ALBERT

Description: Abstract 3036 The ISS introduced by Greipp et al (J Clin Oncol 2005) represents today the most widely used staging system for patients with multiple myeloma (MM) because it is based on two readily available variables: serum albumin and beta2-microglobulin. Serum beta2-microglobulin not only reflects myeloma tumor load but it is also increased in patients with renal dysfunction. Thus, there have been concerns that ISS-3 stage may include MM patients with renal impairment in whom elevated beta2-microglobulin does not reflect tumor burden but rather the degree of renal dysfunction. To address this issue, we assessed the impact of patients' renal function on the prognostic performance of ISS. Our analysis included data from 1516 patients with symptomatic MM that had been entered into the database of the Greek Myeloma Study Group. Renal function was assessed by the estimated GFR (eGFR), which was calculated using the modified MDRD formula (eGFR in mL/min/1.73 m2 = 186 × (Serum Creatinine)−1.154 × (Age)−0.203 × (0.742 if female) × (1.212 if African-American) and the degree of renal dysfunction was staged according to the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (KDOQI) classification of chronic kidney disease (CKD) as follows: stage 1 eGFR ≥90 ml/min; stage 2 eGFR of 60–89 ml/min; stage 3 eGFR of 30–59 ml/min; stage 4 eGFR of 15–29 ml/min and stage 5 eGFR

Description: Interaction between receptor activator of nuclear factor κB ligand (RANKL) and RANK/osteoprotegerin (OPG) plays a dominant role in osteoclast activation and possibly in plasma cell survival in multiple myeloma (MM). We measured soluble RANKL (sRANKL), OPG, and bone remodeling markers in 121 patients with newly diagnosed MM to evaluate their role in bone disease and survival. Serum levels of sRANKL were elevated in patients with MM and correlated with bone disease. The sRANKL/OPG ratio was also increased and correlated with markers of bone resorption, osteolytic lesions, and markers of disease activity. The sRANKL/OPG ratio, C-reactive protein (CRP), and β2-microglobulin were the only independent prognostic factors predicting survival in multivariate analysis. We generated a prognostic index based on these factors that divided our patients into 3 risk groups. The low-risk group had a 96% probability of survival at 5 years, whereas the intermediate-risk and the high-risk groups had probabilities of survival of 52% and 0%, respectively. Not only do these results confirm for the first time in humans the importance of sRANKL/OPG in the development of bone disease, they also highlight the role of this pathway in the biology of plasma cell growth as reflected by its influence on survival.

Description: Abstract 5212 Background: In all Epstein-Barr (EBV)-associated malignancies, the virus displays a latency program of infection and a restricted pattern of gene expression. Among the products of these genes, latent membrane protein 1 (LMP1) is a potent transforming protein with several different roles. LMP1 has been shown in cell lines to stimulate apoptosis. Survivin, a member of the inhibitor of apoptosis (IAP) family, is an important regulator of the mitochondrial apoptotic pathway, while oxidative stress (OS) is a cellular condition particularly relevant to cell aging. In the present study we enrolled patients with non-EBV-related low grade B-cell lymphoproliferative diseases. The aim was to detect (1) the viral load of EBV-positive patients, (2) the expression of LMP1 oncoprotein, (3) the possible apoptotic properties of LMP1 by correlating the levels of survivin with LMP1 expression, and (4) the levels of oxidative stress in LMP1-positive and negative patients. Patients and Methods: Forty eight Greek patients with EBV-unrelated low grade B-cell leukemic lymphomas, were enrolled in the study (chronic lymphocytic leukemia: 27, marginal zone lymphoma: 12, mantle cell lymphoma: 4, hairy cell leukemia: 2, follicular lymphoma: 2, lymphoplasmacytic lymphoma: 1). The majority of patients (61.2%) were treatment-naïve, while the rest had not received any treatment for at least 6 months. DNA from peripheral blood was tested by quantitative real time (qRT) PCR for the EBV-R gene. RNA from EBV-positive patients was examined by RT-PCR and qRT PCR for LMP-1, while using qRT PCR we measured survivin