ALBERT

Description: There is a high incidence of venous thromboembolic complications in patients with beta thalassemia .Recently, a high incidence of asymptomatic multiple silent cerebral infarcts, presumably of arterial origin, were demonstrated in patients with beta thalassemia major (TM) and intermedia. The etiology remains unclear. Endothelial progenitor cells (EPCs) derive from the bone marrow and contribute to regeneration of endothelial damage. We hypothesized that the change in EPCs contributes to the vascular events in patients with TM and that patients have high markers for arterial thromboembolism. Study aim: To assess the change in the number and function of EPCs in TM patients. Methods: Blood samples were drawn from adult patients with TM before blood transfusion and from healthy volunteers. EPCs were isolated from peripheral mononuclear cells by using a Ficoll density-gradient centrifugation. EPCs' levels were detected by flow cytometry, namely by co-expression of VEGFR-2 in CD34 or CD133 positive cells. EPCs were cultured for 7 days after which functional properties were evaluated by: their capacity to form colonies using light microscopy (a colony forming unit (CFU) defined as a cluster of at least 100 flat cells surrounding a cluster of rounded cells) as well as by using a viability assay (using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay). The adhesion molecule P- selectin and the chemokine SDF1 were measured by using Elisa kits according to the manufacturer instructions. Results: Thirty five patients (median age 35 range 27-47y) and 15 healthy volunteers (median age 34.5 range 30-50y) were included. Lower levels of CD34/VEGFR-2 cells and a trend towards CD133/ VEGFR-2 cells were observed in TM patients (Figure 1A). Viability of EPCs and their ability to perform colonies were markedly reduced in TM patients compared to controls; mean MTT 0.05± 0.05 in TM vs. 0.23±0.08 (p

Description: Abstract 18 Introduction: Long-term survival in pediatric relapsed AML is only 20-30%. Optimal reinduction therapy is unknown, and there is a concern about cardiotoxicity with repeated anthracycline use at relapse. Preclinical in vitro and animal studies, and limited clinical data suggest that liposomal daunorubicin (DaunoXome®, DNX) is less cardiotoxic. These considerations lead to a phase III study, in the setting of the International BFM Study Group. Materials and methods: FLAG was randomised against FLAG/DNX in the 1st reinduction course. The conventional 5-days FLAG only was recommended as the 2nd course. DNX was dosed at 60 mg/m2/day on days 1, 3 and 5. After induction, allogeneic stem cell transplantation was generally recommended, but time-to-transplant could be bridged by high- or low-intensive consolidation therapy. Primary endpoint of the study was early treatment response, based on bone marrow examination shortly before reinduction course 2, and defined as either good (≤20% leukemic blasts) or poor (〉20% leukemic blasts). This endpoint was chosen because of its prognostic value in earlier relapsed AML BFM-trials, and because compliance with an extended protocol guideline was likely to be suboptimal within the context of a highly multinational and multicenter AML Relapse protocol. However, secondary endpoints were defined, including the CR2 rate determined after 2 courses, long-term survival, and toxicity. Patients with AML M3 and those 〉18 years of age at initial diagnosis were ineligible. The study opened in most countries in 2002/2003. The study closed for accrual on April 1, 2009 when the required 360 fully eligible and evaluable patients had been randomized. Early and late relapsed AML was defined as a relapse within or after 1 year from initial diagnosis, but this only influenced treatment in that early relapsed AML patients were eligible for haploidentical SCT, while late relapsed AML patients were eligible for autologous SCT, if a matched or partly mismatched transplant was not possible. Thirteen groups from 20 countries and 〉100 centers have enrolled patients, with informed consent and after approval of the study by regulatory authorities. Data are presented according to intention-to-treat, with a median follow-up of 2.7 years for patients at risk. Results: Overall 4-year probability of survival (pOS) was 35% SE 2%, the overall CR2 rate 62%. The good early responders had a 4-year pOS of 45% SE 3% versus 10% SE 3% for poor responders (p

