- Email: [email protected]
P056 Pathophysiology of patients with myelodysplastic syndrome (MDS) over the age of 80
3. Molecular Mechanisms and Pathophysiology biopsy specimen was evaluated as a relative intensity of membrane and extracellular signal in 10 fields of view at 20X magnification using the Vias immunohistochemistry analyzer (Ventana Biosystems). Cohen’s Kappa was used to determine the optimal threshold of CD31 positive and COX2 positive expression above the mean of the normal controls, with a two-sided p-value of less than 5% considered significant. Statistical analysis was done with SAS (version 9.1 for Windows). Results: For patients with RAEB-1 there was a significant negative correlation between CD31 and COX-2 at expression levels that were 10% above the mean of the normal controls for both markers (kappa = −0.39, p = 0.014), while patients with CMML were found to have a significant negative correlation at expression levels that were 15% above the mean of the normal controls (kappa =-0.24, p = 0.047). No other significant correlations were found, possibly due to the limited sample size. CD31 and COX-2 signal intensities for each MDS subtype were plotted with respect to increasing grade of MDS and are shown below. It was observed that patients with del5q tended to have higher expression of both COX-2 (median relative expression intensity 19% higher) and CD31 (median relative expression intensity 60% higher) compared with normal controls, while in the other lowgrade MDS subtypes (RA and RCMD), neither COX-2 nor CD31 expression levels differed significantly from the normal controls. Micro-vessel density counts of the bone marrow biopsy specimens as well as assessment of soluble markers of angiogenesis and inflammation are currently being performed and will be presented. 1.1
Relative Intensity
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2
AML
CMML-2
CMML-1
RAEB-2
RAEB-1
RCMD
RA
del5q
Normal
CD31 expression in biopsy sections. 0.5
Relative Intensity
0.45 0.4 0.35 0.3 0.25 0.2
AML
CMML-2
CMML-1
RAEB-2
RAEB-1
RCMD
RA
del5q
Normal
COX expression in biopsy sections. Conclusions: This pilot study shows significant heterogeneity in the expression of both CD31 and COX-2 in the bone
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marrow of patients with MDS. There is a strong trend in our data toward lower expression of COX-2 in low-risk MDS, a finding that is in accordance with published microarray data (Pellagatti et al British Journal of Haematology 2004;125:576–583) and with the established observation that MVD is low in this subgroup. Although our sample size is insufficient to permit statistically robust conclusions, it appears that COX-2 expression is higher in del5q MDS. We hypothesize that this may be an indirect result of haploinsufficiency of Egr-1(5q31.1). Egr-1 is a positive regulator of PTEN, which has been demonstrated to down regulate COX-2 expression in LPS-stimulated macrophages. Thus, del5q MDS may have an inherently more pro-angiogenic phenotype than other low-risk MDS, perhaps accounting for the success of anti-angiogenic therapy with lenalidomide in this subgroup. Further study of COX-2 expression relative to markers of angiogenesis and inflammation is planned in an expanded sample size.
P056 Pathophysiology of patients with myelodysplastic syndrome (MDS) over the age of 80 L. Wong, L. Go, W. Linz, W. Koss, J. Song, T. Wajima ° . Texas A&M University Health Science Center and Scott White Clinic and Hospital, Temple, USA *E-mail: [email protected] Background: MDS is common disease in older patients, median age 65. Incidence increases with age and is prevalent in patients over 80. Clinical information is limited in patients over age 80. Retrospective analysis was performed to evaluate this older patient population. Subject and Methods: One hundred forty-eight cases of MDS were analyzed from 1995; 39 cases (27%) were over 80 years (28 males/11 females). FAB classification, IPSS, CBC, co-morbidity conditions, and survival were evaluated. Results: Majority of patients had favorable FAB subset and IPSS MDS: RA 41% and RARS 31%; Low risk 33% and Intermediate-1 41%. The remainder had RAEB 15% and RAEB-t 13% as well as Intermediate-2 20% and High risk 6%. Neutropenia (<1800) 38%, anemia (<10 gm/dL) 38%, and thrombocytopenia (<100k) 30% were found in this group. Charlson index for co-morbid medical conditions (e.g. cardiovascular, hepatic, diabetes, pulmonary, and malignant diseases) showed no clinical correlation in terms of survival. Average survival time was 28 months. Discussion and Conclusion: Our analysis showed patients over 80 divided into three groups: short term survival (<6 months): 10 patients; intermediate (6−38 months): 15 patients; long term (>38 months): 14 patients. No correlation was detected between co-morbid medical conditions and survival. FAB and IPSS were accurate determinants for survival in this elderly patient population.