expression in all patients. Densitometric analysis (DA) was used for semi-quantification of the survivin gene expression. The results were expressed relative to the expression of ABL housekeeping gene. The control group included 30 EBV-negative healthy adults. Oxidative stress was measured in the serum of all patients using the PerOx (TOS/TOC) Kit, by Immunodiagnostik. Non parametric methods (Mann-Whitney test) were used for statistical analysis of the results. Results: Twenty five (25) men and 23 women, with a median age of 74 (51–87 years old) were studied. EBV positivity was detected in 19/48 (39.6%) patients, and LMP1 was expressed in 13/19 (68.4%) EBV-positive patients. Survivin levels were lower in LMP1-positive patients vs LMP1-negative patients (2-tailed p=0.009). The oxidative stress was lower (261.4 μmol/L) in LMP1-positive patients vs LMP1-negative patients (372.3 μmol/L), (2-tailed p=0.014). Discussion: The literature lacks information about the expression of LMP1 in the peripheral blood of patients with non-EBV-related low grade B-cell leukemic lymphomas. Previous studies in LMP1-positive lymphoma cell lines have shown the apoptotic functions of LMP1 during type II latency. In this study LMP1-positive patients express statistically significant lower levels of survivin vs LMP1-negative patients. This finding is in accordance to the hypothesis that LMP1 oncoprotein can induce apoptosis. LMP1-positive patients had lower levels of oxidative stress compared to LMP1-negative patients. According to our findings, in non-EBV-related lymphomas, LMP1 may increase apoptosis and decrease the levels of oxidative stress. Disclosures: No relevant conflicts of interest to declare.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder characterized by the somatic mutation of X-linked gene PIG-A, required for glycosylphosphatidylinositol (GPI)-anchor biosynthesis. This results in absent or decreased expression of all membrane proteins normally anchored by GPI - including CD55 and CD59 - in all circulating cells, leading to an unusual sensitivity of red blood cells (RBCs) to complement lysis and subsequently intravascular hemolysis and hemoglobinuria. According to the “dual pathogenesis” model, there is an immunoregulatory selection in favor of PNH clones to proliferate preferentially over normal hemopoiesis on a microenvironment of bone marrow failure. The incidence of “PNH-like” defect has been also demonstrated in many hematological diseases and on peripheral blood cells (PBC) of normal individuals. Complement system is recognized as having the potential to provoke severe impairment to host tissues. This is extensively demonstrated in autoimmune disease setting. Multiple regulatory and inhibitory enzymes, such as CD55 and CD59, known as complement regulatory proteins, adjust the progression of complement cascade at all levels, protecting the autologous cells. Complement activation and cytopenias have been associated with diminished CD55 and/or CD59 expression on PBC membranes. The aim of this study was to evaluate the presence of “PNH-like” red-cell populations in patients with rheumatic diseases and investigate possible correlations with clinical or laboratory parameters. CD55 and CD59 expression was evaluated in erythrocytes of 113 patients (94 females, 19 males, median age: 64 years) with rheumatic diseases: 38 with rheumatoid arthritis, 25 with systemic lupus erythematosus, 17 with Sjögren’s syndrome, 7 with systemic sclerosis, 12 with vasculitis, 2 with dermatomyositis, 1 with ankylosing spondylitis and 11 with mixed connective tissue diseas, using the sephacryl-gel microtyping system, a semi-quantitative, inexpensive and simple method useful in screening “PNH-like” red-cell defect, with sensitivity comparable with that of flow cytometry. One hundred and twenty-one (121) healthy blood donors of similar age and gender and 10 patients with PNH were also studied, as control groups. In all samples with CD55- and/or CD59- negative RBCs, Ham and sucrose tests were also performed. Interestingly, the majority of patients (104/113, 92%) demonstrated “PNH-like” erythrocytic populations: 47 (41.6%) with concomitant deficiency of CD55 and CD59, 50 (44.2%) with isolated deficiency of CD55 and 6 (6.2%) with isolated deficiency of CD59. In healthy donors, only 2 (1%) had red cells