Description: Introduction Survival of beta thalassemia major (TM) and intermedia (TI) patients have improved significantly over the past few decades as better treatment and follow up became available. However, complications are still common and affect patients’ quality of life, and systemic iron overload, particularly in the heart, liver, and endocrine organs, continues to be the main contributor to severe morbidity and early mortality in these patients. Traditionally, most thalassemia patients worldwide are treated in pediatric clinics by pediatricians. Consequently, in contrast to young patients, less data is available for complications and morbidity among adult TM & TI patients. We aimed to characterize disease and patients’ characteristics in patients 〉35 years of age. Methods We retrospectively reviewed medical charts of all consecutive patients above the age of 35 years with TI/TM who were treated in a tertiary thalassemia center. Liver iron load was defined as at least one of the following: elevation of liver enzymes, signs of steatosis by sonography, and evidence of iron accumulation by T2* MRI. We used descriptive statistics to describe characteristics of disease and patients and the Mann-Whiteny test to compare between patients with TI and patients with TM. Results Between the years 2006 and 2013, 62 adult patients with TM & TI 〉18 years old were followed and treated in our center. Of the 62 patients, 14 (TM, n=10 and TI, n=4) were above the age 35 years and were included in the study. The median age was 37 years (range, 35-50), 66% were male, 50% were of Arab ethnicity, 93% had at least high school education and 78% were employed, 93% were splenectomized. Twelve patients (85%) were regularly transfused every 3-4 weeks. The median pre-transfusion hemoglobin and mean corpuscular volume levels were lower in patients with TI compared to TM (8.1 vs. 10 g/dl, p=.002 and 72 vs. 84, p=.004, respectively). Median erythropoietin level in the transfused patients was 43.3 mlU/ml, indicating reduced RBC production. Median LDH and indirect bilirubin levels were higher in patients with TI compared to TM (603 vs. 330 u/L, p=.004 and 2.02 vs. 1.1 mg/dl, p=.06, respectively) indicating increased hemolysis. Median uric acid level was higher in patients with TI compared to TM (7.7 vs. 4.2 mg/dl, p=.0004), resulting in symptomatic gout in one TI patient. At the time of data collection, all TM and half of TI patients were treated with iron chelation therapy (ICT), deferasirox (n=6, 50%), deferiprone (n=2, 16%) and combination of deferioxamine (IV or SC) and deferiprone (n=4, 33%). Good compliance and adherence to ICT were reported in 75% of the patients. Complications associated with thalassemia are shown in the table. No statistically significant difference was observed in any complication, probably due to relatively small size of study cohort. Four TM patients (40%) had a history of heart failure. Nevertheless, with intensified ICT heart function was normalized in all cases, followed by normalization of T2* cardiac MRI. Four TM patients (40%) had hypothyroidism, two of them were treated with thyroid hormone. None of the patients had diabetes requiring hypoglycemic agents. All TM patients had hypogonadism. All females had amenorrhea and were treated with hormone replacement therapy, and none of them tried to conceive. All males, but one, were treated with monthly testosterone injections, 3 patients fathered children. All TM patients had osteoporosis, all treated with hormonal therapy (testosterone or hormone replacement therapy), in addition to calcium and vitamin D supplements. Three TI patients (75%) had metabolic bone disease; two had osteoporosis and one had osteopenia. All TI patients were treated with calcium and vitamin D supplements. None of the patients underwent allogeneic hematopoietic stem cell transplantation and none developed secondary malignancy. Conclusion we show a data set of adult thalassemia patients with prolonged survival, highlighting the long term co-morbidities of this unique population. Our data emphasizes the unmet needs for long term surveillance for identification of organ specific risk factors and early disease manifestations as well as the needs for global transition from pediatric to adult medicine, and from pediatric clinics to dedicated comprehensive adult thalassemia clinics. Disclosures No relevant conflicts of interest to declare.

