ALBERT

Beschreibung: In order to establish an efficient gd T cell-mediated immunotherapy for hematological malignancies, we tried to clarify whether γδ T cells could be expanded from blood cells of patients with myeloma, lymphoma and acute leukemia by culture with zoledronate and a low dose of IL-2 and whether the expanded patients’ γδ T cells could kill tumor cells including self tumor cells with sparing normal clone cells. In addition, we explored the methods to enhance the anti-tumor cytotoxicity of the expanded γδ T cells by activating them with type I IFN, monocyte-derived dendritic cells (mo-DCs), or ab T cells. Although γδ T cells could be expanded in patients with myeloma, lymphoma and leukemia as well as normal persons, the amplification rates of gd T cells before and after the culture were varied from patient to patient in the patients with hematological malignancies. γδ T cells generated in patients with myeloma and lymphoma showed a potent cytotoxic ability against myeloma/lymphoma cell lines (RPMI8226, Daudi) as shown in γδ T cells generated in normal persons. In addition, γδ T cells generated in a patient with myeloma and acute leukemia showed a cytotoxic ability against self myeloma or leukemia cells freshly prepared from bone marrow. However, the same γδ T cells were not cytotoxic to normal lymphocytes of the patients. Then the expanded γδ T cells were stimulated with type I IFN, mo-DCs, or αβ T cells and the activation (CD69 expression) and cytotoxicity against tumor cells were examined. By the stimulation with type I IFN, the expression of CD69 and Trail of γδ T cells was increased and the cytotoxic ability of γδ T cells was enhanced at dose-dependent manner of type I IFN. CD69 expression on γδ T cells was enhanced by co-culture with both immature and mature mo-DCs in a cell-number-dependent fashion. CD69 expression was enhanced after the addition of mo-DCs of either autologous or allogeneic origin. Activation of γδ T cells with mo-DCs enhanced anti-tumor cytotoxicity of γδ T cells against RPMI8226 and CML blastic crisis cell line (C2F8) in an effector-to-target ratio-dependent manner. Although CD69 expression of γδ T cells was enhanced by the co-culture with allogeneic ab T cells, autologous ab T cells couldn’t activate γδ T cells. However, autologous ab T cells stimulated with IL-2 or PHA could induce the activation of γδ T cells. The activation of γδ T cells with stimulated αβ T cells required cell-to-cell interaction. These findings suggested that αβ T cells stimulated by allogeneic γδ T cells could activate the same allogeneic γδ T cells. The present data demonstrated that γδ T cells, which could be expanded in vitro from blood cells of the patients with myeloma, lymphoma and leukemia by culture with zoledronate and IL-2, possess an enough cytotoxic ability against tumor cells including self tumor cells with sparing normal cells. These findings suggested that in vitro generated patients’ γδ T cells could be applied to γδ T cell-mediated immunotherapy for hematological malignancies. Besides, potent γδ T cells activated by type I IFN, mo-DCs or activated αβ T cells were considered to be applicable for γδ T cell-mediated immunotherapy.

Beschreibung: Vascular endothelial growth factor (VEGF), which induces angiogenesis and increases vascular permeability, is a major growth factor mediating tumor progression. In this study, we employed immunohistochemical-staining method to detect the expression of VEGF in lymph nodes taken from39 non-Hodgkin’s lymphomas patients and analyzed the relation of the expression levels to malignant aggressiveness, treatment response, histological grade, clinical stage and prognosis. The patients had been observed for at least 5 years or until death. 9 patients with benign lymphadenopathy were acted as control. The expression of VEGF was assessed according to the percentage of immunoreactive cells in a total of 1000 neoplastic cells (quantitative analysis). Immunoreactivity was graded positive, more than 10% of carcinoma cells stained and negative, no detectable staining or less than 10% of carcinoma cells stained. Furthermore, the qualitative intensity of staining for VEGF was assessed using a scale of 0–3+. The expression analysis of VEGF revealed that in 31 out of 39 (79.49%) specimens VEGF staining was positive. The VEGF staining was always cell membrane. Significant associations were found between the expression of VEGF and histological grade, Ann Arbor stage, prognosis (according to International Prognostic Index, IPI) and chemotherapy response. Among 8 cases of low grade, 7 had lower-level expression and 1 had higher-level expression, but among 31 cases of intermediate and high grade, 13 had lower-level expression and18 had higher-level expression (P=0.044

Beschreibung: We examined a large cohort (N=2,457) of chronic lymphocytic leukemia (CLL) patients evaluated by the CLL Research Consortium (CRC) and found 63 (2.6%) used IGHV3-21. Comparing the Ig heavy chain third complementarity determining region (HCDR3) of the IGHV3-21 cases: 25/63 cases (39.7%) had a conserved amino acid motif (motif 1: DANGMDV) in the otherwise highly variable Ig HCDR3, as described by Tobin et al. Blood 2003. All but one of these Ig heavy chains (IgH) were paired with a lambda light chain encoded by IGLV3-21. In addition, we found that 3/63 cases (4.8%) had a previously unrecognized conserved HCDR3 amino acid motif (motif 2: DPSFYSSSWTLFDY). In contrast, these IgH invariably were paired with kappa immunoglobulin light chains (IgL) encoded by IGKV3-20. Similarly to that noted for CLL cases that use IgH encoded by unmutated IGHV1-69 (Widhopf et al. Blood Epub First Edition 2007), the pairing of IgH encoded by IGHV3-21 with IgL appears governed by the HCDR3. The non-stochastic pairing of IgH with IgL argues strongly that antigen plays a role in selecting the Ig expressed in CLL. To examine for the antigen(s) recognized by the most common Ig encoded by IGHV3-21, we isolated IgH and IgL genes expressed by IGHV3-21/IGLV3-21 CLL cases and generated recombinant antibodies, which we examined for binding to antigen(s) present on microarray of self or environmental antigens. We found that Ig encoded by IGHV3-21/IGLV3-21 had apparent specific binding for protein L, a multi-domain cell-wall protein isolated from Peptostreptococcus magnus, a Gram-positive commensal bacteria that comprise a large portion of the human bacterial gut flora. Prior studies identified that protein L is a superantigen capable of binding human Ig kappa light chains encoded by IGKV genes of the I, III, and IV subgroups, but not human Ig lambda light chains. The specific binding of IGHV3-21/IGLV3-21 to protein L suggested that protein L might play a role in the development of CLL cells that express such Ig. To test this hypothesis, we examined the capacity of various recombinant antibodies to bind protein L by ELISA. We found that lambda IgL encoded by IGLV3-21 could bind to protein L with similar activity, independent of whether this lambda IgL paired with the native IgH, IgH encoded by IGHV3-21 lacking the DANGMDV HCDR3 motif, or even irrelevant IgH encoded by IGHV4-39 that are not found paired with IGLV3-21 in the Ig expressed in CLL. Moreover, Ig formed by pairing IgH encoded by IGHV3-21 that has the DANGMDV HCDR3 motif with an IgL encoded by an IGLV that was irrelevant to IGLV3-21 did not bind protein L. These results reveal a previously unrecognized capacity of human IgL encoded by IGLV3-21 to bind the protein L superantigen of Peptostreptococcus magnus, a bacteria commonly found in the human gastrointestinal tract. However, because the binding of IGLV3-21 does not depend upon the non-stochaistic pairing of IgH and IgL observed in CLL, we reason that the capacity of IGLV3-21 to bind protein L cannot account for the selected Ig repertoire expressed in CLL, suggesting that it actually does not play a role in CLL leukemogenesis. This finding suggests that caution should be exercised when defining an antigen that is found capable of binding the restricted Ig expressed in CLL as the driving factor responsible for leukemogenesis.

Beschreibung: Background: Heavy chain disease (HCD) is a rare lymphoproliferative disorder characterized by a monoclonal heavy chain (HC) unattached to a light chain (LC). IgGHCD or γHCD typically presents as a lymphoproliferative disorder with lymphadenopathy and hepatosplenomegaly. Myeloma has been described associated with γHCD but only with a second intact Ig paraprotein. This report describes a unique presentation of multiple myeloma with monoclonal free γ3HC and kappa free light chains. Case: A 34 year old gentleman presented with mild persistent neutropenia following two episodes of pneumonia, 18 months previously. He admitted to persistent night sweats but no other significant history. Baseline investigations revealed a mild anaemia, neutropenia and a large IgG paraprotein with no associated light chain. Bone marrow aspirate and trephine confirmed myeloma. The patient was treated with cyclophosphamide, thalidomide and dexamethasone and has had a very good partial remission. He is awaiting a sibling allogeneic peripheral blood stem cell transplant. Investigations and results: Serum Electrophoresis confirmed a large IgG paraprotein (23g/l) with no associated light chain in the serum and identified as γ3 subclass by radial immunodiffusion. Western blot showed the γ3HC was truncated with a large deletion. Markedly elevated free kappa (κ) LC (503.58 mg/l [3.30–19.4]) were found in the serum with gross skewing of the kappa/lambda ratio. Urine electrophoresis revealed separate γHC and κ LC paraproteins. Western blot of the fractionated urine protein demonstrated different sized κLC aggregates. Flow cytometry of the marrow aspirate revealed an unusual staining pattern; CD5,19,38,45+ve and CD20,22,23,34,56,138 –ve plasma cells. Cytoplasmic staining revealed 2 distinct populations of plasma cells, the first producing γ3HC and the second only free κLC. Cytogenetics and FISH analysis for 14q, p53 and c-myc abnormalities were normal. Discussion: This is the first description of a Biclonal Myeloma with separate plasma cell populations producing γ3HC and κLC paraproteins. The biclonality confirms the free HC occurs as a result of abnormal synthesis not cleavage. The clinical and immunological findings are clearly different to typical findings in both γ3HCD and Myeloma. HCD has an appalling prognosis and this case is likely to have been ‘smouldering’ for 18 months, evidenced by the 2 pneumonias and persistent night sweats. There is no lymphadenopathy or organomegaly associated with γ3HCD. The immunophenotype of the malignant plasma cells is unique. Other atypical features include frank proteinuria, with a HC in the urine, but normal renal function and no radiological or biochemical evidence of bone involvement. We propose that this unique biclonal myeloma has distinct immunological and clinical features.

Beschreibung: Background: Recent advances in the therapy of multiple myeloma (MM) have greatly increased the treatment options for this uniformly fatal plasma cell malignancy. It is not clear if introduction of novel therapies and the increased use of high dose therapy (HDT) in the past decade have translated into better outcome for patients (pts) with MM. Methods: We examined the outcome of two cohorts of pts seen at our institution. The first cohort consisted of 387 pts who relapsed after HDT and was examined for potential improvement in survival following relapse after HDT. These pts were divided into two groups; those who relapsed before or after December 31, 2000. The second cohort consisted of 2981 patients with newly diagnosed MM seen between January 1971 and December 2006 and was used to examine the trends in overall survival (OS). Results: Among those relapsing after HDT, there were 245 males (63%); median (range) age at HDT was 57 years (32.8–75.4) and the median time to HDT was 8.1 months (1–90 months) from diagnosis. The median time to relapse was 13.2 months (1.1 months-10.3 years) from HDT. In this cohort, a clear improvement in OS from time of relapse was seen over the past decade, with those relapsing after 2000 having a median OS of 23.9 months (95% CI; 19.8, 27.6) compared to 11.8 months (95% CI; 8.7, 14.9) for the rest (P 〈 0.001) (Figure 1). In a multivariate analysis, the effect of the date of relapse on survival was independent of other prognostic factors such as relapse 5.5 mg/dL. Pts who were treated with one or more of the newer drugs (thalidomide, lenalidomide, bortezomib) had longer survival from relapse (30.9 months (95% CI; 23.6, 38.2) compared to 14.8 months (95% CI; 11.3, 18.4); P 〈 0.001) for others. Among the newly diagnosed MM cohort, the median age at diagnosis was 66 years (20.2 – 97 years), and 1,770 (59.4%) were males. The median follow up for the entire group was 27.4 months (0–29.4 years); and at the time of analysis 558 patients (18.7%) were alive with a median follow up of 32.7 months (0–29.4 years). Pts diagnosed in the last decade had an improved OS (44.8 months) compared to those diagnosed before this period (29.9 months; P 〈 0.001) (Figure 2). The improvement in survival seen in the last decade among newly diagnosed patients was predominantly among those younger than 65 years (60.3 mos vs. 33.3 mos improvement in median survival); compared to those over 65 at diagnosis (26.5 mos vs. 32 mos). Conclusion: In this study, for the first time, we demonstrate definite proof for improved outcome in patients with myeloma, both in the relapsed setting as well as at diagnosis, a change that is likely to continue with increased use of these drugs. Figure Figure Figure Figure

Beschreibung: Absence of shear stress due to disturbed blood flow at arterial bifurcations and curvatures leads to endothelial dysfunction and proinflammatory gene expression, ultimately resulting in atherogenesis. KLF2 has recently been implicated as a transcription factor involved in mediating the anti-inflammatory effects of flow. We investigated the effect of shear on basal and TNF-α–induced genomewide expression profiles of human umbilical vein endothelial cells (HUVECs). Cluster analysis confirmed that shear stress induces expression of protective genes including KLF2, eNOS, and thrombomodulin, whereas basal expression of TNF-α–responsive genes was moderately decreased. Promoter analysis of these genes showed enrichment of binding sites for ATF transcription factors, whereas TNF-α–induced gene expression was mostly NF-κB dependent. Furthermore, human endothelial cells overlying atherosclerotic plaques had increased amounts of phosphorylated nuclear ATF2 compared with endothelium at unaffected sites. In HUVECs, a dramatic reduction of nuclear binding activity of ATF2 was observed under shear and appeared to be KLF2 dependent. Reduction of ATF2 with siRNA potently suppressed basal proinflammatory gene expression under no-flow conditions. In conclusion, we demonstrate that shear stress and KLF2 inhibit nuclear activity of ATF2, providing a potential mechanism by which endothelial cells exposed to laminar flow are protected from basal proinflammatory, atherogenic gene expression.

