ALBERT

Description: Background: Thrombosis is an important cause of morbidity and mortality in Antiphospholipid Syndrome (APS) and in SLE patients with antiphospholipid antibodies (aPL). APL recognize β2 glycoprotein I (β2GPI)-bound to receptor (s) in endothelial cells (EC) and other target cells (i.e. platelets, monocytes) and trigger an intracellular signalling and a pro-coagulant and pro-inflammatory phenotype [i e.expression of tissue factor (TF), vascular cell adhesion molecule-1 (VCAM-1)] that lead to thrombosis. There is in vitro evidence that annexin A2 (A2), a receptor for tissue plasminogen activator (tPA) and plasminogen – and possibly other proteins such as toll-like receptors or the receptor for apolipoprotein E2′ - may be binding β2GPI on the membrane of target cells. Here, we examined the involvement of A2 in aPL-mediated pathogenic effects in vivo. We studied the effects of aPL Abs on thrombus formation, VCAM-1 expression in aortas of mice, and TF function in carotid artery homogenates in annexin A2 deficient (−/−) mice. Methods: A2 (−/−) mice and the corresponding wild-type (WT) mice, in groups of 10, were injected i.p. twice (0 and 48 hours later) with IgG from a patient with APS (IgG-APS) or with control IgG (IgG-NHS). Seventy-two hours after the first injection, several procedures were done in each mice: dynamics of thrombus formation (thrombus size), TF function in homogenates of carotid arteries, and c) VCAM-1 expression in the aortas using quantum dot nano crystals and two-photon excitation laser scanning microscopy. In addition, we examined the effect of an anti-A2 antibody on aPL-induced expression of intercellular cell-adhesion molecule (ICAM-1), E-selectin and TF acvitity on cultured endothelial cells (EC). Results: The titers of aCL and anti-β2GPI Abs in the sera of the mice at the time of surgery were medium-high positive in A2 (−/−) mice and in wild type mice injected with IgG-APS. Thrombus sizes were significantly larger in WT mice injected with IgG-APS when compared to similar type of mice treated with IgG-NHS (p=0.003). The size of thrombus in A2 (−/−) mice injected with IgG-APS was significantly smaller than mean thrombus size in WT mice injected with IgG-APS (p:0.0005). However, thrombus size in A2 (−/−) mice was larger in mice injected with IgG-APS when compared to same type of mice treated with control IgG-NHS (p=0.003), indicating a partial but significant abrogation of the thrombogenic effect. TF activity was significantly larger in WT mice treated with IgG-APS when compared to mice injected with IgG-NHS. Importantly, TF activity in carotid arteries homogenates of annexin A2 (−/−) mice injected with IgG-APS was significantly decreased (by 52%) when compared to wild type mice treated with IgG-APS. The expression of VCAM-1 in aorta of annexin A2 (−/−) ex vivo was also significantly reduced compared to LPS-treated mice (positive control) (p= 0.01). Interestingly, anti-A2 antibody significantly decreased aPL-induced expression of ICAM-1, E-sel and TF on cultured EC. Conclusions: Altogether these data indicate for the first time that A2 is involved in vivo pathogenic effects of aPL Abs. These findings may have important implications to devise new targeted and more specific therapeutic approaches to block the pathogenic effects of aPL Abs in patients with APS and SLE.

