates.

A higher percentage of apoptotic cells (apoptotic index or AI) is consistently found in bone marrow (BM) biopsies compared to BM aspirates of patients with myelodysplastic syndrome (MDS). Most studies have only investigated the low-density fraction (LDF) mononuclear cells from BM aspirates following density separation for AI determination. In the present study, both LDF and high-density fraction (HDF) cells for AI were examined by electron microscopy (EM) in 10 MDS patients and 4 healthy donors. Matched BM biopsies were subjected to AI detection by in situ end labeling (ISEL) of fragmented DNA. The results indicate that in LDF and HDF cells, AI is consistently higher in MDS patients (8.5% vs 1.5%, respectively; P =.039) compared to healthy donors (27% vs 4%, respectively; P =. 004). The BM biopsy AI was also higher in MDS patients than in healthy donors (3+ vs 0+, respectively; P =.036). In addition, in MDS patients, more apoptotic cells were found in HDF cells than in LDF cells (27% vs 8.5%, respectively;P =.0001). All stages of maturation, ranging from blasts to terminally mature cells belonging to all 3 lineages, were represented in the dying cells in both compartments. Using EM, typical Pelger-Huett-type cells appeared to be apoptotic granulocytes. Both LDF and HDF cells should be examined for an accurate estimation of apoptotic cells because AI would be underestimated if only the LDF cells were studied. Ultrastructural studies consistently show a higher AI in BM biopsies compared to BM aspirates despite the correction factor of HDF cells provided by AI. This may represent the actual extant state, which could conceivably be due to a higher concentration of proapoptotic signals in the biopsies. (Blood. 2000;96:1388-1392)

Signal antonymy unique to myelodysplastic marrows correlates with altered expression of E2F1.

Myelodysplastic syndromes (MDS) have previously been reported to show competitively high rates of apoptosis and proliferation in the bone marrow (BM). Using a double-labelling technique in the present study, we demonstrated that a significantly high number of S-phase cells were simultaneously apoptotic (signal antonymy; SA) in MDS (mean +/- s.e.m. 53.5 +/- 6.7%, n = 24, P < 0.001). In contrast, SA was negligible in all other specimens studied, including normal control BM (n = 13) from non-Hodgkin's lymphoma (NHL) patients, BM from patients with de novo acute myelogenous leukaemia (1'AML; n = 5), or secondary AML that had transformed from MDS (2'AML; n = 10), or the solid tumours from patients with NHL (n = 9) or head and neck squamous cell carcinoma (HNSCC; n = 10). Subsequently, the expression of a transcription factor, E2F1, was studied in density-separated BM aspirate mononuclear cells from MDS patients (n = 9) and a normal control. Two separate sets of primers were used that recognized the regulatory retinoblastoma (Rb) protein-binding region and the functional DNA-binding region of E2F1. Interestingly, although the latter manifested the expected band (280 bp) in all samples, the Rb-specific primers showed the expected band (380 bp) in the normal and in 4/9 MDS specimens. Two other MDS specimens also showed a smaller band ( approximately 325 bp), whereas 3/9 MDS patients showed exclusively the smaller band. The levels of SA were significantly higher in those MDS cases that showed the smaller Rb-specific band either alone or in addition to the expected band (median 19.5%, n = 4, P = 0.037) than in those showing exclusively the expected band (median 0.4%, n = 3). Our present studies show SA as a characteristic feature of MDS and, importantly, demonstrate its link with an altered expression of E2F1 in some MDS patients.

Biological characteristics of myelodysplastic syndrome patients who demonstrated high versus no intramedullary apoptosis.

Spontaneous intramedullary apoptosis was measured in bone marrow (BM) biopsies of 175 patients with myelodysplastic syndromes (MDS) using in situ end-labeling (ISEL) of fragmented DNA. Two groups of high (n=71) versus low (n =43) levels of apoptosis were identified while 61 patients were ISEL-negative. Semiquantitative assessment of 3 cytokines, the number of macrophages and in vivo labeling indices (LI) were also determined from consecutive sections of the biopsy. Patients with high apoptosis levels tended to have a high LI (p=0.013), more macrophages in their BM biopsies (p=0.006) and higher tumor necrosis factor alpha (TNF-alpha) levels (not significant) compared to patients with no apoptosis. In addition, low risk MDS patients had significantly lower rates of apoptosis (p = 0.047) and lower levels of TNF-alpha (p = 0.055) compared to high-risk MDS patients. We conclude that the genesis of cytopenias in MDS is of multifactorial origin and that cytokine-associated apoptosis clearly identifies a distinct biological subgroup of patients who may benefit selectively by use of anti-cytokine therapies.

Apparent expansion of CD34+ cells during the evolution of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the proliferative characteristics of CD34+ cells of 33 untreated MDS patients (19 RA, 5 RARS, 7 RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML). All patients received a 1-h infusion of the thymidine analogue iodoor bromodeoxyuridine intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry technique by monoclonal anti-CD34 (QBend/10) and anti-IUdR/BrdU antibodies was developed and performed. By this technique we recognised CD34+ and CD34- cells actively engaged in DNA synthesis or not. As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid cells (mean value: RA/RARS 1.67%; RAEB(t) 8.68%; sAML 23.83%) as well as in the percentage of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%; RAEB(t) 0.43%; and sAML 3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI: RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to an exponential increase in the proportion of CD34+ cells of the proliferating compartment during MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These CD34+ cells appeared to proliferate more slowly than their more mature CD34 negative counterparts, since we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 h). This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth advantage leading to apparent expansion.