Beschreibung: Tumor cell–associated tissue factor (TF) is a powerful determinant of metastatic potential. TF may increase metastasis by supporting thrombin-mediated proteolysis, through intracellular signaling events mediated by the TF cytoplasmic domain, through TF/fVIIa/fXa–mediated activation of protease-activated receptors, or through a combination of these processes. To better define the relationship between tumor cell-associated TF and circulating hemostatic factors in malignancy, we generated a set of C57Bl/6-derived tumor lines genetically lacking TF, expressing wild-type murine TF, or expressing a mutant TF lacking the cytoplasmic domain. Comparison of the metastatic potential of these cells in immunocompetent mice with genetic deficits in prothrombin, platelet function, or fibrinogen revealed that TF supports metastasis through mechanisms independent of the cytoplasmic domain, but dependent on each of these distal hemostatic factors. TF was neither required for primary tumor growth nor necessary for initial localization of embolized tumor cells within the lungs. Rather, tumor cell fate studies indicated TF supports metastasis by increasing the survival of micrometastases. One mechanism linking TF to metastasis is through a fibrin(ogen)-dependent and platelet-dependent restriction in natural killer cell–mediated clearance of micrometastases. However, TF also supported the early success of micrometastases through an additional mechanism independent of natural killer cells, but coupled to circulating prothrombin.

Beschreibung: GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Beschreibung: Polycythemia vera (PV) and essential thrombocythemia (ET) are two common types of myeloproliferative disorders (MPD). The prevalence of PV and ET in the United States (US) has not been well documented. Recent breakthroughs in the molecular etiology of these disorders and the accelerated development of targeted pharmacotherapeutics to treat the MPD have significantly increased the need to accurately define the affected population. In the present study, we obtained detailed demographic and health claims data from major commercial insurance payers in Connecticut and the Center for Medicare and Medicaid Services to estimate the prevalence of PV and ET. Health claims data from one payer and the actual diagnoses made by physicians who submitted claims with MPD-related ICD-9 codes were utilized to develop claim-based statistical algorithms to predict the probability that an individual with claims suggestive of MPD truly has PV or ET. Specifically, logistic regression was used to develop the algorithms, and area under the receiver operating characteristics curve (AUC) was used as the measure of goodness-of-fit for each model. Different models were fit, and the model with the highest AUC was selected. For PV, the best-fitting model included age as a continuous variable and the frequency of two PV-related ICD-9 codes (238.4 and 289.6 combined) as a continuous variable, and the AUC was 0.95. For ET, the best-fitting model included age as a continuous variable and the frequency of code 289.9 as a categorical variable (=0, =1, or ≥2), and the AUC was 0.72. For both models, the addition of gender or the frequency of the non-specific code 238.7 did not improve AUC. Subsequently, the algorithms were applied to health claims from multiple payers to estimate the number of PV and ET patients in Connecticut. The total number of Connecticut residents included in the study was close to 2,900,000, which represented about 83.2% of the estimated population of the entire state in July 2003. As of 2003, the age-standardized prevalence of PV and ET in Connecticut was 22 per 100,000 and 24 per 100,000, respectively. Applying the age-specific prevalence of PV and ET to the entire US population resulted in an estimated total of 65,243 patients with PV and 71,078 patients with ET in the US in 2003. This study is the first to assess the prevalence of PV and ET in a very large US population. Given the large number of individuals afflicted with these diseases and the fact that demographic changes alone will further increase the burden of these diseases in the foreseeable future, it is imperative to conduct more systematic research into the etiology and treatment of PV and ET.

Beschreibung: Background: Neurologic complications of multiple myeloma (MM) are numerous, however little is known about strokes occurring in the course of MM and its treatment. Methods: 2877 MM patients were seen for an initial evaluation at our institution over a 5-year period (1998–2002). Patients were identified using discharge summaries by combining ICD-9 codes for MM and stroke. They were included if they had clinical and radiological features of acute stroke. Ischemic stroke was defined as a new acute neurologic deficit lasting 〉 24 hours, irrespective of diffusion-weighted MRI results. Diagnosis of hemorrhagic stroke was made in patients with new acute neurologic deficit plus evidence of hemorrhage on CT scan. We retrospectively reviewed medical records for demographics, type of MM and treatment, stroke type, clinical features, relevant imaging and laboratory data, and outcomes. Results: The 11 patients that we identified included eight with ischemic strokes and three with subarachnoid hemorrhages (SAH). There were no patients with intraparenchymal hemorrhage. The overall incidence of stroke in this cohort was 76/100,000 per year, whereas the incidence of ischemic stroke was 56/100,000 per year. The mean age of patients with ischemic stroke was 59 years. Seven had one or more stroke risk factors and an equal number had received thalidomide. The latter were given small doses of coumadin as part of the treatment protocol; at the time of the stroke, INR range was 0.9–1.4. Based on clinical, radiological and other laboratory features, of the eight patients with ischemic strokes, two were presumed cardio-embolic (one had infective endocarditis), five were thrombotic, and one was caused by hypoperfusion. Two patients had findings of severe stenosis/occlusion in vessels corresponding to the infracted brain territory (internal carotid and basilar arteries). Two patients had documented normal plasma viscosity and three had evidence of extra-cranial thrombosis at the time of the stroke. In the three patients with SAH, hemorrhage occurred in the setting of trauma and thrombocytopenia, although one patient had an incidental anterior communicating artery aneurysm. Seven patients were left with minor or no deficits and four died, two in each of the groups. Conclusion: Overall, strokes did not appear to be more common in MM patients than in the general population, and the pathophysiology was likewise, not different than in patients without MM, with the exception of one patient with infective endocarditis, which is probably directly related to MM or its treatment. The highly selected referral population at our myeloma institute, and the retrospective nature of the study probably underestimate the incidence of stroke. The role of thalidomide in stroke remains unknown. Thrombocytopenia and trauma are probable risk factors for subarachnoid hemorrhage in MM patients. Fatal outcomes were frequent, but when death did not occur, the deficits were minor.

Beschreibung: Recent advances in the diagnosis, molecular pathogenesis, classification and therapy have been made in the field of myelodysplastic syndrome (MDS) and juvenile myelomonocytic leukemia (JMML) in childhood. We report a retrospective analysis of children with MDS and JMML diagnosed between 2001 and 2006 in Korea. In total, 135 patients were enrolled from 19 major hospitals with pediatric hematology oncology clinics: MDS, 96 (primary MDS, 77; constitutional anomalies with MDS, 13; treatment-related MDS, 6) and JMML, 39. The incidence of MDS/JMML was around 22.5/year, which is about 6% of childhood leukemia. Various classification systems including FAB, WHO, IPSS, CCC system, and pediatric adjustment of the WHO classification were applied. The median ages at diagnosis were 68 and 10 months in MDS, and JMML, respectively. Males dominated in JMML. Cytogenetic abnormalities were observed in 43% of MDS (monosomy 7, 5; trisomy 8, 3) and in 10% of JMML. Treatment was chosen by each institute’s preference: 34 patients with MDS received AML-type intensive chemotherapy, with complete remission rate of 82.0%. The 5-year Kaplan-Meier overall survival rate was 54% each for MDS and JMML. Survival of MDS patients was influenced by the marrow blast % (P = 0.007) and disease category (P= 0.006). Stem cell transplantations (SCT) were undertaken in 56 patients (MDS, 29; JMML, 27). The sources of stem cells were: bone marrow, 36; umbilical cord, 18; peripheral blood, 2). Matched related transplants were 9 cases. Conditioning was various, but BuCy based regimen was used in 68.4%. Acute GvHD ≥ Grade II was found in 43.8% and chronic GvHD in 35.1%. The 5-year Kaplan-Meier overall survival rate was 55% for MDS, and 57% for JMML. Survival after unrelated transplant was comparable with that of matched related transplants. This analysis inspired the necessity of nation-wide prospective studies in Korea, including morphologic study by a central pathology review board, epidemiologic study, molecular pathophysiologic study, and therapeutic trials incorporating SCTs, or new drugs.

Beschreibung: The somatic mutation JAK2 V617F has been identified as a pathogenic factor in typical chronic myeloproliferative diseases (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMF). In typical forms of myelodysplastic syndromes (MDS), JAK2 V617F mutation is rarely present (2–5%); on the contrary, it has been found with higher prevalence in patients with RARS-T (i.e. MDS/MPD-U with platelet count 〉600×109/L and ringed sideroblasts more than 15%) and in a subgroup of MDS patients with isolated 5q deletion and a proliferative bone marrow. In this study we analysed the JAK2 V617F mutational status in 53 MDS patients (26 males, 27 females; median age at the time of the study 76 years, range 45–91). Patients were classified as follows: 4 cases 5q- syndrome, 3 RCMD, 5 MDS/MPD, 1 MDS-U, 23 RA, 12 RARS, 5 RAEB. DNA was extracted from purified granulocytes; all samples were analyzed by allele-specific polymerase chain reaction (PCR), according to Baxter el al (2005). DNA samples were further subjected to direct sequencing for confirmatory testing. The JAK2 V617F mutation was present in 3 cases, with an overall frequency of 5%. With respect to MDS subtype, 1 patient had RA and 2 RARS. Among the 12 RARS patients, the two V617F postive displayed thrombocytosis (680×109/L and 649×109/L), whereas none of the 10 RARS V617F negative patients showed high platelet counts (median Plt 157×109/L, range 5–422×109/L). In one JAK2 mutant case, thrombocytosis required treatment with hydroxyurea. Moreover, the two V617F positive RARS patients displayed higher WBC count (6.2×109/L and 8.5×109/L) than the V617F negatives (median WBC 4.05×109/L); no difference was observed in Hb levels. The JAK2 positive RA patient had 10% of sideroblasts in bone marrow, normal platelet and WBC count and no proliferative characteristics; since the occurrence of the mutation may be an early event and preceed the classical manifestations of MDS/MPD, a longer follow-up is necessary to determine its possible prognostic significance. Considering the V617F negative MDS cases, only one patient, diagnosed as MDS/MPD, showed a platelet count 〉600×109/L. In conclusion, we confirmed recent reports showing that JAK2 V617F is present with low prevalence (about 5%) in MDS; in particular, the JAK2 mutation identifies a subset of MDS patients with and “overlap” syndrome, characterised by proliferative bone marrow morphology and frequent thrombocytosis and leucocytosis, who may benefit from JAK2 specifically targeted therapies.

Beschreibung: The simultaneous manifestation of different lymphomas in the same patient or in the same tissue is defined composite lymphoma. Although reports of synchronous or metachronous Hodgkin’s lymphoma (HL) and Non Hodgkin’s lymphoma (NHL) are not uncommon in the literature, the biologic relationship of the 2 malignancies is often unclear. Primary cutaneous B-cell lymphomas (pCBCLs) have been recognized as distinct clinicopathologic entities; they represent a wide spectrum of lymphoproliferative disorders separated from of B-cell NHL secondarily involving the skin and cutaneous B-cell pseudolymphomas. As regarding pCBCLs, a concomitant diagnosis of HL has been described very rarely. This is the case of a caucasian man affected by primary cutaneous follicolar B cell lymphoma (pCFCL). He presented grouped red plaques located on the nape, abdomen, shoulders, arms and even some little tumors surrounded by erythematous papules. Complete staging procedures did show no evidence of extracutaneous disease. A subcutaneous interferon therapy was started, in some months the patient reached a complete remission, and a maintainance therapy was continued for about 2 years. After 15 years, at the age of 58, the patient presented a red to violaceous infiltrated solitary plaque on the back, appeared about 2 months before. The lesion was completely excised and the biopsy showed a diffuse dermal infiltrate, not involving the epidermis, structured in follicle with reactive germinal centers; they were surrounded by small sized monomorphic lymphocytes with irregular nuclei and pale cytoplasm, showing the following immunophenotype pattern: CD20+, CD3−, IRTA+, CD10−, BCL6−, BCL2+, low proliferative index. A plasmacellullar CD138+ and CD79a+ population was at the periphery of the infiltrates, with monotypic expression of cytoplasmic k chain. The whole picture was interpreted as primary cutaneous marginal zone B-cell lymphoma (pCMZL). The association with Borrelia burgdorferi infection, sometimes described in pCMZL, wasn’t demonstrated. The patient presented a large right axillary lymph node that was excised and found to be unexpectly infiltrated by HL, mixed cellularity subtype. The patient underwent a standard baseline staging procedure with total body CT scan and bone marrow trephine biopsy; the latter resulted negative; the t(11;14) and t(14;18) rearrangements weren’t demonstrated in bone marrow; the CT did show no other suspected masses nor lymphoadenopathy, besides the clinically evident right axillary lymph node. A 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) revealed sites of hyperactivity in the same right axillary region, but extending to the subclavian region and the thoracic region. The patient started chemoterapy (ABVD regimen), and at the end of 4 courses a whole body CT scan and FDG-PET resulted both negative. Now he is being treated with radiotherapy. If the nodal malignancy would have been diagnosed first, the skin lesion probably could have been misinterpreted as a secondary localization of HL; if the node biopsy wouldn’t have been performed we could diagnose B-cell NHL secondarily involving the skin (stage IV). Nevertheless the cutaneous and nodal infiltrates had a completely different general picture and phenotype. This case probably reflects a HL after 15-year remission of a pCBCL of low grade and the relationship between HL and the preceding pCFCL is not clear: casual or related to genetic predisposition for oncogenic events or favoured by an immunodeficiency state related to the first disease and the previous immunomodulatory therapy.