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Posters
Management of patients over 80 should be based on MDS staging and not their concurrent medical conditions. P057 Apoptosis and telomere length in radiationassociated myelodysplastic syndrome D. Bazyka ° , I. Ilyenko, O. Belayev, O. Lyaskovska, O. Sevko. Department of Clinical Immunology, Kiev, Ukraine *E-mail: [email protected] Objective: Myelodysplastic syndromes (MDS) in radiation exposed are characterized by a combination of ineffective hematopoiesis with late radiation post-effects. Increase of apoptosis and changes of telomere length were demonstrated in irradiation experiments and exposed humans indicating possible modifying role in myelodysplasia pathways after irradiation. Patients and Methods: Peripheral blood samples were studied in 23 patients MDS-RA (17 Chernobyl cleanup workers, mean dose 0.42 Sv; 6 non-exposed) and comparison groups: acute myeloid leukemia (n = 4), Chernobyl cleanup workers without MDS (n = 10) 20−22 years after exposure, nuclear industry staff (n = 10, mean dose 6.3±0.37 mSv), and nonexposed controls (n = 10). Early and late apoptosis was detected in Annexin-V test, telomere length − by PNA flow-FISH assay. Results: MDS-RA samples have demonstrated high levels of granulocyte and lymphocyte apoptosis and significantly lower relative telomere length (RTL) index in comparison with healthy controls. Comparison of MDS-RA in radiation exposed with other irradiated groups has shown the absence of differences in spontaneous level of early apoptosis while the late apoptotic cells fraction (AnnexinV+PI+ cells) was significantly bigger in RA. Lower RTL indices and no difference in apoptosis were shown between MDS-RA subgroups due to the fact of previous irradiation. Mean RTL indices in the exposed RA subgroup were lower than in comparison groups except the group of cleanup workers. Correlations with age were demonstrated with early apoptosis but not RTL. AML cases in comparison with MDS showed marked decrease of cell numbers at all stages of apoptosis but no difference of group RTL from control due to individual variation. Summary: This study shows a possibility of relationship between apoptosis induction and the telomere region changes in MDS-RA as well as in non-MDS patients at the remote period after radiation exposure while RA is associated with cell entry to the late apoptosis stages.
P058 Intracellular ROS in bone marrow cells in myelodysplastic syndrome: technical and methodological considerations L. Chan1 ° , R. Buckstein2 , M. Reis3 , A. Chesney3 , A. Lam3 , M. Cheung3 , E. Piliotis3 , L. Gu1 , R. Wells3 . 1 Sunnybrook Research Institute, Toronto, Canada; 2 Sunnybrook Hospital, Canada; 3 Sunnybrook Health Sciences Centre, Canada *E-mail: [email protected] Intracellular oxidative stress has diverse biological effects, including apoptosis, DNA methylation and mutagenesis, and cell signaling pathways. Reactive oxygen species (ROS) have come under scrutiny as a participant in the pathophysiology of MDS, particularly in the context of transfusion-related iron overload, where catalysis by labile iron of the production of hydroxyl radicals is thought to be the mechanism through which tissue damage is mediated. The accumulation of reactive oxygen species (ROS) has also been shown to promote proliferation and senescence of haematopoietic stem cells, and is known to be carcinogenic − hence, effects on ROS might account for the reported adverse effects of iron overload on haematopoiesis and leukaemia-free survival in MDS. A flow cytometric assay based on the conversion of DCFHDA to DCF by ROS is commonly used to measure intracellular ROS (iROS). Although the assay is simple in concept and execution, it yields substantial interexperimental variation and is therefore difficult to apply to studies of bone marrow in MDS. We have therefore investigated this method to develop methodology that will yield valid and robust results. We measured the iROS in peripheral blood lymphocytes (PB lym) from healthy volunteers (N = 4). We found that the intra-experimental variation in PB lym iROS is remarkably small between healthy volunteers (range 208– 221; 4 measurements in a single experiment), indicating that PB lym iROS is a sufficiently robust parameter to use in normalizing iROS data collected in different experiments. In contrast, a series of measurements made on separate days on a single subject revealed that the measured iROS of PB lym varied considerably (range 141–437; 25 experiments). These data suggest that the observed inter-experimental variation is a consequence of the instability of DCFH-DA reagent. We investigated the feasibility of utilizing cultured cells as a normalizing control for iROS measurement. However, HL-60 cells assayed in four separate experiments showed significant variation in iROS (range 442–1513); this heterogeneity persisted even when PB lym iROS was used to normalize the data (range 3.25−6.69). Finally, we investigated the robustness of iROS measurements in PB lym specimens that had been maintained in culture overnight or cryopreserved. Both of these treatments resulted in a significant increase in iROS (31−77%).