with concomitant CD55/CD59 negativity and 3 (2%) with isolated CD55 or CD59 deficiency. “PNH-like” erythrocytic clones never surpassed 25% of the total red-cell population, while the most common proportion of deficiency for both antigens was 10%. All PNH patients exhibited simultaneous CD55/CD59 deficiency. Moreover, it should be high-lightened that we found an unprecedented relation between patients' hemoglobin (Hb) and CD55 expression on RBCs (rs= -0.205, p=0.029), while there was a significant difference (δ) when the mean concentration of Hb was compared between patients with normal expression of CD55 and those with deficiency of this protein (δ=-1.4534 g/dl, p=0.0151). There was no clinical or laboratory evidence of hemolysis in our patients. There was no association between the presence of “PNH-like” red-cell populations and cytopenias or specific treatment for the autoimmune disorder. Positive Ham and sucrose tests were found only in PNH patients. In conclusion, this study provides evidence supporting the presence of erythrocytes with CD55- and/or CD59- deficiency in patients with rheumatic diseases. The pre-existence of small PNH clones in the bone marrow of these patients, that acquire a survival advantage to proliferate against normal hemopoietic tissue and become detectable with our methodology, may be the underlying cause for this phenomenon. Moreover, it was demonstrated that CD55- deficiency on RBCs influences the levels of Hb, in these patients. Further studies, using molecular techniques, will be required, to clarify the exact pathophysiologic mechanisms for this deficiency. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Microscopic peripheral blood and bone marrow cell evaluation remains a milestone in the diagnostic of hematology. Many factors contribute to the lack of standardization of this diagnostic test, such as differences in the procedure, staining, degree of skill in interpretation and glossary. However the new WHO classification highlights the importance of morphological aspects, quantitative as well qualitative, for a better stratification of patients in the diagnosis of haematological malignancies, particularly myeloid malignancies and above all myelodysplasia (MDS). In this high technology era, we have the opportunity to exchange, via the internet, images and information without geographic limitation, sparing time and resources. This represents a great opportunity to try to get a consensus for blood cell morphology. The European LeukemiaNet (ELN) Network of excellence is an EU project funded by the 6th FP, involving 174 centres from 28 countries. Its major goal is the construction of a cooperative network for improving leukemia diagnosis, care and research. Within the activities of the Diagnostic Platform (WP10), focused on Flow Cytometric and Morphological panels, a European Morphology Consensus Faculty (EMCF), composed of 21 expert morphologists from 13 European countries, has been organized with the following goals: to harmonize the identification of hematological cells for a common European morphological diagnostic pathway, in terms of cell lineage, maturation level, normality/abnormality and glossary, to take into account specific national skills, competences and methods, to provide the patients with the same morphological diagnosis all over Europe. The first step was to create a consensus-based cell library of meaningful blood cell images identified and named by top level European morphologists and agreed by EMCF, as a valuable tool of traininig. We are presenting the final results of the first study of the EMCF. Methods: From May to July 2007 we collected from all the EMCF members 164 images containing 438 labelled blood cells with the following distribution: Granulocytic series n=126 (28.5%), Erythroid series n=77 (17.5%), Monocytic series n=35 (8%), Lymphoid series n=107 (24.,5%), Megakaryocytic series n=23 (5.,5%), Blasts (not otherwise specified) n=29 (6.5%) Other n= 41 (9.5%). All the images were uploaded to a restricted web page together with an Excel file containing the author’s proposal for each cell. Faculty Members were asked to fill the Excel file with their agreed or personal alternative definition for each labelled cell. On January 2008 cells lacking a full agreement were submitted to a Delphi scoring procedure: a minimum