Beschreibung: Tyrosine kinase inhibitors (TKI) selective for Bcr-Abl, such as dasatinib, imatinib, and nilotinib have had remarkable success in the clinic, potentially shifting the prognosis of chronic myelogenous leukemia (CML) to a manageable chronic disease. With the increase in longevity of CML patients, there is rising concern of co-morbidities that may be influenced by chemotherapy (Force et al., Nature Rev.2007;7:332–340). Recently, congestive heart failure (CHF) and direct cellular cardiotoxicity have been reported in CML patients on imatinib therapy (Kerkela et al., Nature Medicine2006;12:908–916). Ultrastructural mitochondrial abnormalities in cardiomyocytes were observed in CML patients with severe CHF and, interestingly, similar abnormalities were observed in cardiomyocytes of imatinib-treated mice, thus providing a prospective in vivo animal model for imatinib-induced cardiotoxicity. Furthermore, correlative findings of mitochondrial membrane potential loss, decreased cell viability, and increased apoptosis resulted from an array of cell-based assays in imatinib-treated primary rat cardiomyocytes, consequentially affording a supportive, if not predictive, in vitro cardiomyocyte toxicity model. Since imatinib-induced inhibition of the native form of c-Abl kinase was speculated to cause the observed cardiotoxicity and c-Abl is a shared target of dasatinib, imatinib, and nilotinib, the in vitro cardiotoxicity potential of dasatinib and nilotinib at pharmacologically relevant concentrations (0.09 μM and 5 μM, respectively) and up to 10-fold higher concentrations were compared side-by-side with imatinib in primary rat cardiomyocytes. Dasatinib did not significantly affect mitochondrial membrane potential, cell viability, apoptosis, or cellular ultrastructure in vitro, whereas imatinib significantly affected these parameters. Nilotinib at pharmacologically relevant concentration demonstrated decreased cell viability, but differed from imatinib in that mitochondrial membrane potential integrity was not affected under identical experimental conditions. Results suggest that at pharmacologically relevant concentrations, dasatinib does not induce cardiotoxicity, as does imatinib and nilotinib, and the molecular mechanisms of the observed cardiotoxicities may differ between imatinib and nilotinib. Of indirect relation, results from assessing another cardiovascular liability, namely hERG K+ channel blockade, demonstrated that dasatinib, imatinib and nilotinib differentially inhibited the hERG currents in vitro with IC50 of 14.3, 15.6 and 0.66 μM, respectively. These in vitro findings occurred at concentration levels approximately 150, 3 and 0.1-fold the expected human Cmax for the three TKIs, respectively. Thus, although TKI therapies may share similar targeting and clinical indications, differentiating specific toxicity profiles may be predictive of differences in potential clinical adversities.

Beschreibung: Factors regulating which patients become alloimmunized to red blood cell (RBC) antigens are poorly understood. Using a murine model of transfusion, we recently reported that viral-like inflammation with polyinosinic polycytidylic acid [poly (I:C)] significantly enhances RBC alloimmunization. Herein, we tested the hypothesis that poly (I:C) exerts this effect, at least in part, at the level of antigen-presenting cells (APCs). Using a novel in vivo method, we report that in the noninflamed state, most transfused RBCs were consumed by splenic macrophages, with only trace consumption by splenic dendritic cells (DCs). To a lesser extent, RBCs were also consumed by APCs in the liver. However, unlike soluble antigens, no RBCs were consumed by APCs in the lymph nodes. Inflammation with poly (I:C) induced significant consumption of transfused RBCs by splenic DCs, with a concomitant increase in costimulatory molecule expression. Moreover, this resulted in increased proliferation of CD4+ T cells specific for the mHEL RBC alloantigen. Finally, splenectomy abrogated the enhancing effects of poly (I:C) on RBC alloimmunization. Together, these data provide additional insight into the nature of transfused RBCs as an immunogen and provide a mechanism by which viral-like inflammation enhances alloimmunization to transfused RBCs.

Beschreibung: Dietary plant polyphenols are known to have antitumor, antiinflammatory, and antioxidant activity and as such may sensitize tumor cells to chemotherapeutic agents. We have evaluated the effects of the green tea polyphenol epigallocatechin gallate (EGCG) alone and in combination with other drugs on human myeloma cells. The compound is currently under investigation in several phase I/II clinical trials including for treatment of patients with early stage chronic lymphocytic leukemia. EGCG inhibited the in vitro growth of human myeloma cell lines in a time and dose-dependent manner. IC50 concentrations were between 12.5 μM and 50 μM as measured in a colorimetric tetrazolium (MTS) based assay and by trypan blue exclusion. Excess amounts of IL-6, bone marrow stromal cells, or overexpression of Mcl-1 and Bcl-xL could not protect from EGCG induced cytotoxicity. Pretreatment of INA-6 cells with EGCG resulted in a dose-dependent inhibition of IL-6 induced STAT3 tyrosine phosphorylation. In accordance with the essential role of STAT3 for INA-6 cell survival, EGCG induced apoptosis as determined by flow cytometry upon 7-amino-actinomycin D/annexin-V staining. In cell lines not dependent on exogenous IL-6, EGCG induced growth inhibition was abolished by pretreating the cells with 200 U/ml catalase, an enzyme which reduces reactive oxygen species (ROS). The combination of EGCG with doxorubicin, dexamethason, or rapamycin did not result in increased growth inhibition. In contrast, growth inhibition by bortezomib was antagonized with EGCG at concentrations that were not inhibitory when used alone (1–10 μM). In conclusion, EGCG exerts its effects on myeloma cells through several mechanisms including inhibition of IL-6 mediated signalling pathways via STAT3 and induction of oxidative stress. Notably, at pharmacologically achievable concentrations, EGCG antagonized bortezomib activity. Thus, the intake of natural polyphenols (high consumption of green tea or taking green tea extracts) may be critical during therapy with bortezomib.

Beschreibung: PU.1 (Sfpi1) is an ets family transcription factor required for the proper generation of both myeloid (macrophages and neutrophils) and lymphoid lineages (B and T lymphocytes)(Scott 1994, McKercher 1996). Graded expression of exogenous PU.1 in murine PU.1-deficient fetal liver hematopoietic progenitors demonstrated that increased levels of PU.1 are required to initiate development of macrophages (DeKoter, 2000). We have studied the effects of graded expression of PU.1 on its occupancy in chromatin and on the development of myeloid cells in vitro. We measured changes in gene expression, PU.1 occupancy and histone modifications in PU.1-null hematopoietic progenitor cells stably expressing PU.1 fused to the ligand-binding domain of the estrogen receptor (PU.1-ER) (Walsh 2002). The level of active PU.1-ER was regulated with graded levels of the ER inducer tamoxifen. In vitro, intermediate levels of tamoxifen produced cells with granulocyte characteristics in the suspension cell fraction and macrophage-like characteristics in the attached fraction, whereas high levels of PU.1 produced mostly attached macrophage-like cells. Expression of granulocyte-specific PU.1 target mRNAs including gelatinase B (Mmp9) and myeloperoxidase (Mpo) were observed to be expressed only with intermediate levels of tamoxifen. In contrast, expression of macrophage PU.1 target mRNAs including Cd14, F4/80 and Cd68 mRNAs were observed to be gradually upregulated upon PU.1-ER activation, with the maximum expression at the highest levels of tamoxifen. Thus, the expression levels of PU.1 target genes and phenotypic characteristics of the cells are dependent on PU.1 levels. Interestingly, macrophage-like cells can be produced from granulocytic-like cells by changing tamoxifen levels and vice versa. Chromatin immunoprecipitation analysis revealed specific PU.1 occupancy within regulatory regions of the genes predominantly expressed in macrophages including Cd14 and Cd11b after treatment with high levels of tamoxifen. Specific PU.1 occupancy within regulatory regions of the granulocyte specific genes including MMP9 was observed at intermediate levels of tamoxifen. Suprisingly, chromatin immunoprecipitation analysis revealed specific PU.1 occupancy within regulatory regions of the lymphocytic PU.1 target genes including Interleukin-7 receptor (Il-7r) and RAG1 at intermediate levels of tamoxifen even though expression of these genes was not detected. Accumulation of acetylated K9 and methylated K4 of histone H3 in gene loci of macrophage and granulocytic markers such as Cd14, Cd11b, and Mmp9 correlated with their mRNA expression. However, lymphocyte-specific regulatory regions including that of Il-7r gene were hypoacetylated in H3K9 despite a marked PU.1 recruitment suggesting additional factors may be required for PU.1 mediated transactivation. To identify these molecules we have tested PU.1-dependent transcription factors: Egr2, Nab2, Cebpa and Gfi-1 and found that upon increasing PU.1 levels, expression of Egr2/Nab2 and Gfi-1/Cebpa changed in a reciprocal manner and these changes preceded expression of the lineage specific markers. We are currently testing if PU.1 directly regulates expression of Egr2, Nab2, Cebpa and Gfi-1 during granulocytic/macrophage differentiation.

Beschreibung: CD26 (dipeptidyl peptidase IV, DPP IV) is a multifunctional type II cell surface glycoprotein that is widely expressed on T and natural killer cells, as well as on epithelial, endothelial and acinar cells of different tissues; its expression on B cells is very low but it is greatly upregulated following activation. We evaluated, by means of flow cytometry, the expression of CD26 in various types of B-cell lymphoid tumors: Follicular Lymphoma (Fo-Ly, 12 cases), Mantle cell Lymphoma (MCL, 12 cases) Multiple Myeloma (MM, 20 cases), Hairy cell Leukemia (HCL, 12 cases), B-cell Chronic Lymphocytic Leukemia (B-CLL, 112 cases), CD5 negative B-cell Chronic Lymphoproliferative Diseases (CD5neg-B-CLPD, 20 cases) and Diffuse Large cell Lymphoma (DLCL, 12 cases). CD26 expression was absent or low of Fo-Ly and MCL, high on MM and HCL, variable on B-CLL, CD5neg-B-CLPD and DLCL. Fluorescence intensity of positive cells was dim in B-CLL and CD5neg-B-CLPD, heterogeneous in DLCL, and bright in HCL and MM. Interestingly, in CLL patients, CD26 expression was significantly correlated with CD49d and CD38 expressions (p

Beschreibung: Due to the lack of large population-based studies, the incidence of polycythemia vera (PV) has not been well documented in the United States. In 2001, PV became reportable to the Surveillance, Epidemiology, and End Results (SEER) program, which consists of high-quality population-based cancer registries supported and sponsored by the National Cancer Institute. In the present analysis, we accessed the SEER data to estimate the incidence of PV in the United States during 2001 – 2003. Since the addition of PV to SEER reporting is fairly recent, and the diagnosis of PV is different from that of many other types of cancer, especially solid tumors, under-reporting is possible. Therefore, we also used health claims data from Medicare services to estimate the incidence of PV among individuals 65 years and older. SEER data suggest that the age-adjusted incidence rate of PV was 0.87 per 100,000 per year (95% confidence interval 0.83 – 0.91) during 2001 – 2003, which is lower than the estimates from other studies (Ania BJ et al. Trends in the incidence of polycythemia vera among Olmsted County, Minnesota residents, 1935–1989. Am J Hematol1994;47(2):89–93. Johansson P. Epidemiology of the myeloproliferative disorders polycythemia vera and essential thrombocythemia. Semin Thromb Hemost2006;32(3):171–3). The incidence of PV increased with age and was higher in men and white individuals than in women and other racial groups. The SEER-derived age-adjusted incidence rate of PV was 4.1 per 100,000 per year among individuals 65 years and older during 2003, which is considerably lower than the estimates made from nationwide Medicare claims data in the same age group during the same year, suggesting possible under-reporting to SEER. The addition of PV to SEER reporting represents a major step in generating population-based epidemiologic data for this understudied malignancy. However, it is critical to ensure the completeness in the ascertainment and reporting of PV so high-quality SEER data can be utilized to facilitate much needed research on the etiology and outcomes of PV, as well as other myeloproliferative disorders.