Description: Antiphospholipid antibodies (aPL) recognize β2Glycoprotein (β2GPI)-bound to receptor (s) in target cells and trigger a pro-coagulant/pro-inflammatory phenotype [i e.:expression of tissue factor (TF), vascular cell adhesion molecule-1 (VCAM-1)] that lead to thrombosis. The interaction of β2GPI with target cells may involve more than one protein. Investigators have shown that dimeric β2GPI binds to apolipoprotein E receptor 2′ (apoER2′) in platelets, in the absence of anti-β2GPI antibodies, increases their activation and induces enhanced thrombosis and TF activity in mice. However, the role of apoER2′ in vivo in Antiphospholipid Syndrome (APS) is not completely understood. Here, we examined the in vivo effects of dimeric β2GPI and of anti-β2GPI antibodies (IgG-APS) in apoER2′ deficient (−/−) mice and in normal mice pre-treated with recombinant soluble domain 1 of apoER2′ (BD1). In vivo, dynamics of thrombus formation (thrombus sizes), TF activities in carotid artery homogenates and in peritoneal macrophages and ex vivo expression of VCAM-1 in aortas and of TF activity in peritoneal macrophages were examined in the various types of mice after two i.p. injections with 40 μg of recombinant dimeric β2GPI – or with the corresponding monomer control – or with 500 μg IgG-APS (isolated from a patient with APS by protein G Sepharose) or with control IgG (IgG-NHS). Mice injected with IgG-APS had significant titers of anticardiolipin (aCL) and anti-β2GPI antibodies in their sera. In vivo, IgG-APS increased significantly the size of the induced thrombi as well as the TF activities in carotid arteries and in peritoneal macrophages in C57BL/6J (wild type) mice when compared to same type of mice treated with IgG-NHS. Similarly, ex vivo expression of VCAM-1 in mouse aortas and of TF in peritoneal macrophages, detected by two photon excitation laser scanning microscopy were increased in normal mice treated with IgG-APS when compared to control mice. The pre-treatment with 40 μg of BD1 i.p., significantly reduced those effects. Importantly, dimeric β2GPI (in the absence of anti-β2GPI antibodies) or IgGAPS did not increase significantly thrombus size, TF activities in homogenates of carotid arteries or in peritoneal macrophages, or ex vivo expression of VCAM-1 and TF in mice lacking apoER2′. Conclusions: Altogether these data show that dimers of β2GPI mimic pathogenic effects of anti-β2GPI antibodies in mice. Most importantly, apoER2′ is a mediator of those effects in vivo. These findings may provide insights not only for a better understanding of the pathophysiology of APS but may be important in the development of new targeted therapies, by means of interfering with the binding of β2GPI-aPL complexes with their receptor(s) in target cells in vivo.

Description: Background: Nilotinib, a potent and highly selective BCR-ABL inhibitor, has been approved in more than 50 countries including Mexico. Nilotinib is indicated for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph+ CML) patients (pts) in chronic (CML-CP) or accelerated phase (CML-AP) resistant or intolerant to prior therapy including imatinib (IM). Before the approval of nilotinib by FDA or EMEA a Compassionate Use Program (CUP) of nilotinib was initiated in Mexico. Methods: The pts included in this program were adults pts with Ph+ CML, IM-resistant or –intolerant in all phases of the disease. IM resistance and intolerance were defined according to current guidelines (NCCN and ELN). Physical examination, EKG, bone marrow aspiration, karyotyping and BCR-ABL mutation screening were performed in all pts before starting nilotinib. All pts signed an informed consent. Nilotinib was administered orally at a dose of 400 mg twice daily (BID). No dose escalation was allowed. All pts were monitored with karyotyping. Patients that achieved CCyR were evaluated with quantitative RT-PCR for molecular evaluation. The results of CUP of nilotinib in Mexico are reported in this abstract including the cytogenetic, qRT-PCR and mutational analysis in a highly-resistant CML patient population. Results: Between October 2006 and June 2007, 47 pts were included in the nilotinib CUP in Mexico. The median age was 41.7 years (range 22–68) and 20 (44%) were men. Of the 47 pts, 28 (59.6%) had advanced phase CML which includes 7 (14.9%) blastic phase (BP) and 21 (44.7%) accelerated phase (AP) pts. 19/47 pts (40%) were in chronic phase (CP) of CML. Most patients were resistant to IM (86%) and 14% were IM-intolerant/resistant. The median duration since CML diagnosis was 73.8 months (range 14–183). The median duration of prior IM use was 27.6 months. All pts had been treated with hydroxiurea, interferon, and/or cytarabine prior to IM. At