ALBERT

Description: Cognitive radio networks (CRNs) have emerged as a promising paradigm that can solve the shortage of spectrum resources, providing the ability to adapt and opportunistically exploit the spectrum holes. The rendezvous process allows cognitive users to find a common channel and establish a communication link. In this paper, we focus on the design of fast blind rendezvous algorithms to guarantee that every node should be able to rendezvous in all common available channels. We follow a systematic approach by first proposing a role-based algorithm that ensures maximum rendezvous diversity and then extending it to a common strategy through the use of multiples radios. Both proposals guarantee rendezvous under symmetric and asymmetric models. Simulation results show that our proposals outperform other recently developed rendezvous protocols with similar approaches in terms of expected time to rendezvous and maximum time to rendezvous.

Description: A methodology to assess input-variable sensitivity for sediment transport relations is presented. The Mean Value First Order Second Moment Method (MVFOSM) is applied to two bedload transport equations showing that it may be used to rank all input variables in terms of how their specific variance affects the overall variance of the sediment transport estimation. In sites where data are scarce or nonexistent, the results obtained may be used to i) determine what variables would have the largest impact when estimating sediment loads in the absence of field observations and ii) design field campaigns to specifically measure those variables for which a given transport equation is most sensitive; In sites where data are readily available, the results would allow quantifying the effect that the variance associated with each input variable has on the varaince of the sediment transport estimates. An application of the method to two transport relations using data from a tropical mountain river in Costa Rica is implemented to exemplify the potential of the method in places where input data are limited. Results are compared against Monte Carlo simulations to assess the reliability of the method and validate its results. For both of the sediment transport relations used in the sensitivity analysis, accurate knowledge of sediment size was found to have more impact on sediment transport predictions than precise knowledge of other input variables such as channel slope and flow discharge.

Description: Introduction: Multiple myeloma (MM) is a neoplasm of B lymphoid line that is characterized by clonal proliferation of malignant plasma cells in the bone marrow, producing monoclonal paraprotein (M) in blood and/or serum. Interleukin-6 (IL-6) is one of the key molecules related to growth, survival and proliferation of MM cells. Tocilizumab (TCZ) is a humanized monoclonal antibody directed against IL-6 receptor (IL-6R). When radiolabeled and used for tumor imaging, intact IgG exhibits high liver uptake. Antibody fragments (Fab´s) are quickly eliminated from blood and normal tissues (except kidneys), achieving high tumor-to-blood and tumor-to-normal tissue ratios with renal clearance. The aim of our work was to develop a 99mTc radiolabeled TCZ Fab´s fragment and to perform its chemical and biological evaluation in order to be used as a potential MM imaging agent for staging and restaging. Methods: Antibody fragmentation was carried out with papain and, once purified, Fab´s(TCZ) fragments were identified and derivatized with NHS-HYNIC-Tfa as bifunctional coupling agent. MALDITOF/TOF was used to confirm all procedures. A mixture of Tricine/SnCl2.2H2O was added to Fab´s(TCZ)-Tfa-HYNIC and radiolabeled with 99mTcO4-. Radiochemical purity and in-vitro stability in saline, serum and different concentration of L-cysteine up to 4 h were analyzed by ITLC and HPLC. In-vitro binding assays were performed using U266 and MM1S cell lines up to 120 min. Biodistribution and SPECT/CT images were evaluated on healthy Balb/c mice and MM1S tumor-bearing Balb/c nude mice at 0.5, 2 and 4 h. Results: Radiolabeling of HYNIC-Tfa-Fab´s(TCZ) was carried out in a fast, reproducible, easy, stable way showing high radiochemical purity and high specific activity. In vitro binding assays confirm that after its derivatization and radiolabeleing, Tfa-HYNIC-Fab`s(TCZ) does not interfere with the epitope recognition. In vivo biodistribution studies on healthy Balb/c mice and MM1S tumor-bearing Balb/c mice showed that 99mTc-HYNIC-Fab´s (TCZ) has significant renal uptake with neglectable uptake in other organs, indicating renal clearance. Tumor uptake was 12.84±1.80 %ID/g followed by 8.94±0.61 %ID/g and 3.05±1.49 %ID/g at 2 and 4 h, respectively. U266 tumor-to-muscle ratios were 5.79, 8.61 and 2.71 at 0.5, 2 and 4 h, respectively.Tumor uptake for MM1S tumor-bearing Balb/c nude mice was 10.05±1.32 %ID/g, 8.59±2.36 %ID/g and 3.88±0.68 %ID/g at 0.5, 2 and 4 h, respectively. MM1S tumor-to-muscle ratios were 6.32, 4.61 and 3.08 at 0.5, 2 and 4 h, respectively. Biodistribution data of 99mTc-HYNIC-Fab´s(TCZ) on U266 tumor-bearing Balb/c nude mice showed good tumor uptake and retention 0.5 h after its injection SPECT/CT images on healthy Balb/c mice and MM1S tumor-bearing Bal/c nude mice of 99mTc-HYNIC-Fab´s(TCZ) showed renal uptake and a discrete tumor uptake at 4 h p.i (Figure 1). Conclusions: Labeling Fab´s(TCZ) with 99mTc using HYNIC was performed in an easy, fast, stable and reproducible way preserving its biological activity. Biodistribution and SPECT/CT imaging assays allowed us to observe and evaluate its potential role as a diagnostic molecular imaging agent for MM. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4081 Introduction: Multiple myeloma (MM) accounts for 10% of hematological malignancies and 1% of cancer. It represents the second most common hematologic malignancy. The disease is characterised by plasma cell infiltration of the bone marrow, osteolytic bone lesions and the presence of a monoclonal paraprotein in the serum or urine. Interleukin-6 (IL-6) is a key molecule in the pathogenesis of MM. Its secretion occurs from the tumoral plasma cell as well as from the microenvironment. This results in myeloma cell proliferation. Tocilizumab, a humanized anti IL-6 receptor monoclonal antibody that blocks IL-6 cell-to-cell signaling, is currently being studied in MM. Most MM patients experience bone lesions. In spite of the new imaging techniques, the distinction between malignant and benign lesions remains sometimes difficult in clinical practice. The development of a more specific imaging agent may improve the accuracy of this diagnosis. Objective: Development of 99mTcHYNIC-Tocilizumab conjugate. Chemical and biological evaluation as a potential imaging agent in multiple myeloma. Methodology: 10 mg Tocilizumab (Roche) were added to 20 mg HYNIC in NaHCO3 1M, incubated for 30 min at room temperature and purified by means of PD10 column. 1 ml fractions were measured at 280nm. Conjugation of HYNIC-Tocilizumab was evaluated by mass spectrometry MALDITOF and acrylamide gel electrophoresis. 20 uL Tricine/stannous chloride in a 1:1 mass relation were added to 1 mg HYNIC-Tocilizumab in 100 uL of 0.9% NaCl. 2 mL of a solution containing 370 MBq 99mTcO4- was added. Radiochemical purity was controlled by three chromatographic systems: ITLC/NaCl 0.9%, Whatman 1MM/MEK, ITLC in BSA/EtOH-NH3-H2O (2:1:5) as stationary and mobile phase, respectively. Binding to IL-6 receptor in U266 myeloma cells was evaluated in vitro at 15, 30, 60 and 120 min. Tocilizumab was derivatized with FTIC and purified by PD10 column. Laser scanning confocal microscopy was done with an excitation/emission wavelength of 488/530 nm. Fluorescent images were obtained. Biodistribution studies were performed at 24 h in CD1 normal mice (n=3). Each mice was injected with 37 MBq of 99mTcHYNIC-Tocilizumab and sacrificed 24 hs after injection. Organs of interest were collected. Results: Mass spectroscopy MALDITOF (MH+Tocilizumab 147626 Da, M+HYNIC-Tocilizumab 148375 Da) confirmed derivatization of the antibody. This represents 5 molecules of HYNIC per molecule of the antibody. Poliacrylamide gel electrophoresis confirmed that the antibody retained its integrity after derivatization. Radiochemical purity was 88±3 %. In vitro cell binding assays confirmed the binding ability of the radiolabeled antibody to IL-6 receptor. Specificity of binding was supported by competition experiments using unlabelled antibody. Confocal microscopy proved the ability of the fluorescent antibody to recognize the IL-6 receptor in U266 myeloma cells. Biodistribution at 24 h showed blood (8.0±2.0) %act/g, liver (11±1.5) %act/g, kidney (4.0±1.0)%act/g and spleen (6±1.4) %act/g uptake with hepatic and renal elimination. Animal models of MM are being evaluated using xenografted MM cells. Conclusion: 99mTcHYNIC-Tocilizumab labeling was performed. The labeled antibody retained adequate biochemical and biological properties, which included maintenance of specific binding to IL-6 receptor. These findings indicate that this radiolabeled antibody could be used as an imaging agent in multiple myeloma. Acknowledgments: OIEA, Laboratorios Roche, Dr Gustavo Arroyo. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy; more than 90% are B cell lymphomas and express CD-20 antigen. Rituximab® (RTX) is an IgG1κ chimeric anti-CD20 mAb that binds specifically to the CD20-antigen on B lymphocytes. Our group previously reported that 99mTc-RTX represents a promising molecular imaging agent for NHL [1]. When used for tumor imaging, intact IgG exhibits high liver uptake. Antibody fragments (Fab´s) are quickly eliminated from blood and normal tissues (except kidneys), achieving high tumor/blood and tumor/normal tissue ratios with renal clearance. The development of radiolabeled Fab´s directed against specific targets may become a new strategy for NHL staging and surveillance. Objective: To radiolabel Fab´s (RTX) with 99mTc and to perform its chemical and biological evaluation. Methodology: We performed antibody fragmentation with papain and, once purified, fragments were identified by MaldiTOF/TOF and derivatized with Suc-HYNIC as a bifunctional coupling agent. A mixture of Tricine/SnCl2.2H2O was added to