Beschreibung: Introduction: Filgrastim is widely used for mobilizing CD34+ cells into the peripheral blood that are easily collected by apheresis for allogeneic transplantation. With case reports documenting splenomegaly with life-threatening complications in normal donors, we prospectively evaluated spleen size using ultrasonography and clinical examination during PBPC mobilization and collection in a single-arm trial. Methods: Subjects ≥18 yrs eligible to be PBPC donors per institutional guidelines enrolled. Splenic assessments were done before, during, and after PBPC mobilization. Filgrastim dose and schedule and leukapheresis (LK) procedures were per institutional practice. The primary endpoint was fold change from baseline in splenic volume in post-baseline measurements during mobilization (measured by ultrasound [US]). Spleen size by US was measured in 3 dimensions similarly by all centers: longitudinal (craniocaudal), transverse, and diagonal (perpendicular to transverse in transverse image) diameters. Splenic volume was estimated by taking the cross-product of 3 dimensions and multiplying by 0.52, approximating the volume of an ellipse. Physical examination was performed on US days, assessing spleen palpability. US and palpation results were blinded from each other at assessment times. Timepoints included baseline (before first filgrastim dose), first LK (done before LK, typically day 4 or 5 of filgrastim), 2 and 4 days after first LK, and 7 days after last LK. Timepoints in the post-amendment cohort (n=219) were reduced to facilitate enrollment and were baseline and day of first LK (before LK). Results: 309 donors enrolled, median age 44yrs (range 18 to 74), 56% male. Mean daily filgrastim dose was 11.4mcg/kg (SD=3.0). Median number of LK was 1.5 (range 1 to 4). In all donors, the median increase in each measured dimension on first LK day was 1.4cm, 1.4cm, and 0.6cm (12.8%, 12.6%, and 15.0%), and the median fold volume increase from baseline to first LK was 1.47, resolving to near baseline 1 week after last LK. There was no apparent relationship between volume fold change and filgrastim dose, ANC, or CD34+ yield. Of 861 splenic palpation assessments reported in all donors, 98% were reported as nonpalpable (842 assessments), and 2% were palpable (19 assessments, 2 at baseline). Reporting of palpable spleens did not correlate with increased spleen size. Tenderness or guarding upon splenic palpation was reported in 2 donors with a spleen considered palpable and in 6 donors with nonpalpable spleens. No donor experienced a splenic rupture. Adverse events related to filgrastim were generally mild to moderate. Conclusion: During PBPC mobilization with filgrastim in normal donors, the spleen increased a median of approximately 50% from baseline to day of first LK and returned to near baseline 1 week after last LK. Size change was not associated with significant clinical sequelae. Timepoint Median fold change from baseline in splenic volume (Q1, Q3) *statistically significant (p

Beschreibung: Introduction: Fludarabine treatment has been shown to be beneficial for patients with Chronic Lymphocytic Leukemia (CLL), and fludarabine-based combinations may even further improve outcomes in patients with CLL. However, most CLL patients eventually become fludarabine refractory, a state which is associated with a relatively short survival. Treatment of fludarabine-refractory patients is challenging, with a median survival of about 10 months. Recently, 2 phase II clinical trials (Chanan-Khan et al. JCO 2006 and Ferrajoli et al. ASH 2006) demonstrated the clinical efficacy of lenalidomide, an immunomodulatory agent, in relapsed/refractory CLL patients. We conducted a subset analysis to examine the efficacy of lenalidomide in patients who are fludarabine refractory. Methods: All patients enrolled on the 2 phase II single agent lenalidomide clinical trials were evaluated and patients with fludarabine-refractory disease (progressed while on or within 6 months of fludarabine-based therapy) were assessed for clinical efficacy of lenalidomide. Lenalidomide was given orally either at 10 mg daily for 28 days followed by 5 mg increments every 28 days to a maximum dose of 25 mg or given at 25 mg on days 1–21 of each 28-day cycle. Response was assessed using the NCI-WG 1996 criteria. Results: A total of 80 patients were collectively enrolled in these clinical studies. Among these, 29 were identified to have fludarabine-refractory disease. Important clinical characteristics of these patients are reported in Table 1. The overall response rate in fludarabine-refractory patients was 34.5% (10/29). Complete remission was observed in 2 (6.8%) patients. Conclusion: Lenalidomide is a novel agent with immunomodulating properties demonstrating clinical efficacy in relapsed or refractory CLL patients. Interestingly, clinical responses to single agent lenalidomide were noted despite refractoriness to fludarabine (a subset of CLL patients with poor survival and limited therapeutic options). This observation of the clinical benefit of lenalidomide independent of responsiveness to prior fludarabine is encouraging and warrants further evaluation. Table 1 Ferrajoli et al. Chanan-Khan et al. Fludarabine-refractory (N=12) Fludarabine-refractory (N=17) ORR, overall response rate; PFS, progression-free survival; OS, overall survival. Median age, years (range) 62 (51–82) 68 (53–75) Sex, female/male 4/8 4/13 Median no. prior therapies (range) 4 (3–15) 4 (1–10) Median beta microglobulin (range) 5 (3–10) 5 (2–10) Advance Rai Stage (III/IV), n (%) 7 (58.3) 13 (76.4) ORR, n (%) 3 (25.0) 7 (41.2) Median PFS, months 12 14.9 Median OS, months All alive (range, 7–19) 23

Beschreibung: Dyskeratosis congenita (DC) is a congenital disorder characterized by very short telomeres. Clinical presentation includes a diagnostic triad (lacey reticular pigmentation, nail dystrophy, and leukoplakia), aplastic anemia (the main cause of premature death), myelodysplastic syndrome/leukemia and solid tumors. Allogeneic HCT is the only curative option for the hematologic complications of DC but has been associated with a high risk of peri-transplant morbidity and early death. Only about fifty HCT for DC have been performed to-date, and the five year survival after related donor HCT has been about 75%, but only approximately 35% when an unrelated donor was used. To improve survival in DC patients by decreasing transplant mortality, we introduced a reduced intensity regimen including cyclophosphamide (50 mg/kg), fludarabine (200 mg/kg), low dose total body irradiation (200 cGy), and (in patients 3 and 4) campath 1H (1 mg/kg). To decrease the risk of graft rejection, grafts were not T-cell depleted. We report outcomes in four consecutive patients, two adults and two children, all of whom engrafted with donor hematopoiesis: Age (years) Sex Graft and HLA match NC dose (×108/kg) CD34 dose (×108/kg) Follow-up (months) Donor chimerism 24 M URD dUCB 4/6, 4/6 0.55, 0.39 0.5, 0.43 1 (dead*) 83% 29 F REL PBSC 6/6 13.93 4.43 20 (alive) 100% 5 F URD BM 7/8 1.38 1.55 16 (alive) 100% 2 M URD BM 8/8 5.92 2.31 3 (alive) 100% Legend: M, male; F, female; NC, nucleated cell; URD, unrelated donor; REL, related donor; BM, bone marrow; dUCB, double umbilical cord blood; PBSC, peripheral blood stem cells; *Patient had autologous recovery after the first dUCB and died of sepsis 1 month after the second dUCB; HLA matching is reported for antigen level HLA-A, B and allele level DRB1 for cord blood, and allele level typing for HLA-A, B, C, DRB1 for PBSC or BM. The most recent donor chimerism is reported. To decrease the risk of graft rejection and prevent graft versus host disease (GvHD) patients received cyclosporine and mycophenolate mofetil. Patient 2 developed limited chronic GvHD and patient 4 developed grade III skin acute GvHD. Both were treated successfully with systemic and topical steroids. Our data suggest that this conditioning regimen results in a low rate of transplant related complications without compromising engraftment. Critically, early fatal pulmonary and vascular complications, common in post-transplant courses in DC patients, were not observed. This highlights the need to avoid drugs that are associated with pulmonary toxicity such as busulfan, and to limit radiation to the lung in patients with DC. This new less intensive conditioning regimen appears to result in a low rate of transplant related complications, and yet has adequate immunosuppressive activity to permit engraftment from alternative donors in DC patients.

Beschreibung: Extensive chronic graft-versus-host disease (ecGVHD) is characterized by fibrosis similar to that of patients with systemic sclerosis (scleroderma). Since stimulatory autoantibodies against the platelet-derived growth factor (PDGF) receptor (PDGFR) have been found in patients with scleroderma and are responsible for the activation of skin fibroblasts, we tested the hypothesis that these autoantibodies are also present in patients affected by ecGVHD. Serum from 39 patients subjected to allogeneic stem cell transplantation for hematologic malignancies (22 with ecGVHD and 17 without cGVHD) and 20 healthy controls was assayed for the presence of stimulatory autoantibodies to the PDGFR by incubating purified IgG with mouse-embryo fibroblasts lacking PDGFR α or β chains or with the same cells expressing PDGFR α. Stimulatory antibodies to the PDGFR were found selectively in all patients with ecGVHD but in none of the patients without cGVHD. Higher levels were detected in patients with generalized skin involvement and/or lung fibrosis. Antibodies recognized native PDGFR, induced tyrosine phosphorylation, accumulation of reactive oxygen species (ROS), and stimulated type 1 collagen gene expression through the Ha-Ras-ERK1/2-ROS signaling pathway. The biologic activity of these autoantibodies suggests a role in the development of fibrosis and argues for a common pathogenetic trait in ecGVDH and scleroderma phenotypes.

Beschreibung: Background: The initial genetic event in ∼85% of follicular lymphomas (FL), the most common B-cell lymphoma in North America, is the t(14;18)(q32;q21) resulting in over-expression of the anti-apoptotic protein Bcl-2. The secondary events associated with disease progression are not well understood. Alterations affecting the p arm of chromosome 1 are evident by standard karyotype analysis in ∼20% of FL. We have further examined the relationship between 1p deletion and FL using high resolution genomic analyses. Methods: The prevalence of 1p alterations was investigated in 139 cases of indolent and transformed FL using whole genome tiling path BAC array Comparative Genomic Hybridization (array CGH). Array-based single nucleotide polymorphism analysis was performed on a subset of cases using Affymetrix 500K SNP arrays. Results: Array CGH identified a minimum region of deletion spanning ∼0.5MB within 1p36.32 in 51 cases (37%). In 38 cases (27%) this loss was exhibited in the transformed sample but not the pre-transformation sample. The majority of cases displayed heterozygous deletion, while two cases showed homozygous deletion. The mechanisms of loss included simple deletions, unbalanced translocations with various partner chromosomes and eleven cases with an unbalanced t(1;1)(p36;q12). The Affymetrix 500 SNP array analyses showed copy neutral loss of heterozygosity or acquired uniparental disomy (aUPD) in three of ten cases that were negative for loss by aCGH. Contained within the 1p36.32 minimally deleted region are only a few candidate genes including tumor necrosis factor receptor superfamily 14 (TNFRS14), which has been implicated in growth inhibition of HT-29 human colon adenocarcinoma cells and induction of Fas-mediated apoptosis in non-Hodgkin’s lymphoma. Conclusions: Our data indicate that loss of heterozygosity at 1p36.32 through deletion or aUPD constitutes the most common secondary cytogenetic event in FL. LOH at 1p36 may represent an important step in the progression of indolent to transformed FL. Further studies have been initiated to investigate other possible gene inactivation events such as methylation and mutation.

Beschreibung: CD4+CD25+Foxp3+ regulatory T cells (CD25+ Treg cells) direct the maintenance of immunological self-tolerance by active suppression of autoaggressive T-cell populations. However, the molecules mediating the anergic state and regulatory function of CD25+ Treg cells are still elusive. Using differential proteomics, we identified galectin-10, a member of the lectin family, as constitutively expressed in human CD25+ Treg cells, while they are nearly absent in resting and activated CD4+ T cells. These data were confirmed on the mRNA and protein levels. Single-cell staining and flow cytometry showed a strictly intracellular expression of galectin-10 in CD25+ Treg cells. Specific inhibition of galectin-10 restored the proliferative capacity of CD25+ Treg cells and abrogated their suppressive function. Notably, first identified here as expressed in human T lymphocytes, galectin-10 is essential for the functional properties of CD25+ Treg cells.