Description: Introduction & Objective Adoptive T cell therapy with tumor-specific CD8+ T cells is a promising treatment option for a variety of malignant diseases. However, it is unclear which subset of CD8+ T cells characterized by distinct functions is most suitable for achieving effective and durable anti tumor responses. So far CD8+ T cells have been considered to act predominantly as cytotoxic effector cells in cellular anti-tumor immunity. In this respect cytolytic molecules such as perforin and granzymes and apoptosis-inducing receptors of the tumor necrosis family such as FasL, TNFα and TRAIL have been regarded as major CD8+ T cell effector mechanisms. Methods & Results We here demonstrate in an experimental tumor model that CD40L, the key molecule for “immunological help”, is expressed by up to 50% of tumor-specific CD8+ T cells in B6 mice challenged with SV40 T antigen+ cancer cells. To study the influence of CD40L on anti-tumor CD8+ T cell immunity in vivo we challenged Rag1-/- mice with cancer cells and transferred wt or CD40L-/- CD8+ T cells. Transfer of wt CD8+ T cells prevented the establishment of a solid tumor, whereas injection of CD40L-/- CD8+ T cells alone or in addition with wt CD4+ T cells resulted in a non-controlled tumor development similar to non-treated tumors. The requirement of CD40L on CD8+ T cells for tumor rejection was further confirmed by injecting cancer cells in mice that lack CD40L expression only on mature CD8+ T cells. CD40Lflox x E8Icre mice were more susceptible to tumor formation than wt mice. Furthermore we demonstrated that CD8+ T-cell derived CD40L had to interact with CD40 on cancer cells, an eminent signal to induce apoptosis in various cancer cell types. Conclusion Our results reveal a crucial functional relevance of CD40L expressed by CD8+ T cells in anti-tumor immunity. Various cancer cell types express CD40 and its engagement induces pro-apoptotic or growth-inhibitory signals in a variety of cancer cells. Therefore CD40 agonists are recognized as promising agents for therapeutic interventions. We here introduce CD40L+ CD8+ memory T cells as a new major physiological source of CD40L, essential for rejection of tumors. Our data reveal that the presence or absence of CD40L+ CD8+ T cells represents a crucial element in control of CD40 expressing cancers disclosing novel treatment approaches in adoptive T-cell therapies novel treatment approaches. Disclosures: No relevant conflicts of interest to declare.

Description: Patients with T-cell ALL are at increased risk of CNS relapse and require more intensive CNS directed therapy. CRT is considered to be the most effective therapy, but is associated with serious adverse late effects and secondary brain tumors. We evaluated retrospectively, in the context of the ALL-BFM-based protocols, the impact of CRT or extended T.I.T. on outcome within High Risk (HR: Prednisone poor responders) and non-HR (Prednisone good responders) subgroups. In INS 89, modified ALL-BFM 86/90, all Non-HR patients received extended T.I.T (×18) as CNS preventive treatment, and VP-16 was added systemically in between the HD-MTX (5g/m2). In INS 98, based on ALL-BFM 95, VP-16 was omitted and pulses of VCR+DEXA were added in maintenance. Patients with T-ALL Non-HR and WBC 〉100000, were assigned to receive CRT 12 Gy, following the results of the AIEOP-ALL 91 (Conter J.C.O. 15:2786–2790, 1997) reporting inferior outcome in this group when comparing T.I.T only to BFM with irradiation. T-ALL HR patients were treated without modification and were all assigned to CRT 18 or 12Gy. Results: Between 1989 and 2002, 145 T-ALL children aged

Description: Fusion genes derived from the platelet-derived growth factor receptor beta (PDGFRB) or alpha (PDGFRA) play an important role in the pathogenesis of BCR-ABL–negative chronic myeloproliferative disorders (CMPDs). These fusion genes encode constitutively activated receptor tyrosine kinases that can be inhibited by imatinib. Twelve patients with BCR-ABL–negative CMPDs and reciprocal translocations involving PDGFRB received imatinib for a median of 47 months (range, 0.1-60 months). Eleven had prompt responses with normalization of peripheral-blood cell counts and disappearance of eosinophilia; 10 had complete resolution of cytogenetic abnormalities and decrease or disappearance of fusion transcripts as measured by reverse transcriptase–polymerase chain reaction (RT-PCR). Updates were sought from 8 further patients previously described in the literature; prompt responses were described in 7 and persist in 6. Our data show that durable hematologic and cytogenetic responses are achieved with imatinib in patients with PDGFRB fusion–positive, BCR-ABL–negative CMPDs.

Description: One of the diagnostic criteria of essential thrombocythemia (ET) is the absence of the Philadelphia chromosome (Ph-neg). On the molecular level, Ph-neg ET patients may carry BCR-ABL transcript. The natural history of BCR-ABL positive Ph-neg ET patients is undetermined. We examined the BCR-ABL status by reverse transcriptase two-step nested polymerase chain reaction in bone marrow aspirates of 25 Ph-neg ET patients. We found 12 BCR-ABL positive and 13 BCR-ABL negative patients in the study group. The comparison showed that the two groups had similar clinical and laboratory characteristics, except for a significant increased patients' age and decreased polymorphonuclear cell count in the BCR-ABL positive group. During a median follow-up of 20 and 22.5 months for the BCR-ABL negative and positive groups, respectively, there was neither blastic transformation nor unrelated death in both groups. We conclude that it is important to look for BCR-ABL transcript in Ph-neg ET patients and to follow them closely to investigate the nature of this translocation in this group of patients.

