Sairah Alvi

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.

Biological significance of proliferation, apoptosis, cytokines, and monocyte/macrophage cells in bone marrow biopsies of 145 patients with myelodysplastic syndrome.

Labeling index (LI), apoptosis, levels of 2 pro-apoptotic cytokines tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), and the number of monocyte/macrophage cells that are the likely source of the cytokines were simultaneously measured in plastic-embedded bone marrow (BM) biopsy sections of 145 patients with myelodysplastic syndromes (MDS). TNF-α was correlated with TGF-β (P = .001) and with monocyte/macrophage cells (P = .003). Patients with excess blasts in their marrows had a higher TGF-β level (P = .01) and monocyte/macrophage number (P = .05). In a linear regression model, TGF-β emerged as the most significant biological difference between patients who have excess of blasts and those who do not (P = .01). We conclude that in addition to TNF-α, TGF-β also plays a significant role in the initiation and pathogenesis of MDS, and that a more precise definition of its role will likely identify better preventive and therapeutic strategies.

Increased incidence of mitochondrial cytochrome c‐oxidase gene mutations in patients with myelodysplastic syndromes

Summary.  Mitochondria (mt) play an important role in both apoptosis and haem synthesis. The present study was conducted to determine DNA mutations in mitochondrial encoded cytochrome c‐oxidase I and II genes. Bone marrow (BM) biopsy and aspirate, peripheral blood (PB) and buccal smear samples were collected from 20 myelodysplastic syndrome (MDS) patients and 10 age‐matched controls. Cytochrome c‐oxidase I (CO I) and II (CO II) genes were amplified using polymerase chain reaction and sequenced. CO I mutations were found in 13/20 MDS patients and the CO II gene in 2/10 normal and 12/20 MDS samples, irrespective of MDS subtype. Mutations were substitutional, deletional and insertional. CO I mutations were most common at nucleotide positions 7264 (25%) and 7289 (15%), and CO II mutations were most common at nucleotide positions 7595 (40%) and 7594 (30%), suggesting the presence of potential ‘hot‐spots’. Mutations were not found in buccal smears of MDS patients and were significantly higher in MDS samples compared with age‐matched controls in all cell fractions (P < 0·05), with bone marrow high‐density fraction (BMHDF) showing a higher mutation rate than other fractions (P < 0·05). MDS marrows showed higher levels of apoptosis than normal controls (P < 0·05), and apoptosis in BMHDF was directly related to cytochrome c‐oxidase I gene mutations (P < 0·05). Electron microscopy revealed apoptosis affecting all haematopoietic lineages with highly abnormal, iron‐laden mitochondria. These results suggest a role for mt‐DNA mutations in the excessive apoptosis and resulting cytopenias of MDS patients.

Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes.

We used bone marrow biopsies instead of mononuclear cells to maintain long-term cultures from 103 patients belonging to all five sub-categories of myelodysplastic syndromes (MDS), as well as 12 normal controls. By week 4, 30-50% confluency was reached and could be maintained for up to 12 weeks with 100% confluency. The four prominent cells were fibroblasts, macrophages, endothelial cells and adipocytes. Immunohistochemical and electron microscopic studies provided lineage confirmation. Normal hematopoiesis was well supported by MDS stroma. Neither the FAB nor cytogenetics was co-related with the potency of growth. MDS stroma appears to be both morphologically and functionally normal.

Pilot Study of Pentoxifylline and Ciprofloxacin with or without Dexamenthasone Produces Encouraging Results in Myelodysplastic Syndromes

Forty-three patients with myelodysplastic syndromes (MDS) were treated with a combination of pentoxifylline and ciprofloxacin (PC) with the addition of dexamethasone (PCD) in 18 patients who failed to respond to PC. There were 15 females and 28 males, and the median age was 67 years. A total of 18 patients either showed a hematopoietic improvement, a partial or complete cytogenetic response or a combination of both for an overall response rate of 42%. Seven PC only patients responded, four showing hematologic improvement, two cytogenetic responses and one patient showing a combined response. This 16% response rate to PC was increased to 61% by the addition of dexamethasone with 11/18 patients showing a response. Four of the 7 patients who responded initially to PC were given dexamethasone after at least 12 weeks of PC therapy, and only 1 showed a further improvement in response. Thus, we conclude that the combination of PCD provides an encouraging novel approach to treating MDS. The mechanism of action is probably related to the suppression of a veriety of cytokines which in turn attenuate the excessive intramedullary apoptotic death of hematopoetic cells in MDS, an observation which has been speculated to be the basis of the paradox of variable cytopenias despite cellular marrows in MDS. Larger numbers of patients need to be treated and followed for longer periods to determine the true efficacy of this therapy, especially the nature and duration of the cytogenetic responses.