Cell cycle kinetic studies in 68 patients with myelodysplastic syndromes following intravenous iodo- and/or bromodeoxyuridine.

Sixty-eight patients with myelodysplastic syndromes (MDS) received sequential infusions of iodo- and/or bromodeoxyuridine for cell kinetic analysis. Bone marrow biopsy sections were treated by appropriate antibodies and a labeling index (LI), duration of S-phase (Ts), and total cell cycle time (Tc) of myeloid cells were determined. The mean LI was 28.4%, Ts was 11.8 hours and Tc was 40.7 hours. The %LI decreased as the disease evolved from refractory anemia toward transformation to acute leukemia (p = 0.04). Double-labeling of biopsy sections for apoptosis and proliferation showed that 30-90% of S-phase cells in MDS patients were simultaneously apoptotic or "antonymous." We conclude that MDS are highly proliferative disorders in which the ineffective hematopoiesis is probably the result of excessive apoptosis rather than slow proliferation.

Measurement of apoptosis, proliferation and three cytokines in 46 patients with myelodysplastic syndromes.

Extensive apoptosis or programmed cell death (PCD) of both hematopoietic (erythroid, myeloid, megakaryocytic) and stromal cells in myelodysplastic syndromes (MDS) cancels the high birth-rate resulting in ineffective hematopoiesis and has been demonstrated as the probable basis for peripheral cytopenias in MDS by our group. It is proposed that factors present in the microenvironment are inducing apoptosis in all the cells whether stromal or parenchymal. To investigate this hypothesis further, bone marrow biopsies from 46 MDS patients and eight normal individuals were examined for the presence of three cytokines, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and granulocyte macrophage-colony stimulating factor (GM-CSF) and one cellular component, macrophages, by the use of monoclonal antibodies immunohistochemically. Results showed the presence of TNF-alpha and TGF-beta in 41/46 and 40/46 cases of MDS respectively, while only 15 cases showed the presence of GM-CSF. Further a significant direct relationship was found between the degree of TNF-alpha and the incidence of PCD (p= 0.0015). Patients who showed high PCD also had an elevated TNF-alpha level. Thus, the expression of high amounts of TNF-alpha and TGF-beta and low amounts of the viability factor GM-CSF may be responsible for the high incidence of PCD leading to ineffective hematopoiesis in MDS. Future studies will be directed at attempting to reverse the lesion in MDS by using anti-TNF-alpha drugs such as pentoxifylline.

Indication of an involvement of interleukin-1 beta converting enzyme-like protease in intramedullary apoptotic cell death in the bone marrow of patients with myelodysplastic syndromes.

Our previous studies using in situ end labeling (ISEL) of fragmented DNA revealed extensive apoptotic cell death in the bone marrows (BM) of patients with myelodysplastic syndromes (MDS) involving both stromal and hematopoietic cells. In the present report we show greater synthesis of interleukin-1 beta (IL-1 beta) in 4 hour cultures of density separated BM aspirate mononuclear (BMAM) cells from MDS patients as compared to the cultures of normal BM from healthy donors or lymphoma patients (1.7 +/- 0.37 pg/10(5) cells, n = 29 v 0.42 +/- 0.24 pg/10(5) cells, n = 11, respectively, P = .049). Further, these amounts of IL-1 beta in MDS showed a significant correlation with the extent of apoptosis detected by ISEL in corresponding plastic embedded BM biopsies (r = .480, n = 30, P = .007). In contrast normal BMs did not show any correlation between the two (r = .091, n = 12, P = .779). No significant correlation was found between the amounts of IL-1 beta and % S-phase cells (labeling index; LI%) in MDS determined in BM biopsies using immunohistochemistry following in vivo infusions of iodo- and/or bromodeoxyuridine. Neither anti-IL-1 beta antibody nor IL-1 receptor antagonist blocked the apoptotic death of BMAM cells in 4 hour cultures (n = 5) determined by ISEL (apoptotic index; AI%), although the latter led to a dose-dependent accumulation of active IL-1 beta in the culture supernatants. On the other hand, a specific tetrapetide-aldehyde inhibitor of ICE significantly retarded the apoptotic death of BMAM cells at 1 mumol/L in 5/6 MDS cases studied (AI% = 2.99 +/- 0.30 in controls v 1.58 +/- 0.40 with ICE-inhibitor, P = .05) and also reduced the levels of active IL-1 beta synthesized (5.59 +/- 2.63 v 2.24 +/- 0.93 pg/10(6) cells, respectively). In normal cells, neither IL-1 blockers nor the ICE inhibitor showed any effect on the marginal increase in apoptosis observed in 4 hour cultures. Our data thus suggest a possible involvement of an ICE-like protease in the intramedullary apoptotic cell death in the BMs of MDS patients.