the time of starting nilotinib 17/47 pts (36.12%) had BCR-ABL mutations (P-loop mutations in 3 pts, IM binding mutations in 5 pts, catalytic domain mutations in 5 pts, and A-loop mutations in 4 pts). Only 3/47 pts (6.38%) had T315I mutation. The median duration of exposure to nilotinib was 304 days (range 19–632). Most pts tolerated nilotinib well and only one severe adverse event (AE) was reported (long-term myelosupression). At the time of data cut-off (July 31, 2008), 25 pts (53.2%) remain on therapy. Reasons for discontinuation of therapy were: lack of efficacy in 7/47 pts (14.9%) including 3 pts with T315I mutation; disease progression in 13/47 (27.5%); adverse events in 1/47 (2.1%); lost of follow-up in 1/47 (2.1%). The cytogenetic and molecular evaluation was performed after 12 and 18 months of treatment with nilotinib, respectively. The rate of overall hematological response (HR) was 79%. The major cytogenetic response (MCyR) according the phase of the disease were as follows: 57% in CP, 40% in AP and no MCyR was observed in BP. Of the 47 pts, 11 (23.4%) achieved CCyR, 5 (10.63%) achieved molecular responses including 2 (4%) with complete molecular response (CMR) and 3 (6%) with major molecular response (MMR). The current status of pts according baseline mutational analysis was: mutation detected/alive (8/47, 17%); no mutation detectable/alive (22/47, 46.8%); mutation detected/deceased (9/47, 19.2%); no mutation detectable/deceased (8/47, 17%). Conclusions: Nilotinib showed efficacy in IM-resistant or -intolerant CML pts in Mexico regardless of the presence or absence of BCR-ABL mutations. Nilotinib induced complete cytogenetic and molecular responses in some of the advanced and resistant CML patient population.

Description: INTRODUCTION: Several studies have reported the presence of expanded T-cell clones in patients with multiple myeloma (MM), which could be involved in an anti-tumour response and extended survival. The Human Leukocyte Antigen (HLA) system seems to play an essential role in MM control and could influence disease control. This feature has been poorly studied and there are only few data favouring higher incidence of some HLA specifities such as B18 and B5 in myeloma patients. AIM: To compare HLA-DRB1 phenotypic frequencies in smoldering MM versus symptomatic MM patients and control individuals. PATIENTS: A total of 181 patients with a diagnosis of MM were analysed. According to their behaviour patients were classified into two subsets: 128 symptomatic MM who were homogeneously treated according to the GEM-2000 protocol (Spanish Myeloma Group/PETHEMA protocol) and 53 patients with the diagnosis of smoldering MM according to the criteria of the International Myeloma Working Group and who were free of therapy for at least 1 year following diagnosis. Additionally, 1818 healthy donor individuals from the Castilla y Leon registry for hematopoietic stem cell-transplantation were included as control population. All three populations involved Caucasian individuals. METHODS: After genomic DNA extraction, HLA-DRB1 typing at low-resolution level (two digits) was carried out using the PCR-rSSO methodology according to the standards of the European Federation of Immunogenetics. Allele frequencies were estimated by direct counting. Comparisons of allele and phenotype frequencies between populations were performed with the two-sided Fisher’s exact test using GraphPad Prism 4.0 Software. The strength of associations was estimated by the odds ratio (OR), and their 95% confidence intervals (CI) were calculated by Cornfield methods (values of p 〈 0.05 were considered statistically significant). P-value was corrected (Pc) for the number of valid comparisons made (Bonferroni correction). RESULTS: DRB1 phenotypic frequencies were not significantly different among MM patients and healthy control individual. In contrast, when the two MM cohorts were analyzed, DRB1*01 phenotypic frequencies were significantly higher in the smoldering patients as compared to symptomatic MM patients (37.7% vs. 14.1, p=0.0011, Pc=0.0143, OR: 3.7, 95% CI: 1.76–7.81). Furthermore, DRB1*01 phenotypic frequencies were significantly higher in the smoldering patients as compared to the healthy control individuals (37.7% vs. 21.7%, p=0.0106, Pc〉0.05, OR: 2.19, 95% CI: 1.24–3.86). In addition, symptomatic MM patients showed a higher incidence in DRB1*07 phenotypic frequencies as compared to control population (38.3% vs. 27.6%, p=0.0111, Pc〉0.05, OR: 1.63, 95% CI: 1.12–2.36). CONCLUSIONS: The present data suggest that HLA-DRB1*01 phenotype is associated with indolent MM and this may reflect a better ability to efficiently present myeloma-related antigens to immunocompetent cells.