Beschreibung: Background: Romidepsin is a bicyclic peptide that inhibits Class I and II HDACs. Piekarz et al noted responses to romidepsin in CTCL (ASCO, 2004). This pivotal phase II study sought to confirm the activity. Methods: This single arm, open label study enrolled CTCL (Stages 1B–1VA), including MF and Sézary syndrome (SS) patients (pts) from ∼40 sites in Europe, Russia, Ukraine, Georgia and the US. Pts with biopsy-proven CTCL (centrally reviewed) who failed ≥1 prior systemic therapy received romidepsin at 14 mg/m2 as a 4-hour IV infusion on Days 1, 8, and 15 q 28 days for up to 6 cycles but could continue if deriving benefit. Eligibility criteria included adequate organ function and ECOG PS ≤ 1. Exclusions included significant CV abnormality or treatment with QTc-prolonging or CYP3A4-inhibiting drugs. The primary endpoint is response rate measured by a combination of imaging, circulating malignant T-cell counts and a weighted scoring instrument to determine skin involvement (confirmed by photography). Target accrual of 64 evaluable pts (i.e. received 2 courses) has been reached and the study will close. Results: 92 pts were eligible with 68 evaluable for efficacy per protocol. Responses in evaluable pts are 1 CR, 3 CCRs, 20 PRs, 40 SD and 4 PD for an ORR of 35% (duration 2–21 months). Of pts who received ≥1 dose, the ORR is 26% (24/92) but includes 5 too early to be assessed. Median time to response is 8 weeks (range 4 – 20). Responses by stage at entry in evaluable pts, as available: Stage IB-IIA 7/23 (30%); Stage IIB-IVA 15/37 (41%). In pts with pruritus at baseline i.e. score of ≥ 30 mm on a 100 mm visual analogue scale (VAS), relief of ≥ 30 mm from baseline or a VAS score of 0 (no itching) for at least 2 cycles, was seen in 18/38 pts (47%). In those pts with severe pruritus (VAS score ≥70 mm), 14/24 (58%) experienced relief of itching. Adverse event (AE) data are available for 75 pts. AEs were reported in 54/75 (72%) of dosed pts but Grade (G) 3/4 events in only 12/75 (16%). Most frequent AEs are nausea/vomiting (G2) fatigue (G2/3), myelosuppression (G2/3), and asymptomatic ECG changes (transient mild QTc prolongation and nonspecific ST-T wave abnormalities). Thirteen pts withdrew due to AEs but there were no treatment-related deaths although 4 pts died of PD and 1 from right ventricular failure. Serious AEs considered possibly, probably or likely related to treatment were reported in 12 pts. Of these, 8 had ≥G3 events: tumor lysis, cardiac tamponade, sepsis, constipation, oral candidiasis, dermatitis, hyperglycemia/vomiting/nausea and bradyarrhythmia/atrial fibrillation. Conclusions: This study confirms the efficacy of romidepsin in treatment-refractory CTCL including relief of pruritus and an encouraging response rate with 4 CCR (1 pathology-confirmed). The low rate of discontinuation due to AEs and prolonged treatment duration of some patients illustrate that toxicity has been manageable.

Beschreibung: Waldenstrom’s Macroglobulinemia (WM) is an incurable B-cell malignancy characterized by bone marrow (BM) infiltration with a spectrum of clonally related cells, including small lymphocytes and lymphoplasmacytic cells (CD19+) as well as mature plasma cells (CD138+). The molecular pathogenesis of the disease remains to be defined. We therefore analyzed the gene expression profiles of CD19+ and CD138+ BM mononuclear cells from 30 untreated patients with WM and compared their gene expression profile to their normal counterparts from 10 healthy donors using Affymetrix microarrays (U133 plus 2.0). Since the microenvironment plays an important role in the pathogenesis of WM, we also profiled and compared gene expression profiling for CD19 and CD138 depleted BM mononuclear cells from the same patients and healthy donors. Gene expression analysis was performed using dChip software. Unsupervised hierarchical cluster analysis demonstrated distinct gene expression patterns between WM cells versus their normal counterparts. In supervised hierarchical cluster analysis selecting for genes with 〉 2 fold change in expression and a False discovery Rate (FDR) 〈 2%, a set of 1171, 582 and 360 genes were found to be differentially expressed between WM patient and healthy donor CD19+, CD138+, as well as CD19/CD138 depleted (microenvironmental) cells, respectively. Among the most significantly over-expressed genes in the CD19+ compartment in WM patients were: BCL2, TNFRSF13B, TNFRSF17, IGLL1, CCR2, CLLU1, whilst the AP1 family genes JUND and FOSB were among the most significantly down-regulated genes in both CD19+ and CD138+ cells in WM patients. Other interesting transcripts which were over-expressed in CD138+ cells from WM patients included those from genes involved in transcription regulation (ZKSCAN1, ZMYM1, ZNF189, ZNF19, and ZNF559) and interferon response (IFI16 and IFIH1). Of considerable interest was our observation that microenvironmental cells in WM patients demonstrated an overactive transcriptional profile composed of genes which are associated with immune and inflammatory responses including the Toll like receptors (TLR 1,5,7,8), interferon and cytokines (IFI16, IFNAR1, IL-10R, IL-8R), genes encoding extracellular matrix components (Fibronectin and Hepatocyte Growth Factor) as well as genes involved in apoptotic signaling (TNFSF10, TRAF4). These studies provide the first comprehensive molecular characterization of WM, dissecting the molecular features of the two immunophenotypically distinct populations of malignant cells, and providing for the first time evidence for a distinct molecular profile in BM microenviromental cells.

Beschreibung: Introduction: Event-free survival (EFS) for children with ALL is approximately 80%. Despite substantial success in achieving second and subsequent remissions, survival of patients with relapsed ALL (rALL) remains dismal. We propose that progress depends on identification of novel drug combinations with more activity in rALL than those commonly employed. The literature suggests a 40% CR rate for second and subsequent relapse (Br. J Haematol2005;131:579). However, CR rates depend on the number of prior therapeutic attempts and duration of any prior response, complicating identification of promising regimens. Limited patient numbers and a large number of potential candidate regimens discourage randomized trials. We surveyed local experience with rALL to establish a robust benchmark for evaluation of novel drug combinations. Methods: The TACL Consortium (www.tacl.us) was formed to develop novel drug combinations for patients with rALL. We initiated a review of all patients with rALL treated between 1995 and 2004 at eight TACL institutions. Detailed data on therapy, response, and duration of response were collected on all patients. Results: Of 313 rALL patients, 62% were males, 27% were older than 10 years at diagnosis, 26% had initial white blood counts (WBC) at diagnosis 〉=50,000/uL, and 46% were high-risk by NCI risk criteria. Re-induction attempts ranged between 1 and 9 and most commonly employed combinations of traditional ALL agents. Limiting analyses to patients with marrow involvement, we obtained 86% CR’s for 1st relapse (95% confidence interval 80%–90%), 44% for second relapse (35%–53%), and 30% for third relapse (19%–43%). CR rates declined with the number of prior treatment attempts (see Table, p

Beschreibung: Introduction: DIC is a complication that occurs during serious infection with Gram-negative bacteria. Endotoxin is a component of the bacterial cell wall that elicits a cytokine-mediated cascade of tissue factor-dependent hypercoagulable reactions. The resulting hypercoagulable state may be inhibited by potent anticoagulation. Rivaroxaban is an oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. The aim of this study was to examine the effects of rivaroxaban in a rat model of endotoxin-induced DIC. Methods: Rivaroxaban (0.1–10 mg/kg p.o.) or vehicle control (PEG400/H2O 60/40%, 5 mL/kg p.o.) were administered 30 minutes before endotoxin injection (lipopolysaccharide O55 B5 [LPS], 250 μg/kg i.v.) to conscious rats. Blood samples were withdrawn from anesthetized animals by heart puncture 4 hours after LPS injection and fibrinogen, platelet count, thrombin-antithrombin (TAT) complex levels and IL-6 were measured. Results: The induction of DIC was indicated in the placebo + LPS group vs vehicle control by decreased fibrinogen (2.12±0.08 vs 2.69±0.10 g/L) and platelet count (571±28 vs 904±30×109/L) an increase in TAT (75±9 vs 2±1 μg/L) and IL-6 (8.6±1.23 μg/L vs 0.028±0.020 μg/L). Pretreatment with rivaroxaban (0.1–10 mg/kg p.o.) dose-dependently ameliorated the effects of LPS on fibrinogen, platelets and TAT. Rivaroxaban 10 mg/kg p.o. normalized fibrinogen (2.58±0.07 g/L) and TAT (5.6±1.2 μg/L) and increased platelet count (703±19×109/L) (Table). Rivaroxaban also slightly reduced the plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) (4.24±0.67 μg/L). Conclusions: These results show that direct, selective inhibition of FXa by rivaroxaban effectively normalized the hypercoagulable reactions of endotoxemia with a slight modulating effect on the generation of pro-inflammatory active cytokines, such as IL-6, in the rat DIC model. Further research into the use of rivaroxaban in the management of DIC is therefore warranted. Effects of rivaroxaban in an endotoxin-induced DIC model in rats 4 hours after LPS injection. Results show mean ± SEM. Vehicle control Placebo + LPS Rivaroxaban 1 mg/kg + LPS Rivaroxaban 10 mg/kg + LPS DIC, disseminated intravascular coagulation; IL-6, interleukin-6; LPS, lipopolysaccharide O55 B5; SEM, standard error of the mean; TAT, thrombin-antithrombin ## p

Beschreibung: Introduction: Radioimmunotherapy (RIT) is a new treatment for B non Hodgkin’s lymphoma (NHL) patients. 90Y ibritumomab tiuxetan (Zevalin®) consists of a murine monoclonal antibody to CD20, conjugated to a metal chelator tiuxetan for retention of the beta emitter 90Y. Thus Zevalin® delivers radiation to B-NHL, combining the tumor targeting attributes of a monoclonal antibody and the beta radiation of 90Y. Zevalin® is approved for the treatment of follicular lymphoma (FL) refractory to or relapsed after rituximab, on the bases of clinical trials where it achieved a response rate as high as 83%. Several ongoing registrational trials are evaluating the efficacy of Zevalin® in other NHL, as diffuse large B cell (DLCL) and mantle cell lymphoma (MCL). We are here evaluating the effect of Zevalin® as consolidation therapy in NHL patients that achieved a complete clinical response (CCR) with chemotherapy. Methods: In B cell NHL patients that achieved a CCR after 1st or multiple lines anthracyclines based chemotherapy +/− Rituximab, minimal residual disease was evaluated by PCR on bone marrow samples, for the following rearrangements: JH, Bcl-1, Bcl-2. Patients received Zevalin® 6-9 weeks post chemotherapy. Evaluation of molecular response was assessed after a follow up period at 12 weeks. The aim of the study was the role of Zevalin® in inducing a complete molecular response (CMR). Results: 23 B-NHL patients (13 FL, 6 MCL, 4 DLCL; male:female 13:10, median age 63, range 42–73. See table) in a CCR after chemotherapy (documented by TC scan and/or PET-scan negative for abnormal lesions or glucose captation) have been enrolled. 10 patients had a pathological rearrangement before RIT, while 13 were already in a CMR condition. Zevalin® was completed in all 23 patients and the post infusion evaluation was performed after 12 weeks. In the follow-up period thrombocitopenia was commonly documented, but it was not associated to bleeding or need of platelet transfusion, but in one singular case. After 12 weeks from RIT a new molecular evaluation was performed on bone marrow samples. All the 23 patients have completed the 12 weeks follow-up: 8 of 10 (80%) patients positive before RIT achieved a CMR with Zevalin® administration. The 13 PCR negative patients maintained the CMR. The 21 PCR negative patients are now under follow-up to evaluate the molecular disease free survival after Zevalin® RIT. Conclusion: Zevalin® is an efficient consolidation therapy in B cell NHL patients after chemotherapy. In this series of patients Zevalin® administration allowed to convert 8 of 10 CCR to CMR. In the remaining 13 patients Zevalin® maintained the CMR. Zevalin® addition to medication treatment is feasible and associated with manageable hematological toxicity. Pts disease sex age previous chemotherapy lines molecular response before RIT molecular response after RIT 1 FL M 68 1 POS NEG 2 FL F 53 1 NEG NEG 3 FL M 54 1 NEG NEG 4 FL M 51 4 NEG NEG 5 DLCL F 66 2 POS NEG 6 DLCL F 67 1 NEG NEG 7 FL F 42 1 POS POS 8 FL M 52 1 POS NEG 9 FL F 54 3 NEG NEG 10 FL M 57 2 POS NEG 11 FL F 62 2 POS NEG 12 FL M 58 2 POS NEG 13 FL F 69 2 NEG NEG 14 MCL M 62 1 POS NEG 15 MCL M 66 1 POS POS 16 MCL M 66 2 NEG NEG 17 MCL M 67 1 POS NEG 18 FL F 67 2 NEG NEG 19 DLCL F 67 3 NEG NEG 20 MCL M 70 2 NEG NEG 21 FL M 61 4 NEG NEG 22 DLCL M 43 2 NEG NEG 23 MCL F 73 2 NEG NEG