Presence of activation-related m-RNA for EBV and CMV in the bone marrow of patients with myelodysplastic syndromes

The bone marrow (BM) in myelodysplastic syndromes (MDS) undergoes pathobiological changes that mimic an inflammatory process, and hence, an infectious etiology was suspected in these disorders. In the present report, we examined the bone marrow mononuclear cells (BMMNC) of 19 MDS patients and seven normal donors for the expression of one latency-related (Latency membrane protein 1 (LMP-1) and immediate early protein (IEP)) and one activation-related (BZLF and DNA-Pol) m-RNA each for two herpes viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), respectively. Reverse transcriptase polymerase chain reaction was used for this purpose. The latency-related transcripts (EBV-LMP-1 and CMV-IEP) were present in all the MDS and normal specimens. Intriguingly, 10/19 MDS specimens ( approximately 53%) and 2/7 normal donors ( approximately 28%) were positive for active EBV-BZLF (P=0.0067), while 2/19 MDS specimens ( approximately 11%) with 1/7 normal ( approximately 14%) showed active CMV-DNA-Pol (P=0.1588). Later, from another set of MDS patients (n=7) and normal donors (n=4), BM stromal cultures were established, which, at a 75% confluency, were overlaid with cord blood mononuclear cells (CBMNC). IEP was detectable in the CBMNC before and after co-incubation with MDS, as well as normal stroma. So, it was also present both in MDS and normal stromal cells. The other three were absent both in MDS and normal stromal layers. In CBMNC though, active EBV-BZLF and CMV-DNA-Pol m-RNA were detectable in one of seven MDS co-cultures each, albeit from different patients. None of the normal co-cultures showed active virus, either in stroma or CBMNC. Thus, the present report demonstrates, for the first time, the presence of active herpes viruses in the BMMNC of MDS patients and reveals the ability of the MDS stroma to support the viral activation.

Involvement of Cyclin D1 and E2F1 in Intramedullary Apoptosis in Myelodysplastic Syndromes

An unusually high incidence of apoptosis in S-phase cells is characteristically found in the bone marrow (BM) of patients with myelodysplastic syndromes (MDS). Previously, E2F1, c-myc, and Cyclin D1 have been shown to bring about both S-phase changes and/or apoptotic changes. We have already found a stoichiometric imbalance between pRb and E2F1 causing deregulated E2F1 activity in these disorders. In the present study, we investigated the status of Cyclin D1 in relation to E2F1 and apoptosis in 19 patients with a confirmed diagnosis of MDS in comparison with 6 healthy donors. Cyclin D1 was localized immunohistochemically using a specific monoclonal antibody (1:150 dilution) in plastic-embedded BM sections. The nuclear localization of Cyclin D1 graded on a subjective rating scale of 0 (negligible staining) to 8+ (highest), demonstrated negligible levels in normal marrows (median 1+), and in 11/19 evaluable MDS marrows. In contrast, 8/19 MDS biopsies showed an almost four-fold increase in Cyclin D1 localization (p< or =0.001). A western blot analysis of E2F1 in corresponding bone marrow (BM) aspirate mononuclear cells (MNC) demonstrated that the MDS patients with elevated Cyclin D1 expression also had a significant increase in E2F1 protein (p< or =0.03). Additionally, these patients revealed higher levels of mRNA of one of the E2F1 transcriptional target genes, dihydrofolate reductase (DHFR, p=0.01). Subsequently, the relationship of Cyclin D1 with apoptosis was elucidated in a colocalization experiment in BM biopsy sections using immunohistochemistry for Cyclin D1 and in situ end labeling of DNA (ISEL) for apoptosis. The percentage of ISEL-positive apoptotic cells was several fold higher in MDS as compared to normal BMs (p=0.009). Interestingly, 7-41% (median 20%) of the apoptotic cells in different MDS BMs revealed co-localization of Cyclin D1 in their nucleus, whereas in normal BMs co-localization was virtually absent (p=0.008). Thus, it is possible that in a subset of MDS patients, apoptotic death of bone marrow cells may involve Cyclin D1/E2F1 pathway.

Novel in Vitro Culture and Ultrastructural Techniques to Study the Biology of Myelodysplastic Syndromes (MDS)

MDS is a heterogeneous clonal stem cell disorder with trilineage dysplasia resulting in ineffective, monoclonal hematopoiesis ([Resegotti et al., 1993]). Extensive studies over the decade have led to better understanding of cell proliferation, differentiation, cytokine production and apoptosis in MDS ([Greenburg et al., 1987]; [Shetty et al., 1996]; [Clark et al., 1990]; [Yoshida et al., 1993]; [Raza et al., 1995]). However there has been limited information in the literature regarding long term bone marrow cultures and ultrastructural studies performed on MDS. In the past, it has been difficult to grow MDS bone marrow (BM) cells as the progenitor cells have a great propensity to undergo programmed cell death ([Ohmori et al., 1991]; [Aizawa et al., 1999]; [Raza et al., 1996]). On the other hand the role of electron microscopy has not been clearly established in MDS either, other than few individual cases on BM aspirate because of the failure to overcome the technical impediment of maintaining the ultrastructural details in decalcified tissues ([Guccion et al., 1992]; [Gerrad et al., 1992]; [Cohen et al., 1997]; [Payne et al., 1987]). As a result very little is known about the association between the hematopoietic cells and their microenvironment especially in a disease like MDS where both the parenchymal and stromal cells may be involved in the disease process ([Coutinho et al., 1990]; [Sawada et al., 1998])). The present chapter focuses on novel techniques where primarily the MDS core bone marrow biopsy was used to maintain long term bone marrow cultures followed by ultrastructural studies, hence preserving the BM architecture and the geographical distribution of the hematopoietic and stromal cells as they existin vivo.A number of novel and interesting observations were made which are summarized here.