Description: Abstract 3579 Children with Down syndrome (DS) have an increased risk of developing B-cell precursor acute lymphoblastic leukemia (BCP-ALL), characterized by a low frequency of the common genetic aberrations, and a high frequency of CRLF2 and JAK aberrations. Because this disease is relatively rare, the clinical outcome, treatment-related mortality (TRM) and prognostic factors of DS-ALL patients treated in contemporary protocols are uncertain. Previous studies demonstrated poorer survival and a high rate of treatment related mortality (TRM), but most studies are small since DS ALL patients comprise only 1–2% of all protocol patients. We therefore conducted a large retrospective study of 653 children with DS-ALL treated in clinical trials of 16 collaborative study groups between 1995 and 2005. All genotypes obtained from conventional karyotyping, FISH or RT-PCR were centrally reviewed and assigned to specific cytogenetic groups. The 310 girls and 343 boys have a median age of 5.0 years (range, 1.2 – 17.9) and a median white blood-cell count (WBC) of 10.2 × 109/L (range, 0.2 – 459). The 827 non-DS BCP-ALL control patients from the Dutch Childhood Oncology Group treated in the same era had similar WBC (8.8 × 109/L; p=0.25) but were younger (4.6 years; p〈 0.001). The median follow-up time was 6.8 years for DS-ALL survivors and 7.3 years for non-DS-ALL survivors. DS patients have a higher 8-year cumulative incidence of relapse (CIR) (26±2% vs. 18±1%; p=0.001) and higher 2-year TRM (7±1% vs. 1.0±

Description: Chronic lymphocytic leukemia (CLL) lymphocytes manifest anomalous motility and cap formation. Since these processes involve cytoskeletal proteins, vimentin from intermediate filaments of normal and CLL lymphocytes was investigated using hetero- and monoclonal antisera. The antisera reacted predominantly with a 60-kD polypeptide, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total lymphocyte proteins. When lymphocytes were stained by indirect immunofluorescence, normal lymphocytes demonstrated well defined cytoplasmic fibrils that capped spontaneously after contact with a glass surface and incubation at 37 degrees C. This capping was dependent on energy and intact microfilaments. Lymphocytes from patients with CLL showed several patterns. In one group, the initial staining was weak, and few capped cells were present after incubation. Lymphocytes from other patients had either normal or aberrantly organized fibrils in which capping was diminished. In another group, a fibrillar pattern with normal or increased capping was seen. In total, 47% +/- 5.1% (mean +/- SE) of normal lymphocytes capped after a 1-hr incubation at 37 degrees C (n = 12) compared to 21% +/- 5.1% for CLL lymphocytes (n = 20, p less than 0.002). Purified subpopulations of normal B and T cells did not differ from unfractionated normal lymphocyte populations. These results demonstrate an anomalous vimentin capping response in CLL lymphocytes. They also show that the arrangement of vimentin in these cells differs from that of normal lymphocytes.

Description: Methotrexate (MTX) is one of the leading drugs in the treatment of leukemia, but extensive metabolism to 7-hydroxymethotrexate (7-OHMTX) can limit its therapeutic efficacy. In this study we investigated whether 7-OHMTX itself can provoke antifolate resistance that may further disrupt MTX efficacy. For this purpose we developed resistance to 7-OHMTX as well as MTX in two human leukemia cell lines (CCRF-CEM and MOLT-4) by stepwise exposure to increasing concentrations of 7-OHMTX and MTX. Consequently, both leukemia cell lines displayed marked levels of resistance to 7-OHMTX (〉10 fold) and MTX (〉75 fold), respectively. The underlying mechanism of resistance in the MTX-exposed cells was a marked decrease (〉10-fold) in reduced folate carrier (RFC)-mediated cellular uptake of MTX. This was associated with transcriptional silencing of the RFC gene in MTX-resistant CCRF-CEM cells. In contrast, the molecular basis for the resistance to 7-OHMTX was solely due to a marked decreased (〉 95%) in folylpolyglutamate synthetase (FPGS) activity which conferred 〉100-fold MTX resistance upon a short term exposure to this drug. This is the first demonstration that 7-OHMTX can provoke distinct modalities of antifolate resistance as compared to the parent drug MTX.