Sefer Gezer

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.

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.

Remicade as TNF suppressor in patients with myelodysplastic syndromes.

Remicade, a chimeric human-murine monoclonal antibody capable of neutralizing tumor necrosis factor alpha was given to 37 low-risk myelodysplastic syndromes (MDS) patients in two cohorts; 5 and 10 mg/kg intravenously every 4 weeks for 4 cycles. Median age was 68 years, 33 had primary MDS, 14 had refractory anemia (RA), 14 RA with ringed sideroblasts, 9 RA with excess blasts. Nine patients stopped therapy prior to completing 4 cycles, 3 from cohort 1 and 6 from cohort 2 and response was evaluated using the International Working Group criteria in 28 patients who completed the 4 cycles. Six patients showed disease progression, 14 had stable disease and 8 showed hematologic responses, 3/15 (20%) in cohort 1 and 5/13 (38%) in cohort 2. Two patients had multi-lineage responses, 2 had > 100% increase in absolute neutrophils, 1 had > 1 gm/dl increase in hemoglobin, 1 had reduction in blasts from 7% to 1%, and 2 had minor cytogenetic responses (> 50% reduction in + 8 and 20q-metaphases respectively). We conclude that Remicade may have a variety of activities in low risk MDS patients, is well tolerated with a high patient compliance, and may be considered for combination therapy in the future.

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.

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.

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.

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.

Incidence of aplastic anemia in Turkey: A hospital-based prospective multicentre study

The incidence of aplastic anemia among hospitalized adult patients was prospectively determined in this first study in Turkey. New cases of aplastic anemia among patients 14 years and older who were admitted to the study centers were included in a 3 year survey. Seventy-three patients fulfilled the diagnostic criteria, yielding a mean annual incidence rate of 1.14 cases in 10(3) admissions. The male-to-female ratio of the cases (1.6:1) differed from the almost equal ratio of the larger population of Turkey. The median age was 30 years and females were younger at diagnosis. The age distribution of the cases was different from that of the population; showing two incidence peaks in both sexes. The majority of the patients (89%) had severe disease.

High Remission Rate in Acute Myeloblastic Leukemia with Only Two Days of Chemotherapy

Twenty five patients with AML who had neither a history of toxic exposure or myelodysplasia were treated with a remission induction regimen consisting of two pulses of chemotherapy separated by 96 hrs. Each pulse consisted of cytarabine 2gm/m2 (at t=0 and t=12 hrs) with mitoxantrone [30mg/m2] administered immediately after the second cytarabine administration. Amifostine was administered three times a week [on Monday, Wednesday, and Friday] until the outcome of therapy was known. This regimen induced complete remissions in 15 of 17 patients less than 70 years of age and in 5 of 8 patients older than 70 years.