Fab´s (RTX)-HYNIC and radiolabeled with 99mTcO4-. Radiochemical purity was determined by HPLC. The in-vitro radiochemical stability of the radiolabeled Fab´s were analyzed in saline and serum up to 4 h. In-vitrobinding and competition assays were performed using Ramos and Raji cell lines up to 90 min. Biodistribution studies were evaluated in normal Balb/c mice and in Raji tumor-bearing Nude mice at 0.5 and 1 h. Results: Radiochemical purity of radiolabeled Fab´s were ≥90%. The in-vitro radiochemical stability studies showed that the radioconjugate was stable and no significant transchelation was detected. In-vitro binding and competition assays confirm that after its derivatization and radiolabeling, Fab´s (RTX) retained its specificity of binding to CD-20 antigen. This results confirm that Fab´s (RTX) affinity for CD20+ NHL cells remained unaffected after its derivatization. In-vivobiodistribution studies show that radiolabeled Fab´s has renal uptake with neglectable uptake in other organs, indicating that the primary route of clearance is renal. Lymph-node/muscle ratios of 4.00 and 2.55 at 0.5 and 1 h post injection, respectively. Conclusions: Fab´s (RTX) were easily and rapidly labeled demonstrating good stability and radiochemical purity. Based on lymph-node uptake and lymph-node/muscle ratios, 99mTc-HYNIC-Fab´s (RTX) may be useful for tumor molecular imaging agent for NHL. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Non-Hodgkin lymphoma (NHL) is the most common hematologic malignancy and the fifth leading cause of cancer death in the world; over 90% are B cell lymphomas and express CD-20 surface antigen [1]. CD-20 antigen is a transmembrane protein of 33-37 kDa protein located on the surface of pre-B and mature lymphocytes B [2, 3]. It is expressed in 90% of B-cell NHL [4]. Anti-CD20 antibody (Rituximab®), a specific chimeric monoclonal antibody directed against CD20 on B lymphocytes and the first monoclonal antibody approved for the treatment of CD20+ NHL. The development of new agents directed against specific targets may improve the sensitivity and specificity of imaging for its staging. Objective To radiolabel Rituximab with 99mTc and evaluate its properties as a potential imaging agent for NHL. Methodology Rituximab was derivatized with succinimidyl-hydrazinonicotinamide (Suc-HYNIC) and MALDI TOF/TOF was used to confirm the level of HYNIC conjugation to Rituximab. This antibody was radiolabeled with 99mTc using a mixture of Tricine/SnCl2.2H2O. Radiochemical purity was determined by ITLC-SG, size exclusion chromatography and HPLC. In-vitro stability was studied in solution; serum and L-Cysteine up to 24 h. In-vitro binding and competition assays were performed with Ramos and Raji NHL cell lines up to 120 min and were analyzed by laser confocal microscopy. Ramos and Raji cells were incubated with buffer to evaluate any degree of autofluorescence. Biodistribution studies were performed in normal Balb/C mice at 1, 4, 24 and 48 h (n = 5). Results HYNIC- Rituximab was efficiently labeled with 99mTc. The in-vitro radiochemical stability studies of the radiolabeled antibody showed that the complexes formed were stable and no significant transchelation was detected. In-vitro binding and displacement assays confirm that after derivatization and labeling, Rituximab retained its specificity of binding to CD-20 antigen. Immunoreactivity of HYNIC-Rituximab to Ramos and Raji cell lines was determined by direct immunofluorescence. We observed a remarkable cell membrane staining of the NHL cells. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). After its incubation with buffer, auto fluorescence was discarded. These results show that Rituximab´s affinity for NHL cells remained unaffected after its derivatization. Biodistribution studies were performed to quantify localization and clearance of the radiolabeled antibody complex in normal tissues. Significant accumulation of radioactivity was found in liver, indicating that the primary route of clearance was hepatic. Other major uptake was not found after evaluating the rest of the organs at the observed time points. Conclusions 99mTc-HYNIC-Tocilizumab was easily and rapidly labeled, with radiochemical purities greater than 90%, retaining its specificity of binding. Our results indicate that 99mTc-HYNIC-Rituximab represents a promising molecular imaging agent for NHL. Acknowledgments ANII, Roche Laboratories, Pro.In.Bio. PEDECIBA Química References [1] Swerdlow AJ. Epidemiology of Hodgkin’s disease and nonHodgkin’s lymphoma. Eur J Nucl Med Mol Imaging 2003; 30 Suppl 1:S2–12. [2] Einfield DA, Beown JP, Valentine MA, et al. Molecular cloning of the human cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains. EMBO J 1988; 7, 711-717. [3] Valentine MA, Meier KE, Rossie S, et al. Phosphotylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase. C J Biol Chem 1989; 264, 11282-11287. [4] Tedder TF, Boyd AW, Freedman As, et al. The B cell surface molecule B1 is functionally linked withBcell activation and differentiation. J Immunol 1985; 135, 973-979. Disclosures: No relevant conflicts of interest to declare.