Relative Intensity
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2
AML
CMML-2
CMML-1
RAEB-2
RAEB-1
RCMD
RA
del5q
Normal
CD31 expression in biopsy sections. 0.5
Relative Intensity
0.45 0.4 0.35 0.3 0.25 0.2
AML
CMML-2
CMML-1
RAEB-2
RAEB-1
RCMD
RA
del5q
Normal
COX expression in biopsy sections. Conclusions: This pilot study shows significant heterogeneity in the expression of both CD31 and COX-2 in the bone
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marrow of patients with MDS. There is a strong trend in our data toward lower expression of COX-2 in low-risk MDS, a finding that is in accordance with published microarray data (Pellagatti et al British Journal of Haematology 2004;125:576–583) and with the established observation that MVD is low in this subgroup. Although our sample size is insufficient to permit statistically robust conclusions, it appears that COX-2 expression is higher in del5q MDS. We hypothesize that this may be an indirect result of haploinsufficiency of Egr-1(5q31.1). Egr-1 is a positive regulator of PTEN, which has been demonstrated to down regulate COX-2 expression in LPS-stimulated macrophages. Thus, del5q MDS may have an inherently more pro-angiogenic phenotype than other low-risk MDS, perhaps accounting for the success of anti-angiogenic therapy with lenalidomide in this subgroup. Further study of COX-2 expression relative to markers of angiogenesis and inflammation is planned in an expanded sample size.
P056 Pathophysiology of patients with myelodysplastic syndrome (MDS) over the age of 80 L. Wong, L. Go, W. Linz, W. Koss, J. Song, T. Wajima ° . Texas A&M University Health Science Center and Scott White Clinic and Hospital, Temple, USA *E-mail: [email protected] Background: MDS is common disease in older patients, median age 65. Incidence increases with age and is prevalent in patients over 80. Clinical information is limited in patients over age 80. Retrospective analysis was performed to evaluate this older patient population. Subject and Methods: One hundred forty-eight cases of MDS were analyzed from 1995; 39 cases (27%) were over 80 years (28 males/11 females). FAB classification, IPSS, CBC, co-morbidity conditions, and survival were evaluated. Results: Majority of patients had favorable FAB subset and IPSS MDS: RA 41% and RARS 31%; Low risk 33% and Intermediate-1 41%. The remainder had RAEB 15% and RAEB-t 13% as well as Intermediate-2 20% and High risk 6%. Neutropenia (<1800) 38%, anemia (<10 gm/dL) 38%, and thrombocytopenia (<100k) 30% were found in this group. Charlson index for co-morbid medical conditions (e.g. cardiovascular, hepatic, diabetes, pulmonary, and malignant diseases) showed no clinical correlation in terms of survival. Average survival time was 28 months. Discussion and Conclusion: Our analysis showed patients over 80 divided into three groups: short term survival (<6 months): 10 patients; intermediate (6−38 months): 15 patients; long term (>38 months): 14 patients. No correlation was detected between co-morbid medical conditions and survival. FAB and IPSS were accurate determinants for survival in this elderly patient population.