of 3 agreements was needed for a cell to be scored in the Delphi questionnaire. By July 2008 we have received the Delphi files for the final evaluation of the study results. Evaluation rules: Full consensus: cells with an agreement of at least 17 out of 21 (〉80%) EMFC members. Delphi questionnaire for all the cells with an agreement 59% of all submitted cells. The main discrepancies in the morphological consensus concern the groups of blast and monocytic series, while for the other groups of cells the distribution is similar. After the Delphi scoring we reached an agreement on all the 216 submitted cells. The EMCF created a new category “Cell to delete” for 8 cells due to a not perfect quality of images. This image library is composed of 430 cells now identified through a consensus method by a European Morphology Consensus Faculty and the cells will be uploaded onto the ELN web site, with a clear identification of those cells identified with a Delphi scoring procedure. Moreover cell names are harmonized and this represents the first morphological European glossary. Future developments: The second extended Faculty including morphologists from 16 European countries has been formed with the aim to apply the consensus glossary to identify a new set of cells submitted by the authors without any suggestions. A meeting is planned to discuss the results.

Description: Abstract 1600 Background: It is well known that Fc gamma receptors expressed on monocytes and macrophages enhance the presentation of antibody-bound antigens. Human Fc gamma receptors are coded by genes located on chromosome 1q23. Fc gamma RIIA (CD32) is a transmembrane protein expressed on neutrophils. Efficacy of phagocytosis by polymorphonuclear neutrophils is known to vary between allotypes of Fc gamma RIIa. Polymorphisms of Fc gamma RIIA have been linked to susceptibility to infections by encapsulated bacteria (N. meningitides, H. influenzae) and severe sepsis by several authors. Moreover, susceptibility to perinatal HIV-1 infection has been attributed to polymorphisms of Fc gamma RIIA, and this is the only association of FC gamma RIIA with viral diseases in the literature. FcγRIIA gene has two condominantly expressed alleles the 131-Arg (R131) and the 131-His (H131) allele. R131 binds IgG2 much less avidly than H131. Aim of the study: To propose Fc gamma RIIA polymorphisms as a genetic risk factor for acquisition and latency of EBV infection in patients with lymphoproliferative diseases. Patients and Methods: Forty Greek patients with EBV-unrelated low grade B-cell leukemic lymphomas (chronic lymphocytic leukemia: 23, marginal zone lymphoma: 11, mantle cell lymphoma: 3, hairy cell leukemia: 2, follicular lymphoma: 1), were included in the study. Patients' sera were tested with ELISA for the presence of EBV-VCA IgG antibodies. DNA from peripheral blood was studied by quantitative real time - PCR for the EBV-R gene, while RNA from peripheral blood was studied by RT-PCR for the EBV-LMP1 oncoprotein. Genomic DNA from peripheral blood was tested for Fc gamma RIIA 131Histidin(H)/arginine(R) SNP, by PCR, followed by restriction fragment length polymprphim (RFLP) using the appropriate enzyme (BstUI, Fermentas, Vilnius, Lithuania). We used Pearson Chi-square for the statistical analysis of the results. Results: All but 2 patients were positive for EBV-VCA IgG antibodies. Nineteen patients (47.5%) were EBV-positive. LMP1 was expressed in 13/19 (68.4%) EBV-positive patients. The vast majority (16/19, 84.2%) of EBV positive patients carried the R131 allele (13 were heterozygous and 3 homozygous), while only 3 were homozygous for the H131 allele. Among 21 EBV-negative patients, only 6 (28.5%) carried the R131 allele (4 heterozygous and 2 homozygous) (2-sided p=0001). R131 allele was present in 11/13 (84.6%) LMP1-positive patients in comparison to 6/21 (28.0%) EBV-negative patients (2-sided p=0.002). Discussion: The high prevalence of FC gamma RIIA polymorphisms among EBV-positive patients indicates a possible pathogenetic role of the FC gamma RIIA in acquisition and chronicity of EBV infection. LMP1 is the major oncoprotein of EBV. The correlation of this polymorphism to LMP1 expression is an indication that it may play a major role in the expression of latency phase proteins, a fact that may further be implicated in the pathogenesis of lymphoproliferative diseases in these patients. We continue the study in our centre, increasing the number of patients enrolled. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3205 Background: Hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disorder characterized by elevated serum L-ferritin and early onset bilateral cataracts. The syndrome, first described in Italy and France in 1995, is caused by mutations in the iron responsive element (IRE) of the L-ferritin gene (FTL-ferritin light chain) on chromosome 19q13.3. The conformational changes in the mutated IRE disrupt the binding of the iron regulatory proteins (IRPs) to the IRE, resulting in loss of the posttranscriptional control in L-ferritin synthesis. L-ferritin is constitutively expressed in tissues, irrespective of cellular iron status and without otherwise disrupting cellular iron metabolism. Despite the five to twenty-fold increase in serum L-ferritin, there is no clinical or laboratory evidence of iron overload. Serum iron and transferrin saturation remain within normal limits. Visual impairment and early cataract formation is the only clinical symptom of HHCS, with lens replacement being the only treatment needed. Extensive and invasive investigations in the undiagnosed hyperfferitinemic patient, as well as unnecessary treatment with phlebotomies, can be avoided with awareness of the syndrome, leading to the correct clinical and molecular diagnosis. Aim: HHCS has been previously reported in a limited number of patients of Greek origin, with the study of three unrelated families in Greece. Papanikolaou et al. (Blood Cells Mol Dis. 2006) detected the C39〉G mutation in the IRE of the FTL in all affected individuals. Our aim was to further investigate the syndrome within the Greek population, both phenotypically and genetically. Methods: We investigated 81 patients with undiagnosed hyperferritinemia referred to the Hepatology Outpatients Clinic of the 1st Department of Internal Medicine at “Laikon” General Hospital in Athens. For all patients full medical history and clinical examination (including ophthalmological evaluation) was performed, and laboratory investigations were tailored accordingly. Where indicated from the medical history and laboratory findings, molecular analysis for mutations in the IRE of the FTL was performed using direct DNA sequencing. Results: From the total of 81 patients referred for undiagnosed hyperferritinemia, 18 unrelated subjects had a positive history of cataract and elevated ferritin. In those subjects ferritin levels ranged from 600 to 2070μg/L (normal values: 12–160μg/L). Serum iron was within normal limits and transferrin saturation ranged from 9 to 40%. Cataract diagnosis was reported as early as 5 years of age and 8 out of the 18 subjects had undergone surgery for lens replacement at the time of referral. From the 18 subjects that were screened for mutations in the IRE of the FTL, mutations were detected in 12 unrelated subjects. Ten out of the 12 individuals were positive for the previously described C39〉G point mutation. The other two subjects were found to carry the A40〉G mutation. The A40〉G mutation has been described in the literature as part of the spectrum of mutations causing HHCS (Beaumont et al, Nat. Genet. 1995), however it has not up to now been reported in subjects of Greek origin. From the 12 subjects positive for either mutation, 8 reported positive family history for either early onset cataracts, hyperferritinemia or both. Where possible, clinical, laboratory and molecular investigations were extended to other members of these families. A total of 40 subjects from the members of the studied families fulfilled the clinical criteria of HHCS. Genetic testing was performed in 12 of them, revealing 11 subjects heterozygous for the C39〉G mutation and 1 heterozygous for the A40〉G mutation. Conclusion: HHCS is a non-iron loading, rare disorder that should be considered in all patients with unexplained hyperferritinemia, in order to avoid unnecessary investigations, potentially hazardous treatment and confer reassurance to the patient. Our findings contribute to the clinical and molecular characterization of the syndrome and extend the study of the disorder in the Greek population. Disclosures: No relevant conflicts of interest to declare.