Beschreibung: INTRODUCTION: Janus kinase 2 gene (JAK2) encodes for a cytoplasmic tyrosine kinase involved in normal hematopoietic growth factor signaling. Point mutations of the JAK2 gene on chromosome 9, specifically V617F, a point mutation at amino acid 617, are associated with myeloproliferative disorders (MPD). The V617F JAK2 mutation has been found in 90% of patients with polycythemia vera, 50–60% of patients with essential thrombocythemia or idiopathic myelofibrosis and 1–5% of patients with other MPD. To our knowledge, previous studies involving the V617F JAK2 mutation were not performed on a control population of normal individuals. Therefore, the prevalence of this mutation has not been established. In this study, we tested volunteer blood donors from a hospital-based blood donation center for the presence of the V617F JAK2 mutation. METHODS: Citrated whole blood was obtained from volunteer blood donors, age 17 and older, who presented to donate whole blood at a hospital-based blood donation center. The donors met all qualifications to donate blood as defined by FDA regulations. DNA was extracted using the QIAagen and QIAamp DNA extraction columns, quantified and diluted to 100ng/ul. DNA was simultaneously amplified and detected using allele specific minor groove binder probes and primers for the V617F JAK2 mutation. The resultant amplification was recorded by real-time, quantitative PCR using an ABI 7500 (Applied Biosystems, Foster City, CA). A 1% limit of detection, determined from sensitivity and specificity studies using a known cell line control, was set as the technically reproducible threshold sensitivity of the test. Samples were defined as negative for the V617F JAK2 mutation if only the wild type allele was detected. Samples that had a mutant allele detected above the 1% limit of detection were defined as positive for the V617F JAK2 mutation. Samples that had a mutant allele detected below the 1% limit of detection were defined as negative for the V617F JAK2 mutation. RESULTS: A total of 181 DNA samples from volunteer blood donors were tested for the V617F JAK2 mutation. The test group consisted of 104 males (mean age 44, range 17–77) and 77 females (mean age 42, range 18–71). Of the 181 donors tested, 171 had only wild type allele detected and were considered negative. Ten donors had high background of the mutant allele detected below the 1% limit of detection and were considered negative. DISCUSSION: To our knowledge, this is the first report documenting the prevalence of the V617F JAK2 mutation in a healthy blood donor population. In this study of 181 volunteer blood donors none had the V617F JAK2 mutation. Although 10 of the 181 donors were found to have mutant allele detected, they were below the 1% technically reproducible sensitivity threshold of the test and were considered negative. We recommend that mutations detected below the technical threshold of 1% of our assay be considered false positives. The results of this study suggest that the V617F JAK2 mutation is not present in a healthy blood donor population and is significant when detected by our method.

Beschreibung: The capacity of CD4+CD25+Foxp3+ (Treg) cells to regulate adaptive and innate immune responses has led to studies investigating their application to regulate allogeneic T cell responses arising during hematopoietic stem cell transplants (HCT). With respect to HCT, a fundamental clinical concern is the reconstitution of the lymphoid compartment in recipients, particularly T cells since this process can be exceedingly delayed. We have previously found that host Treg cells can regulate resistance to engraftment following HCT, demonstrating that such cells survive and function at least transiently in recipients. The present studies investigated the residual host Treg compartment following HCT. Utilizing varying levels of total body irradiation (5.0 – 14Gy), we observed: recipient CD4+CD25+Foxp3+ cells can survive ablative as well as reduced intensity conditioning, the surviving, i.e. residual Treg cells undergo expansion as assessed by BrdU uptake and cell numbers, and these cells comprise the majority of the Treg compartment in recipients for several months post-HCT during which time donor derived Treg cells gradually arise and cede this compartment. Residual Tregs also dominated the compartment following allogeneic HCT of MHC-matched bone marrow depleted of T cells. To assess the functional capacity of the residual Treg cell compartment, the development of autoimmune disease following transplant of IL-2Rβ −/− (CD122−/−) bone marrow into syngeneic recipients with and without residual Tregs was examined. Autoimmune disease symptoms and T cell alterations were prevented in B6-wt but not T cell deficient recipients. Interestingly, the failure to transfer autoimmune disease following IL-2Rβ −/− HCT into lethally conditioned B6-CD4−/− recipients was associated with the presence of a peripheral CD8+FoxP3+ population not detected in B6-wt mice or the B6-wt mice transplanted with IL-2Rβ −/− BM. This finding indicates that in the genetic absence of CD4+ T cells, a CD8 regulatory population appears to emerge. In total, our observations support the notion that functioning host Tregs initially occupy a niche in the transplant recipient permitting lymphopenic expansion and an extended period of contribution to this compartment. Notably, this contribution reflected much greater levels than conventional T cell populations - even in aggressively conditioned recipients. Finally, these findings imply that the presence of host regulatory cells may be important to consider with respect to eliciting anti-tumor responses and vaccination in recipients during the early period post -hematopoietic cell transplantation.

Beschreibung: Imatinib mesylate has revolutionized treatment of Chronic Myeloid Leukemia. Yet for women of child bearing age problems may arise during pregnancy. While continuation of the drug is in the interest of maternal health, the safety of this approach for the fetus has been questioned. Therapy is therefore usually immediately interrupted on recognition of pregnancy. Only three successful pregnancies have been reported to date in which imatinib was continued. A 34-year-old woman of Chinese descent in complete cytogenetic and major molecular remission (i.e. 3-log reduction of BCR-ABL transcripts) of her CML in chronic phase on 400mg imatinib daily continued treatment during conception and throughout pregnancy. After an uneventful full term pregnancy she gave birth to an apparently healthy son. The mother retained her major molecular remission. Routine screening for congenital hypothyroidism of her child however repeatedly revealed an elevated TSH, rising from 11 mU/l at the age of 1 week to15 mU/l at 4 weeks, associated with a relatively low free T4 of 24.8 and 17.4 pmol/l respectively. Thyroglobulin and thyroxine binding globulin concentrations were normal for age. The mother was euthyroid and had no detectable anti-thyroid antibodies. A mild primary hypothyroidism was suspected. An ultrasound showed no developmental defect of his thyroid gland and a 123I scan with a perchlorate discharge test ruled out an intra-thyroidal iodide organification defect. There was no family history of congenital hypothyroidism. The child was put on levothyroxine and is currently doing well. Hypothyroidism has been noticed as a potential side effect of imatinib therapy in thyroidectomized patients receiving levothyroxine and has been ascribed to enhanced non-deiodination clearance of T4 (de Groot et al Clin Pharmacol Ther2005;78:433–8). An induction of uridine diphosphate-glucuronosyl transferases by imatinib was hypothesized as mechanism for an altered levothyroxine metabolism in these patients. Congenital hypothyroidism has to the best of our knowledge not been reported as a potential consequence of imatinib therapy.

Beschreibung: The relative importance of various human leukocyte antigen (HLA) loci and the resolution level at which they are matched has not been fully defined for unrelated donor transplantation. To address this question, National Marrow Donor Program data from 3857 transplantations performed from 1988 to 2003 in the United States were analyzed. Patient-donor pairs were fully typed for HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, -DPB1, and -DPA1 alleles. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 (8/8 match) was the minimum level of matching associated with the highest survival. A single mismatch detected by low- or high-resolution DNA testing at HLA-A, -B, -C or -DRB1 (7/8 match) was associated with higher mortality (relative risk, 1.25; 95% CI, 1.13-1.38; P 〈 .001) and 1-year survival of 43% compared with 52% for 8/8 matched pairs. Single mismatches at HLA-B or HLA-C appear better tolerated than mismatches at HLA-A or HLA-DRB1. Mismatching at 2 or more loci compounded the risk. Mismatching at HLA-DP or -DQ loci and donor factors other than HLA type were not associated with survival. In multivariate modeling, patient age, race, disease stage, and cytomegalovirus status were as predictive of survival as donor HLA matching. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 alleles is associated with higher rates of survival.

Beschreibung: Risk factors for ONJ in MM pts include dental extraction, bisphosphonates (BP) use, older age and longer survival. There is also an increased risk of skeletal related events (SRE) in ONJ pts (Badros, JCO 2006). The current study provides long term follow-up data for ONJ pts with regard to ONJ recurrence, SRE and MM status. The study included 97 pts: 60 from Greece and 37 from the US. Pts’ characteristics are summarized in the table below. Median follow-up time has not been reached; lower limit of the 95%CI was 3.2 yrs. ONJ resolved in 60 of 97 pts (62%), resolved and recurred in 12 pts (12%), and did not heal over a 9 months period in 25 pts (26%). Dental extraction preceded ONJ in 46 of 97 pts (47%) and was more common in pts with a single episode of ONJ (35 of 60, 58%) than in the recurrent and non-healing pts (11 of 37, 30%) (p-value=0.007). The median number of ONJ episodes in the recurrent group was 3 (range, 2–6); recurrence of ONJ was precipitated by re-initiation of BP and by dental procedures in 5 and 4 pts of 12, respectively. There was a trend toward higher ONJ recurrence rate in the US (8 of 37, 22%) versus the Greek (4 out of 60, 7%) pts (p-value=0.053). Surgery was performed more often in the US than in Greece 17 of 37 (45%) versus 19 of 60 pts (32%). BP reinitiation was more frequent in US 16 of 37 (43%) than in Greece 3 of 60 (5%). Non-healing ONJ lesions were managed with antibiotics; 10 of 25 pts developed fistulas and needed surgery; in 9 pts the lesions remained asymptomatic. Twenty-one ONJ pts had SRE including fractures (ribs, vertebrae and long bones, n=13) and avascular necrosis of the femur (n=8). The rate of MM relapse was higher in pts with recurrent and non-healing ONJ (84%) compared to pts with a single episode (62%) (p-value=0.02). The median OS from diagnosis of MM was 10.8 yrs (95% CI; 9.3 yrs- not reached) and did not differ between pts with single, recurrent/non-healing ONJ (p= 0.2). In summary, pts in whom ONJ followed dental procedures were less likely to have recurrence or non-healing, both, although infrequent, were linked to BP re-challenge, mostly in the setting of relapsed MM. Non-healing ONJ lesions remained stable/asymptomatic without extensive intervention. BP should be discontinuation until ONJ lesions heal. The decision to restart BP should be individualized based on MM-SRE risk. ONJ Pts characteristics and outcome AA, African American; ttt, treatment; CR, complete remission; PR, partial remission; PD, progressive disease; Dex, dexamethasone, thal, thalidomide; Len, lenalidomide; Bort, bortezomib; A, pamidronate; Z zoledronic acid. The Fisher’s Exact test was used, all p-values reported are two-sided. ONJ, n= 97 one episode, n=60 recurrent, n=12 non-healing, n=25 age at MM; median (range) 60 (26–77) 61 (26–77) 55 (43–76) 61 (36–73) Sex; male/female 59/38 38/22 8/4 13/12 Caucasian/AA 87/10 54/6 10/2 23/2 Isotype; IgG, A, D, LCH 60/20/1/16 36/11/1/12 7/2/0/3 17/7/0/1 MM ttt at ONJ (n=93); none/dex/thal/len/bort 22/31/26/6/8 11/25/16/4/2 5/1/3/1/1 6/5/7/1/4 MM status at ONJ diagnosis; CR/PR/PD 7/54/33 4/37/17 3/8/1 0/9/15 BP use; AZ/Z 59/35 34/23 10/2 15/10 Dental extraction 46 35 5 5 Restarted BP 19 11 6 2 bone complciations 21 14 3 4 MM course after ONJ; continous remission/Relapse 29/68 23/37 2/10 4/21 MM status at last follow up; CR/PR/PD (died) 3/59/35(28) 3/35/22(20) 0/10/2(2) 0/14/11(6)

Beschreibung: Background: The phosphatidylinositol 3-kinase/mammalian target of rapamycin (mTOR) signal transduction pathway integrates signals from multiple receptor tyrosine kinases to control cell proliferation and survival. Everolimus (RAD001, Novartis Pharmaceuticals) is an oral investigational antineoplastic agent that targets mTOR. Objectives: To learn the anti-tumor activity and toxicity of single-agent RAD001 in pts with relapsed/refractory aggressive NHL. Patients and Methods: Patients were eligible if they had measurable disease, a platelet count 〉75,000, an absolute neutrophil count 〉1,000, and a creatinine and bilirubin

Beschreibung: Background: Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin’s Lymphoma characterized by being incurable with a low response rate and short progression free survival when treated with conventional chemotherapy agents. Lenalidomide (Revlimid®), an immunomodulatory drug, is approved for the treatment of relapsed/refractory multiple myeloma and myelodysplastic syndromes associated with a del(q5) cytogenetic abnormality. Lenalidomide has also shown activity in chronic lymphocytic leukemia and cutaneous T-cell lymphoma. Aim: To determine the activity and safety of lenalidomide in relapsed/refractory MCL. Methods: Patients with relapsed/refractory MCL and measurable disease ≥ 2 cm after at least 1 prior treatment regimen were eligible. Patients received 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continued therapy for 52 weeks as tolerated or until disease progression. Response and progression were evaluated using the IWLRC methodology. Results: Fifteen patients with MCL were enrolled. Median age was 67 (45–84) and 7 were female. Median time from diagnosis to lenalidomide was 5.1 (0.7–12.7) years and median number of prior treatment regimens was 4 (2–6). Eight patients (53%) exhibited an objective response (1 complete response (CR), 1 complete response unconfirmed (CRu) and 6 partial responses (PR)), 2 had stable disease (SD) and 5 had progressive disease (PD). All eight responses were in eleven patients having a tumor burden 〈 50 cm2 and a time since last rituximab therapy of ≥ 230 days. No responses were achieved in four patients having a tumor burden ≥ 50 cm2 or a time since last rituximab therapy of 〈 230 days. Four of 5 patients (80%) with a prior stem cell transplant responded. Progression free survival [PFS] is 5.7 months and ongoing. Seven patients (47%) required at least one dose reduction with a median time to first dose reduction of 2.3 months (1.2–4.9). Grade 4 adverse events were neutropenia (13%), thrombocytopenia (13%), and thromboembolism (13%). Most common Grade 3 adverse events were neutropenia (33%) and leukopenia (20%). Conclusion: Lenalidomide oral monotherapy produced a 53% response rate in relapsed/refractory MCL with manageable side effects.

