Laurie Lisak

The clinical and biological effects of thalidomide in patients with myelodysplastic syndromes

Thirty patients with myelodysplastic syndromes (MDS) were treated with thalidomide at 100 mg/d p.o., increased as tolerated to 400 mg/d for 12 weeks. Levels of apoptosis, macrophage number, microvessel density (MVD), tumour necrosis factor alpha (TNF‐α), transforming growth factor beta (TGF‐β), interleukin 6 (IL‐6), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined in the serum, bone marrow (BM) plasma and BM biopsies before and after therapy. Pretherapy biological characteristics of MDS patients were compared with similar studies performed in 11 normal volunteers. Ten patients demonstrated haematological improvement in the erythroid series, six becoming transfusion independent. Responders had a higher pretherapy platelet count (P < 0·048) and lower BM blasts (P < 0·013). Median time to response was 10 weeks, and four remain in remission beyond a year. Pretherapy MDS BMs showed higher MVD (P < 0·001) and TGF‐β (P < 0·03) and higher serum TNF‐α (P < 0·008) compared with normal control subjects. After therapy, only BM TGF‐β decreased significantly (P < 0·002). Pretherapy haemoglobin was directly related to serum VEGF (P < 0·001) in responders and inversely related in non‐responders (P < 0·05), suggesting the possibility that angiogenesis may be a primary pathology in the former and a consequence of anaemia‐induced hypoxia in the latter. We conclude that thalidomide has important clinical and biological effects in at least a subset of MDS patients, but the precise mechanism of its action remains unknown and requires further study including a larger number of 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.

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

Abstract:  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‐α) 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‐α (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.

The Clinical and Biologic Significance of Abnormal Lipid Profiles in Patients with Myelodysplastic Syndromes

Serum lipid profiles were obtained in 108 patients with myelodysplastic syndrome (MDS) and compared to 28 healthy volunteers. Serum cholesterol and low-density and high-density lipoproteins (LDL and HDL) were found to be significantly lower in MDS patients than in normals (p = 0.0001, 0.0038 and 0.037, respectively). This difference was significant for all MDS categories. Serum cholesterol and HDL were negatively related to biopsy cellularity (p = 0.001 and 0.0001, respectively), and serum triglycerides were negatively related to labeling index (p = 0.0003). No differences were noted in the lipid profiles of MDS patients with normal versus abnormal karyotypes. However, low-risk MDS patients with abnormal karyotypes had significantly lower triglyceride levels compared with the high-risk patients (p = 0.027), as did low-risk patients with normal cytogenetics (p = 0.015). Serum HDL levels were significantly higher for the low-risk group with normal cytogenetics as well (p = 0.003). We conclude that serum cholesterol, LDL, and HDL are significantly reduced in MDS patients, probably indicating excessive intracellular lipid biosynthesis in the expanding clone. These relatively simple measurements could serve as important prognostic markers and reliable indicators of disease activity in individual patients. Prospective studies to determine their utility as independent variables that guide the need for active therapeutic intervention are warranted.

Biologic characteristics of patients with hypocellular myelodysplastic syndromes

Rates of proliferation and apoptosis as well as expression of tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta) and the number of macrophages were measured in bone marrow (BM) biopsies of 33 patients who presented with hypocellular (cellularity < 30%) myelodysplastic syndromes (MDS). Results showed that 2/3 of the patients had high apoptosis, high cytokine levels and large number of macrophages in their biopsies while 1/3 did not. Apoptosis and TNF-alpha levels were directly related (r = 0.583, P = 0.003, n = 24) as was apoptosis and the degree of anemia (P = 0.033, n = 18). A subgroup of patients with abnormalities of chromosomes 5 or 7 had higher platelets (P = 0.026) and higher apoptosis (P = 0.038) when compared with the rest of the group. Eight patients had no evidence of apoptosis and almost no detectable TNF-alpha in their biopsies. We conclude that within the hypocellular variant of MDS, there may be two distinct sub-groups of patients, one who present with high cytokine-mediated intramedullary apoptosis and the other who may be better characterized as having a stem-cell failure defect since they showed no evidence of apoptosis.

Biologic Characteristics of 164 Patients with Myelodysplastic Syndromes

Rates of proliferation, apoptosis and cytokine expression were measured in bone marrow (BM) biopsies of 164 myelodysplastic syndrome (MDS) patients. There were 107 males and 57 females. Median age was 69 years and 101 had refractory anemia (RA), 17 RA with ringed sideroblasts (RARS), 38 with RA and excess blasts (RAEB) and 8 with RAEB in transformation (RAEB-t). Apoptosis measured by in-situ end labeling (ISEL) was directly related to the number of macrophages (p = 0.028, n = 83). Mean tumor necrosis factor alpha (TNF-alpha) and ISEL positivity were higher in RAEB + RAEB-t patients (p = 0.0554 and p = 0.06 respectively) while hemoglobin was higher for RA + RARS group (p = 0.0472). Patients with high apoptosis had lower white blood cell counts (p = 0.0009), lower percentage of blasts (p = 0.0009) and higher number of macrophages (p = 0.0086). We conclude that measurements of apoptosis, proliferation and cytokine expression provide important biological information which helps to distinguish RA + RARS patients from RAEB + RAEB-t patients, and may be of additive prognostic significance.

Pentoxifylline, Ciprofloxacin and Dexamethasone Improve the Ineffective Hematopoiesis in Myelodysplastic Syndrome Patients

Twenty-five patients with a diagnosis of myelodysplastic syndromes (MDS) were randomized to either begin therapy with pentoxifylline, ciprofloxacin and dexamethasone (PCD) immediately (10 patients) or after a 12 week observation period (control arm, 15 patients). PCD was administered with the goal of suppressing cytokine-induced excessive intramedullary apoptosis of hematopoietic cells. No marked fluctuations of blood counts were noted during the period of observation. Twenty-two patients completed at least 12 weeks of therapy: 18/22 showed some type of hematologic response, 9/18 showing an improvement in absolute neutrophil count only (p = < 0.001) and 9/18 showing multi-lineage responses. No unique category of MDS responded better, however 19/25 patients had refractory anemia (RA)/RA with ringed sideroblasts. The median time to response was 6 weeks and 3/18 responding patients maintained their responses beyond a year. We conclude that hematologic improvement in response to PCD therapy supports the validity of this unique anti-cytokine approach. Future trials should combine PCD therapy with established approaches (growth factors/chemotherapy) and also should focus on identifying more effective ways of suppressing the pro-apoptotic cytokines in MDS.

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.