Description: Bone involvement is the commonest clinical manifestation of sickle cell disease (SCD) including chronic disorders, such as osteopenia/osteoporosis. The aim of the present study is to evaluate the bone mineral density (BMD) of patients with SCD/β-thalassemia (S/β-th) in parallel with markers of bone turnover in an attempt to better understand the pathophysiology of bone loss in these patients. We studied 52 patients with S/β-th (23M/29F; median age 40 y). The BMD of the lumbar spine (L) and femoral neck (F) was evaluated by DEXA. Bone remodeling was assessed using the following serum indices: (a) bone resorption markers [C-telopeptide of type-I collagen (CTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)], (b) bone formation markers [bone-alkaline phosphatase (bALP), osteocalcin (OC), and C-terminal propeptide of collagen type-I (CICP)], and (c) osteoclast stimulating factors [soluble receptor activator of nuclear factor κB ligand (sRANKL), and osteoprotegerin (OPG)]. Moreover, all patients had a thorough evaluation of their renal function (creatinine clearance, and cystatin-C serum levels), bone marrow expansion (soluble transferin receptors, sTfR), serum erythropoietin (Epo) and parathyroid hormone (PTH) levels. The same biochemical parameters were also determined in 25 age- and gender-matched controls. According to WHO criteria, 17 patients (32.6%; 8M/9F, median age 45 y) had osteopenia or osteoporosis (median L/T-score: −2.26; L/BMD: 0.9 g/cm2; F/T-score: −1.86; F/BMD: 0.68 g/cm2). In contrast, 30 patients (57.6%; 12M/18F, median age 40 y) had osteosclerosis (median L/T-score: +3.15; L/BMD: 1550.5 g/m2; F/T-score: +0.52; F/BMD: 1113.5 g/cm2). Renal function, Epo, Hb and sTfR levels and the number of crises/year were similar between both groups. All patients displayed increased levels of OPG, bALP, and CICP compared with controls (p

Description: Osteoporosis in thalassemia major (TM) is due to several factors, including bone marrow expansion, iron overload, hormonal deficiency, and increased osteoclast activity. Potent inhibitors of osteoclast function, such as bisphosphonates, have been recently used in the management of TM-induced osteoporosis. The aim of this trial was to determine the efficacy and safety of a third-generation aminobisphosphonate, zoledronic acid, on osteoporosis and bone remodeling in patients with TM. Sixty-six patients with TM-induced osteoporosis (21M/45F; median age 35.5 years) have been enrolled in this study. The majority of patients had pathological fractures and/or bone pain due to osteoporosis at baseline. Patients were randomized to receive zoledronic acid at a dose of 4 mg, iv, in 15 min infusion, every 6 months (23 patients; group A) or every 3 months (21 patients; group B), or to receive placebo every 3 months (22 patients; group C), for a period of one year. All patients were under oral calcium (500 mg) administration during the treatment period. Effects were monitored by measuring bone mineral density (BMD) in comparison with a series of serum bone remodeling indices: i) bone resorption markers [C-telopeptide of type-I collagen (CTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)], ii) bone formation markers [bone-alkaline phosphatase (bALP), osteocalcin (OC), and C-terminal propeptide of collagen type-I (CICP)], and iii) osteoclast stimulating factors [receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG)]. BMD of the lumbar spine, femoral neck and forearm was determined using DEXA, before and 12 months after treatment. The above biochemical parameters were measured at baseline and before the administration of zoledronic acid or placebo in each cycle. We also evaluated these markers in 30 healthy, age and gender-matched, controls. All patients had increased values of CTX, TRACP-5b, bALP, CICP, sRANKL, OPG, and sRANKL/OPG ratio, compared with controls (p