Beschreibung: Methotrexate (MTX) is an antifolate widely used to treat childhood acute lymphoblastic leukemia (ALL). MTX is retained within cells as long-chain polyglutamates (MTX-PGs), after metabolism by the enzyme folylpoly-γ-glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR), and the additional inhibition of thymidylate synthetase (TS). The FPGS gene was shown to be regulated by the transcription factors Sp1 and NFY. We performed DNaseI hypersensitive assays and identified a hypersensitive site mapping closely upstream of exon 1 suggesting that chromatin remodeling may contribute to FPGS gene regulation. Using co-immunoprecipitation and Western blotting we investigated the role of histone modifications and chromatin remodeling on the expression of FPGS and uncovered interactions between NFY, Sp1 and HDAC1. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assay (DAPA) revealed that the HDAC1-NFY and HDAC1-Sp1 complex binds to the NFY and Sp1 binding sites in the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by acetylation of NFY and Sp1 factors and interaction with HDAC1, and/or chromatin remodeling. We then examined the effect of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBu) on the expression of FPGS and other folate-related genes. The level of FPGS, ATP-binding cassette subfamily C (ABCC1), ATP-binding cassette subfamily G (ABCG2), DHFR, γ-glutamyl hydrolase (GGH), solute carrier family 19/folate transporter (SLC19A1), and TS mRNA gene expression was determined by qRT-PCR in NALM6 (Bp-ALL), REH (Bp-ALL, t(12,21)/TEL-AML1), SupB15 (Bp-ALL, t(9,22)/BCR-ABL), and CCRF-CEM (T-ALL) cells treated with NaBu [2mM-5mM]. In all cell lines examined, treatment with NaBu induced 2- to 5-fold the level of FPGS and ABCC1 mRNA expression whereas the level of DHFR, SLC19A1, and TS mRNA expression was decreased. Expression of GGH and ABCG2 mRNAs was increased 2-fold in CCRF-CEM but remained unaltered in Bp-ALL NaBu treated cells. Promoters of butyrate-responsive genes have been shown to contain genetic elements such as Sp1/Sp3 binding sites which interact with HDAC1 to mediate the action of NaBu. On this basis, we hypothesized that pre-treatment of ALL cells with NaBu should lead to induction of FPGS expression and subsequently, higher synthesis of MTX-PG and enhanced MTX cytotoxicity in ALL cells. To test this hypothesis, CCRF-CEM, NALM6, REH, and SupB15 cells were treated sequentially with NaBu (24h) and MTX (4h), and assessed for cell viability. Treatment of NaBu and MTX increased cell death by ∼40% in NALM6, REH, and SupB15 Bp-cells, and ∼60% in CCRF-CCEM cells when compared to treatment with each drug alone. These data suggest that combination of HDACi and MTX may represent a novel therapeutic strategy for treatment of ALL. This strategy may be particularly useful to overcome MTX resistance in patients diagnosed with phenotypes that accumulate low levels of MTX-PGs and for patients after relapse.

Beschreibung: Context: Akt plays a pivotal role in the survival and proliferation of multiple myeloma (MM) cells and functions as a central link between upstream signaling pathways, such as growth factor receptors (e.g. IL-6/IL-6R, IGF/IGF-1R) and the kinase PI-3K with their downstream signaling effectors, such as the multifunctional mTOR-p70S6K cascade. The pivotal role of Akt in these proliferative/anti-apoptotic cascades for MM has provided the impetus for development of small molecule inhibitors against the kinase activity of Akt. We show that the dual Akt/p70S6K kinase inhibitor EXEL-6075 has potent anti-MM activity in preclinical models. Methods/Results: We tested a panel of 15 human MM cell lines for in vitro response to EXEL-6075 using MTT colorimetric survival assays, which showed that the majority of MM cells responded to EXEL-6075 with IC50 values 1-log differential activity against the non-neoplastic tissues tested in our study. This remains an interesting strategy for the treatment of MM. Future in vivo studies will be needed to confirm these interesting in vitro results.

Beschreibung: Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastatic, anti-angiogenic, and anti-thrombotic activities. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with disease stage and inversely correlates with survival. To explore the therapeutic potential of rNAPc2 during tumor growth and metastasis, we tested rNAPc2 efficacy in experimental colorectal cancers in mice. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). Using CD31 and Ki67 immunohistochemisty, we found that rNAPc2 synergized with either 5-FU or bevacizumab in inhibiting microvessel density and tumor cell proliferation in HCT116 human colorectal tumor xenografts. Furthermore, rNAPc2 synergized with CPT-11 in inhibiting hepatic metastasis in nude mice with portal vein injection of HCT116 human colorectal tumor cells. Long-term administration of rNAPc2 also significantly suppressed formation of intestinal adenomas and adenocarcinomas in ApcMin/+ mice. The dosing regimens of rNAPc2 used in these studies were well tolerated up to a three-month period by recipient mice without major hemorrhage or other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.

Beschreibung: Ras mutations occur in 40–60% of multiple myeloma (MM) patients and are implicated in progression to advanced MM (including plasma cell leukemia/extramedullary lesions). The small molecule PRLX (Prolexys Pharmaceuticals) was identified in the context of “synthetic lethal” chemical screening for genotype-selective cytotoxicity against cells transformed with forced expression of mutant Ras (but not against isogenic normal cell counterparts) and was tested for anti-MM activity. In MTT survival assays, 34 of 46 human MM cell lines (74%) responded to 48hr treatment with sub-μM PRLX concentrations (achievable in preclinical pharmacokinetic studies). (24 MM lines had IC50 values

Beschreibung: Salvage chemotherapy followed by high dose therapy (HDT) and autologous stem cell transplantation (ASCT) is the standard of treatment for chemosensitive relapses in diffuse large B cell lymphoma. Improvement of salvage chemotherapy was suggested with the association rituximab, Ifosfamide, etoposide, carboplatinum, R-ICE. What is the optimal chemotherapy regimen and can we reduce the post ASCT relapses rate? The ongoing CORAL trial was designed to answer these questions. DLBCL CD 20+ in first relapse or pts refractory after first line therapy were randomized between rituximab plus DHAP and R-ICE. Stratification was made on centers, prior rituximab exposure and refractory/12months, 108 refractory/early relapses; 97 pts with prior exposure to rituximab; Stage 3-4 107 pts; elevated LDH 88 pts; secondary IPI 0–1 112 pts; sIPI 2-3 63pts. The two arms were well balanced. In the prior rituximab cohort exposure more pts had refractory disease and adverse prognostic factors. However, at inclusion patients characteristics were not significantly different in the stratified subgroups. The overall response rate was 68%, with 41% complete remission rate. Toxicity was similar to what is expected with intensive therapy, 72 SAE were reported with 12 deaths during the initial salvage regimens. In univariate analysis factors significantly affecting response rate (p1 54% vs 77% and prior exposure to rituximab 54% vs 82%. In a logistic regression model only refractory/early relapse and secondary IPI remain significant for response rate. Intent to treat 2 yrs EFS and OS were 50% (CI 42–57%) and 69% (CI 61–75%) respectively. Only 107 pts in this prospective study received, per protocol ASCT. For patients transplanted, 2 yrs EFS was 75% (CI 63–84%) with OS 89%. Two yrs EFS was affected by: prior treatment with rituximab, 34% vs 66% (p=.0001); refractory/early relapse 36% vs 68% (p

Beschreibung: Vascular endothelial growth factor (VEGF)-mediated signaling has at least two potential roles in diffuse large B cell lymphoma: potentiation of angiogenesis, and potentiation of proliferation and/or survival induced by autocrine VEGF receptor-mediated signaling by lymphoma cells. We have recently shown that diffuse large B cell lymphomas expressing high levels of VEGF protein also express high levels of VEGF receptors 1 and 2. We have now assessed a larger multi-institutional cohort of patients with de novo diffuse large B cell lymphoma treated with anthracycline-based therapy to address whether either tumor vascularity as assessed by microvessel density counting, or expression of VEGF protein and its receptors as assessed by immunohistochemistry, contribute to patient outcomes. Increased tumor vascularity (Figure 1a, b, c -- increasing microvessel density) is associated with poor overall survival (p=0.047), and is independent of the international prognostic index (Figure 1d). In contrast, high expression of VEGFR1 by lymphoma cells is associated with improved overall survival (p=0.044) and a trend toward improved progression-free survival (p=0.054). High expression of VEGF by lymphoma cells is also associated with a trend toward improved overall survival (p=0.089). The concordant trend toward improved survival with increased lymphoma cell expression of VEGF and its receptor VEGFR1, together with their coordinate overexpression in a subset of diffuse large B cell lymphomas (p=0.00025, chi value 21.5), suggests the presence of an autocrine VEGF-VEGFR1-mediated feedback loop in these lymphomas. Indeed the combination of high VEGF and VEGFR1 protein expression identifies a subgroup of patients with improved overall (p=0.003) and progression-free (p=0.026) survival; these findings are independent of the international prognostic index (Figure 2). The presence of improved survival in a cohort of patients whose lymphomas potentially depend on autocrine signaling via VEGFR1 suggests that dependence on this pathway may render patients susceptible to the effects of anthracycline-based therapy; indeed, downregulation of VEGF expression at the mRNA level by anthracyclines and other chemotherapeutic agents has been demonstrated in a variety of non-hematolymphoid neoplasms. The relative importance of autocrine VEGF-mediated signaling versus vascularity should certainly be taken into account in clinical trials of anti-VEGF/VEGF receptor therapy in diffuse large B cell lymphoma. Figure Figure Figure Figure

Beschreibung: The importance of bone marrow examination in the evaluation of leukemia, multiple myeloma, anemia, pancytopenia, and other disorders is well established. The objective for this study was to evaluate the ability of a powered bone marrow aspiration device to penetrate the intraosseous medullary space of the iliac crest, and to aspirate bone marrow samples for the ultimate purpose of diagnosing disease and monitoring the course of disease and medical therapy. The device was used to obtain bone marrow samples in accordance with accepted practice guidelines and device’s directions for use. Among other data, insertion success, time to insertion, and complications were recorded. Patient pain levels were rated from 0 to 10 (10=extreme pain). Device operators rated the use of the device from 0 to 10 (10=outstanding). There were 55 patients in the study from three centers. Successful insertion and aspiration of bone marrow samples were achieved in 54 of the 55 patients (98.1%). Mean insertion time was 4.9±3.0 seconds; significantly faster than the 7.3 minutes reported by Kuball et al* (one-sample t-test, p

Beschreibung: B-cell chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the Western world, however, infrequent in the Eastern. Although the median survival is around 10 years, CLL patients have a highly variable clinical course and prognosis. While some patients show an indolent disease and never require treatment, others suffer from a much more aggressive course requiring intensive treatment shortly or immediately after diagnosis. Identification of these subgroups and insight into the prognosis for each individual patient will be important to determine individualized treatment strategies. To explore the prognostic significance of CD38 expression in Chinese patients with CLL, multi-parameter flow cytometry was used to detect the expression of CD38 on CD5+CD19+ cells of 147 patients with CLL. CD38 expression and its association with some other prognostic factors such as Binet stage, lymphocyte count in peripheral blood, serum lactate dehydrogenase (LDH), β2-microglobulin (β2-MG), ZAP-70 expression and cytogenetic abnormalities were analyzed. The Kaplan-Meier method was used to construct survival curves, and results were compared using the log-rank test. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. Out of the 147 CLL patients, positive expression of CD38 was found in 45 (30.6%) cases. CD38-positivity identified a subgroup of CLL patients with aggressive disease of Binet stage at the time of the test (P=0.036). Furthermore, the presence of higher serum LDH and β2-MG levels at diagnosis was strongly correlated with CD38-positive (P=0.016 and P=0.025, respectively). Prognostically unfavorable cytogenetic abnormalities, including 17p13 deletions and 11q22 deletions were significantly more frequent in CD38-positive patients than in CD38-negative ones (P=0.047 and P=0.001, respectively). There was no significant difference between CD38-positive and CD38-negative subgroups in terms of age, sex, or lymphocyte count. In addition, in CD38-positive patients, the percentage of leukemic cells expressing ZAP-70 protein was not significantly higher than in CD38-negative ones (P=0.120). There was no significant difference between CD38-positive and CD38-negative groups in molecular cytogenetic aberrations of del(6q23), del(13q14), 14q32 translocation, or trisomy 12. CD38 expression was associated with poor outcome. Patients with positive expression of CD38 had significantly shorter overall survival (mean, 81 months) than patients without CD38 expression (mean, 179 months) (P=0.015). Univariate and multivariate analysis showed that serum levels of LDH and β2-MG, del(17p13) and CD38 expression were strongly associated with survival. It was showed that the patients with high level of CD38 expression had poorer outcome; CD38 was a good predictor of survival time in Chinese patients with CLL.