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Posters
Management of patients over 80 should be based on MDS staging and not their concurrent medical conditions. P057 Apoptosis and telomere length in radiationassociated myelodysplastic syndrome D. Bazyka ° , I. Ilyenko, O. Belayev, O. Lyaskovska, O. Sevko. Department of Clinical Immunology, Kiev, Ukraine *E-mail: [email protected] Objective: Myelodysplastic syndromes (MDS) in radiation exposed are characterized by a combination of ineffective hematopoiesis with late radiation post-effects. Increase of apoptosis and changes of telomere length were demonstrated in irradiation experiments and exposed humans indicating possible modifying role in myelodysplasia pathways after irradiation. Patients and Methods: Peripheral blood samples were studied in 23 patients MDS-RA (17 Chernobyl cleanup workers, mean dose 0.42 Sv; 6 non-exposed) and comparison groups: acute myeloid leukemia (n = 4), Chernobyl cleanup workers without MDS (n = 10) 20−22 years after exposure, nuclear industry staff (n = 10, mean dose 6.3±0.37 mSv), and nonexposed controls (n = 10). Early and late apoptosis was detected in Annexin-V test, telomere length − by PNA flow-FISH assay. Results: MDS-RA samples have demonstrated high levels of granulocyte and lymphocyte apoptosis and significantly lower relative telomere length (RTL) index in comparison with healthy controls. Comparison of MDS-RA in radiation exposed with other irradiated groups has shown the absence of differences in spontaneous level of early apoptosis while the late apoptotic cells fraction (AnnexinV+PI+ cells) was significantly bigger in RA. Lower RTL indices and no difference in apoptosis were shown between MDS-RA subgroups due to the fact of previous irradiation. Mean RTL indices in the exposed RA subgroup were lower than in comparison groups except the group of cleanup workers. Correlations with age were demonstrated with early apoptosis but not RTL. AML cases in comparison with MDS showed marked decrease of cell numbers at all stages of apoptosis but no difference of group RTL from control due to individual variation. Summary: This study shows a possibility of relationship between apoptosis induction and the telomere region changes in MDS-RA as well as in non-MDS patients at the remote period after radiation exposure while RA is associated with cell entry to the late apoptosis stages.
P058 Intracellular ROS in bone marrow cells in myelodysplastic syndrome: technical and methodological considerations L. Chan1 ° , R. Buckstein2 , M. Reis3 , A. Chesney3 , A. Lam3 , M. Cheung3 , E. Piliotis3 , L. Gu1 , R. Wells3 . 1 Sunnybrook Research Institute, Toronto, Canada; 2 Sunnybrook Hospital, Canada; 3 Sunnybrook Health Sciences Centre, Canada *E-mail: [email protected] Intracellular oxidative stress has diverse biological effects, including apoptosis, DNA methylation and mutagenesis, and cell signaling pathways. Reactive oxygen species (ROS) have come under scrutiny as a participant in the pathophysiology of MDS, particularly in the context of transfusion-related iron overload, where catalysis by labile iron of the production of hydroxyl radicals is thought to be the mechanism through which tissue damage is mediated. The accumulation of reactive oxygen species (ROS) has also been shown to promote proliferation and senescence of haematopoietic stem cells, and is known to be carcinogenic − hence, effects on ROS might account for the reported adverse effects of iron overload on haematopoiesis and leukaemia-free survival in MDS. A flow cytometric assay based on the conversion of DCFHDA to DCF by ROS is commonly used to measure intracellular ROS (iROS). Although the assay is simple in concept and execution, it yields substantial interexperimental variation and is therefore difficult to apply to studies of bone marrow in MDS. We have therefore investigated this method to develop methodology that will yield valid and robust results. We measured the iROS in peripheral blood lymphocytes (PB lym) from healthy volunteers (N = 4). We found that the intra-experimental variation in PB lym iROS is remarkably small between healthy volunteers (range 208– 221; 4 measurements in a single experiment), indicating that PB lym iROS is a sufficiently robust parameter to use in normalizing iROS data collected in different experiments. In contrast, a series of measurements made on separate days on a single subject revealed that the measured iROS of PB lym varied considerably (range 141–437; 25 experiments). These data suggest that the observed inter-experimental variation is a consequence of the instability of DCFH-DA reagent. We investigated the feasibility of utilizing cultured cells as a normalizing control for iROS measurement. However, HL-60 cells assayed in four separate experiments showed significant variation in iROS (range 442–1513); this heterogeneity persisted even when PB lym iROS was used to normalize the data (range 3.25−6.69). Finally, we investigated the robustness of iROS measurements in PB lym specimens that had been maintained in culture overnight or cryopreserved. Both of these treatments resulted in a significant increase in iROS (31−77%).