Description: The proteasome inhibitor bortezomib represents an important advance for the treatment of both previously untreated and treated patients with multiple myeloma (MM). However, nearly all patients eventually relapse or become refractory to bortezomib therapy, so there is a continued need for new therapies. Vorinostat is a potent oral inhibitor of Class I and II histone deacetylases (HDACs) and has demonstrated antiproliferative and proapoptotic activity alone and in combination with bortezomib in preclinical models of MM. Preliminary results from an open-label, multicenter Phase I trial of oral vorinostat in combination with bortezomib have shown that the combination is generally well tolerated and effective in heavily pretreated patients with advanced MM (Weber et al. Haematologica2008;93(S1):0640). Patients (aged ≥18 years with an ECOG performance status 0–2) were sequentially enrolled on escalating doses of vorinostat combination therapy using a standard 3+3 design for ≤8 cycles. Cycles were repeated every 21 days and consisted of vorinostat 200 mg bid or 400 mg daily (Days 1–14) in combination with bortezomib 0.7 or 0.9 mg/m2 i.v. on Days 4, 8, 11, and 15 or escalated to 0.9, 1.1, or 1.3 mg/m2 i.v. on Days 1, 4, 8, and 11. The addition of oral dexamethasone 20 mg on Days 1–4 and 9–12 was allowed for disease progression. We now report the safety and efficacy results for a cohort of patients with relapsed/refactory MM who were previously treated with bortezomib but not within 3 months prior to study enrollment. To date, 13 patients who received prior bortezomib therapy have been enrolled in the trial. Drug-related adverse events (AEs) occurred in 11/13 patients; 90% of these AEs were mild to moderate in severity and 5 patients had serious AEs (7 events). One patient experienced Grade 4 thrombocytopenia, and 8 patients experienced Grade 3 drug-related AEs. The most common drug-related toxicities of any grade were fatigue, nausea and diarrhea. Eleven patients have now discontinued treatment: 6 due to progressive disease and 5 due to AEs. For the 13 patients previously treated with bortezomib, the best response was partial response in 5 patients (duration 99 to 203 days), minimal response in 1 patient (duration 122 days) and stable disease in 7 patients (duration 25 to 320 days). These preliminary data indicate that in this important subset of patients with MM who have relapsed while on, or were refractory to, previous bortezomib therapy, this combination of bortezomib and vorinostat (+/− dexamethasone) administered in a 21-day cycle shows activity, with acceptable tolerability. Further to these promising early findings, data will be presented according to whether patients had relapsed or refractory MM after prior bortezomib. The effect of adding dexamethasone to the combination of vorinostat and bortezomib will also be presented. Supplementary studies in patients clearly resistant to bortezomib are warranted to determine the additional effect of vorinostat in this combination.