Beschreibung: Liposome-mediated gene transfer is known to exhibit a number of desirable features for gene delivery, such as decreased immunogenicity, lack of mutagenesis, efficient transgene expression, internalization of episomal DNA irrespective of size, reproducibility, compositional variability and ease of use. As we and others recently demonstrated, cationic Phosphonolipids (CPs) have pronounced gene delivery potency into human cells. Based on the well-established dependency of gene transfer efficiency upon lipoplex properties, the superior-performing CP EG.308 and a novel cationic lipophosphoramide vechicle (CL) KLN-5, were evaluated for delivery of the human A-gamma globin gene in K562 human erythroleukemia and 5637 human epithelial carcinoma cells. The plasmid vector used for Aγ-globin gene was pAγLuc (3.3 kb HindIII human Aγ-globin gene). Two other vectors were used as controls: pGL3bLuc+, which encodes for luciferase; and, pEGFP-CI, which encodes for the green fluorescent protein. Dynamic Light Scattering experiments underlined the cooperative effect of the liposome vesicle diameter, complex formation medium and charge ratio upon lipoplex size. At optimized lipoplex generation conditions, the initial lipid size was not found to be always predictive of lipofection potency, rather transfection efficiency is a function of the liposome vehicle to generate lipoplexes of enhanced size (300–3500nm). Dot-blot analysis and reporter gene-expression assays revealed that among a number of varying composition solvent-systems, optimal nuclear transgene incorporation and expression was attained in 300mM NaCl. Lipoplexes EG.308- and KLN-5/pAγLuc formed in 300mM NaCl mediated maximal transgene delivery and expression 1 and 3 days-post transfection, respectively (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein, respectively). On day 14, sustained plasmid DNA quantity was still detected in the nucleus. Enhanced plasmid integrity was confirmed by Southern blotting analysis. Both linear and circular pAγLuc DNA delivery resulted in pronounced transgene entry (330pg/ng genomic DNA); however, by introducing the latter, transgene expression was almost 100% higher. As opposed to the effective KLN-5-mediated transfection of K562 cells (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein), transgene targeting and expression in 5637 cells was low (73pg luciferase/ng genomic DNA, 13150 RLU/μg protein). Confocal fluorescence microscopy revealed similar lipoplex-cell membrane association events and overlapping endocytic activity in EG.308- and KLN-5- mediated lipofection of target cells. However, KLN-5/pDNA nuclear co-localization in a greater number of GFP+ transfected cells compared to EG.308/pDNA lipofected ones, support a lipid-specific complex behaviour. These results indicate that effective transgene nuclear targeting is feasible with the tested CP and CL. Human Aγ-globin transgene nuclear incorporation and expression in target cells by CPs lends credence to their use as a vehicle of therapeutic transgene delivery.

Beschreibung: Background: In this study we cloned a vector based on a self-inactivating lentiviral backbone containing MDR1 (multidrug resistance 1 gene) connected by an ECMV-IRES-element with MGMT P140K (O6-BG-resistant mutant of O6-methylguanine-DNA methyltransferase). The chemoprotective potential of this HR′SIN-MDR-IRES-MGMT combination vector was compared to single vectors (HR′SIN-MDR, HR′SIN-MGMT). Methods: HL60 and CD34+ cells were transduced with the various vectors. After chemotherapeutical treatment MTT assays were used to detect chemoresistance levels in HL60 cells, CD34+ cells were held in liquid culture under differentiation conditions and analysed by FACS for MDR1 expression. Results: HL60 cells transduced with the combination vector showed significant chemoresistance to O6-BG/ACNU (IC50 13x higher compared to untransduced control), the IC50 of cells transduced with HR′SIN-MGMT was 35x higher. The IC50 of paclitaxel (MDR1 substrate) was 24x higher in cells transduced with HR’SIN-MDR and 25x higher with HR’SIN-MDR-IRES-MGMT compared to untransduced control. Combined exposure of cells to O6-BG/ACNU and paclitaxel showed a survival advantage of cells transduced with the combination vector (IC50 6.25x higher), for the single vectors the IC50 was 1.63x higher (MDR1) and 2.08x higher (MGMT) compared to untransduced control. Treatment of CD34+ cells with increasing concentrations of doxorubicin (up to 0.8 μM) resulted in a higher fraction of MDR1-positive cells either with HR′SIN-MDR (26.6x) or with HR′SIN-MDR-IRES-MGMT (30.6x) compared to untreated cells. After combination treatment (20μM O6-BG/16μM BCNU and 0.4μM doxorubicin) the fraction of MDR1-positive cells was higher for HR′SIN-MDR-IRES-MGMT (14x) than HR′SIN-MDR (8x) transduced cells. Conclusion: The protective effect of the combination vector is comparable with the single vectors for monotherapy and superior for combined therapy. The combination vector presents simultaneous protective effects of two drug resistance genes, thus reducing the risk of insertional mutagenesis by one transduction process. Consequently our results might help to reduce myelotoxic side effects and increase the chemotherapeutic efficiency.

Beschreibung: Background: The discovery of JAK2V617F mutation has lead to the proposal of new algorithms to diagnose and classify MPD. Separation of PV from ET could become less clear, especially in JAK2V617F patients, if one considers that mutated PV and ET are similar conditions. However, the short-term risk of thrombosis, and long-term risk of evolution to leukemia have not yet been shown to be similar in JAK2V617F ET and PV. Current WHO criteria for PV require either RCM 〉125% of predicted value, or Hb level 〉18.5 g in men and 16.5 g in women. Some investigators recently proposed a threshold for Ht at 52% in men and 48% in women to diagnose PV in JAK2V617F patients. Diagnosis of ET requires excluding PV, and using Hb or Ht values for this purpose could avoid RCM measurement. Aims: To evaluate the prevalence and prognostic impact of erythrocytosis determined by RCM measurement in MPD patients classified as ET on peripheral blood counts. Methods: We reviewed all RCM measurements performed in patients suspected of MPD in 2 nuclear medicine laboratories during a 10-year period. Results: Among 566 patients referred for RCM measurement, 71 had isolated thrombocytosis (i.e. both Hb and Ht 〈 values used for PV diagnosis as defined above). RCM was normal in 38/71, but was 〉125% of predicted value, revealing inapparent erythrocytosis, in 33 cases. Thus, after RCM measurement, final diagnosis was ET in 53.5% (38/71), and PV in 46.5% (33/71) of the patients, respectively. Pts with elevated RCM had significantly higher Ht (p

Beschreibung: We previously reported that histone deacetylase (HDAC) activity is elevated, but is not correlated to the JAK-2 mutation status, in patients with myelofibrosis myeloid metaplasia (MMM) (Blood 107:319b 2005). Now we have studied more patients: totally, 17 with MMM, 19 with other myeloproliferative disorders (MPD), and 16 normal volunteers as controls. Significantly elevated HDAC levels again was shown in patients with MMM compared with other MPD patients and normal volunteer controls (p

Beschreibung: It has long been proposed that Myelodysplastic syndromes (MDS) arise into a context of genomic instability leading to accumulation of multiple mutations. However, the underlying mechanism remains elusive. Two different genetic instability pathways, chromosome instability (CIN) and microsatellite instability (MSI) can be study using repetitive polymorphic markers (STR). Mutations of CIN genes increase the rate of gross chromosomal changes and loss of heterozygosity (LOH), which is an important mechanism of tumor suppressor gene inactivation. MSI is manifested by alterations of the length of STR due to germline mutations or methylation-induced silencing of key DNA mismatch repair genes (MMR). To verify whether both mechanisms might be involved in MDS, microsatellite instability and loss of heterozygosity (LOH) were analyzed with 10 specific STR markers. Bone marrow samples from 21 de novo MDS patients (12 females/9 males) with a mean age of 70.7 years (range 38–93), including 13 RA, 4 RAEB, 1 RAEB-T, 2 RARS and 1 CMML, were analyzed. All patients gave informed consent and the study was approved by our Ethics Committee. Mononuclear cells were separated by density gradient centrifugation through Ficoll Hypaque and depleted of adherent cells by 1 h incubation at 37°C. Myelodysplastic DNA was obtained from non-adherent mononuclear cells, while normal DNA was extracted from polymorphonuclear cells. Ten STR were amplified at different annealing temperatures (55–58°C), depending on the average size of each primer. Four mono- or dinucleotide markers (BAT 25, BAT 26, D2S123 and D18S58) were chosen from the Bethesda reference panel for MSI studies. Six additional loci at CRTL (5q13-14), IRF (5q31), D5S209 (5q31-33), CSF1RT (5q33-35), D7S525 (7q21-31) and TP53 (17p13.1), were selected because of their location on specific genomic points involved in MDS and acute leukemia. PCR products were analyzed by medium size non-denaturing polyacrylamide (15%) gel electrophoresis and silver staining (0.1%). Allele variations between normal and myelodysplastic DNA were scored according international criteria. Six out of 21 cases (28.6%) presented an unstable phenotype with band shifts or losses. No STR alterations were observed with markers BAT 25, BAT 26, IRF and D2S123. Two cases (9.6%) presented LOH at TP53, D5S209 and D7S525. Bethesda panel revealed MSI only in one patient (4.8%) at D18S58. STR at hematological loci allowed the identification of 5 cases (23.8%) with amplifications or deletions of the repeat sequences at CSF1RT, CRTL and TP53. MSI was detected only at 1/9 or 2/9 STR per patient and therefore these cases were classified to exhibit low grade of MSI (MSI-L). The mean frequency of mutated STR/individual showed a significant difference between hematological markers (0.08±0.03) respect to Bethesda STR (0.01±0.03) (Student test, p=0.007), suggesting that colon markers are not useful to evaluate MSI/LOH in MDS. MSI-L was more frequent than LOH in patients with unstable phenotype, suggesting that putative tumor suppressor genes at the loci studied are not involved in MDS genomic instability. Our data suggest that a subset of patients with MSI-L, are not related to MMR mutations but are probably associated to other genetic alterations, critical in MDS development. Moreover, a STR panel specific for MDS should be defined in order to deep evaluate genomic instability in this disease.

Beschreibung: We performed a retrospective analysis of our Spanish database of patients with ET in order to assess the role of different response predictive potential factors to anagrelide treatment. 411 ET patients from 54 sites from February 2005 to August 2006 were included in a retrospective chart review. ET was diagnosed according to PVSG criteria (1997). All patients had started treatment with anagrelide before December 2004, either as a first line or as a second line therapy. The population was divided itself in three groups of risk at diagnosis: of high Risk: those patients older than 60 years or with previous history of thrombosis (40.6%); of low Risk: patients younger than 60 years with thrombocytosis lower than 1.5· 109/L, without cardiovascular risk factors and previous history of thrombosis (35.8%); intermediate risk: those patients who did not reunite the criteria of previously mentioned groups (23.6%). The response to the treatment was defined as complete remission (CR), when a reduction of platelets′ count were equal or less than 400×109/L, or a reduction higher than 50% with respect to the basal numbers; partial remission (PR), when the platelets′ count was between 400× 109/L and 600×109/L with respect to the basal numbers; and no response, those with a lower reduction than PR or increasing their platelets′count. CR was obtained in 219 patients (53.6%; 95%CI = 48.6–58.5) and PR in 113 (27.6%; 95%CI = 23.4–32.2), giving an overall response (OR) rate of 81.2% [95%CI = 77.0–84.9]. The influence of certain factors (such as age, gender, risk group, platelets levels before treatment with anagrelide, previous cytoreductive untreated patients and maximum anagrelide dose), in the objective response was explored. A chi-square test and a multivariate analysis (logistic regression) were performed. A worse response (p = 0.021) was associated with a higher dose (≥ 2.5 mg per day). A trend (p = 0.103) to better response in patients without previous treatment (anagrelide as a first line) was detected. Additionally a significant association (p = 0.02) between the previously treated with hydroxyurea and the presence of evolutive hematological transformations (myelofibrosis and acute myeloid leukemia/myelodysplastic syndrome) was observed. It is difficult to set a cause effect relation in all these findings due to the retrospective design of the analysis. As a conclusion, the response to anagrelide in ET patients was demonstrated to be independent of the age, gender, platelets′ count and level of previous risk.