Description: Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies. (Blood. 2006;108:107-115)

Description: Abstract 1040 Poster Board I-62 Treatment of the elderly patient with acute myelogenous leukemia remains problematic as many have poor performance status precluding aggressive therapy and others have high risk features including abnormal cytogenetics. The success of 5-azacitidine among patients with advanced myelodysplastic syndromes in controlling blast cells and prolonging survival has led to its use in AML. However choosing between potentially curative hospital-based induction chemotherapy and palliative outpatient hypomethylating treatments is difficult. The availability of predictive comorbidity scores may aid in guiding patients in this decision. Methods: Patients with a new diagnosis of AML, ages 60 and older, were offered traditional in-patient induction therapy (7 days cytarabine 100 mg/m2 CI with 3 days idarubicin 12mg/m2) or out-patient therapy (5-azacitidine 75 mg/m2 IV 5 days per month). We report a retrospective review of clinical outcomes among patients treated between June 2003 and August 1, 2009. A review of presenting clinical features, placing patients into prognostic groupings by the Charlson Comorbidity Index (J Chronic Dis 1987; 40:373), Hematopoietic Cell Transplantation – Specific Comorbidity Index (Blood 2007; 110:4606), and the Myelodysplastic Syndrome – Specific Comorbidity Index (Blood 2008; 112: abstr 2677) was performed. Points for a diagnosis of leukemia were excluded from the indices. Results: 99 patients with AML were treated at our center, all with leukemic blasts 〉20% (median 48%). 75 chose traditional 7+3 and 24 chose 5-aza. The median age was younger for those choosing 7+3 (67 vs 77 years, p

Description: Chronic myelogenous leukemia (CML) is caused by a balanced translocation between chromosomes 9 and 22 that creates the BCR-ABL fusion gene (FG) and activates the ABL tyrosine kinase (TK). Imatinib inhibits this TK and causes long-term remission in most CML patients. Relapse during imatinib therapy is often associated with mutations in the BCR-ABL FG that cause resistance to inhibition by imatinib. The usual procedure for detecting imatinib resistance mutations involves reverse transcription, PCR of the BCR-ABL FG, then nested PCR of the ABL TK to generate a template for sequencing. This procedure is suboptimal in a clinical laboratory because of the risk of carryover contamination, and extra time and workload, all due to the use of nested PCR. We describe a procedure for preparing sequencing template from the BCR-ABL FG that does not require nested PCR. The initial PCR employs a mixture of thermostable DNA polymerases that increases the sensitivity of the reaction for limiting concentrations of the desired target. One round of PCR provides enough template for sequencing. The PCR products are sequenced directly without gel purification, using three internal primers plus the downstream PCR primer as sequencing primers. The internal primers provide additional specificity resulting in a high quality sequence even if the PCR produces undesired products. Using this methodology we analyzed a series of eleven CML patient specimens selected because of a four-fold or greater increase in expression of the BCR-ABL FG during treatment with imatinib. The expression of BCR-ABL RNA was measured using a reverse transcription Q-PCR assay. The leftover cDNA from the expression assay was used as template for the PCR to generate the sequencing template. Five patients lacked a detectable mutation, and six had a mutation in the BCR-ABL TK. The six mutations found, G279E, F317L, L248V, M351T, F359C, and H396R, had all been seen previously and associated with resistance to imatinib. We conclude that detection of imatinib resistance mutations in the BCR-ABL FG is feasible without using nested PCR, although with a reduced analytical sensitivity. Elimination of the nested PCR step makes the procedure easier, quicker and less likely to suffer from carryover contamination. This method is a viable alternative for the detection of imatinib resistance mutations in the clinical laboratory.

Description: Introduction: Multiple myeloma (MM) is the second most common hematologic malignancy after non-Hodgkin’s lymphoma and despite recent advances in therapy, including the introduction of thalidomide, bortezomib, and lenalidomide, remains incurable. Vorinostat is an inhibitor of Class I and II histone deacetylases, which play key roles in the regulation of both transcriptional and post-transcriptional activity in a variety of tumor types, including MM. This histone deacetylase inhibitor has demonstrated anti-proliferative activity as monotherapy and synergistically with other agents in a variety of tumor types, including MM, where it was well tolerated in Phase I trials. Lenalidomide is a potent structural analog of thalidomide and demonstrates clinical efficacy in the treatment of MM as a single agent and to a larger degree, in combination with dexamethasone. Preclinical data suggest that the addition of vorinostat to lenalidomide and dexamethasone has at least additive, and possibly synergistic, therapeutic potential, with the anti-tumor mechanisms of vorinostat and dexamethasone being distinct from the immunomodulatory effects of lenalidomide. This Phase I, multicenter, open-label, non-randomized study assessed the safety and tolerability of vorinostat in combination with lenalidomide and dexamethasone in patients with relapsed or refractory MM. Methods: The primary objective was to determine the maximum tolerated dose (MTD). Secondary and exploratory objectives included: assessment of safety and tolerability; determination of clinical activity of the combination, and evaluation of in vivo molecular and biologic effects of the combination in patients with MM through analysis of gene expression. Patients aged ≥18 years with an established diagnosis of relapsed or refractory MM were enrolled sequentially into 1 of 5 dosing levels (table). Patients received vorinostat daily, administered orally for 14 days with 7 days on (Days 1–7 and 15–21), combined with oral lenalidomide daily for 21 days, and oral dexamethasone 40 mg/day (Days 1, 8, 15, and 22; cycles were repeated every 28 days); use of concomitant prophylactic acetylsalicylic acid was recommended. Dose-limiting toxicities (DLTs) were assessed in the first treatment cycle. Barring DLT, dose escalation continued until the MTD was established. Response, safety, and tolerability were evaluated. Adverse events (AEs) were recorded throughout the study. Dosing Regimen Dose Level Vorinostat Dose (mg q.d.) 7 days on 7 days off (Days 1–7 and Days 15–21) in each 28-day cycle Lenalidomide Dose (mg q.d.) × 21 days (Day 1 through Day 21) in each 28-day cycle Dexamethasone Dose (mg q.d.) On Days 1, 8, 15, and 22 in each 28-day cycle 1 300 10 40 2 400 10 40 3 400 15 40 4 400 20 40 5 400 25 40 Results: Enrolment is ongoing and tolerability of treatment has been good so far. Of 7 patients assessed to date, 6 patients (86%) have reported ≥1 AE, and 3 patients’ (43%) AEs were considered drug-related. The most frequently reported AE was anemia (n=4, 57%). Serious AEs were reported by 2 patients (29%), none of which were considered drugrelated. No patients have discontinued due to AEs or SAEs, and no DLT has been observed to date. Of 6 evaluable patients, the best responses were: partial response in 1 patient, minimal response in 1 patient and stable disease in 2 patients and progressive disease in 2 patients. Currently, 4 patients remain on treatment and 3 patients have discontinued treatment due to progressive disease. Conclusion: Vorinostat with lenalidomide and dexamethasone represents a novel combination therapy for the treatment of relapsed or relapsed, refractory MM. Preliminary results suggest that the combination is well tolerated to date, is active and has the convenience of oral administration.

Description: Bruton tyrosine kinase (Btk) is critical for B-cell development. Btk regulates a plethora of signaling proteins, among them nuclear factor-[κ]B (NF-κB). Activation of NF-κB is a hallmark of B cells, and NF-κB signaling is severely compromised in Btk deficiency. We here present strong evidence indicating that NF-κB is required for efficient transcription of the Btk gene. First, we found that proteasome blockers and inhibitors of NF-κB signaling suppress Btk transcription and intracellular expression. Similar to Btk, proteasome inhibitors also reduced the expression of other members of this family of kinases, Itk, Bmx, and Tec. Second, 2 functional NF-κB–binding sites were found in the Btk promoter. Moreover, in live mice, by hydrodynamic transfection, we show that bortezomib (a blocker of proteasomes and NF-κB signaling), as well as NF-κB binding sequence-oligonucleotide decoys block Btk transcription. We also demonstrate that Btk induces NF-κB activity in mice. Collectively, we show that Btk uses a positive autoregulatory feedback mechanism to stimulate transcription from its own promoter via NF-κB.