ALBERT

Description: Background: Next generation transcriptome sequencing (RNA-Seq) is emerging as a powerful experimental tool for the study of alternative splicing and its regulation, but requires ad-hoc analysis methods and tools. PASTA (Patterned Alignments for Splicing and Transcriptome Analysis) is a splice junction detection algorithm specifically designed for RNA-Seq data, relying on a highly accurate alignment strategy and on a combination of heuristic and statistical methods to identify exon-intron junctions with high accuracy. Results: Comparisons against TopHat and other splice junction prediction software on real and simulated datasets show that PASTA exhibits high specificity and sensitivity, especially at lower coverage levels. Moreover, PASTA is highly configurable and flexible, and can therefore be applied in a wide range of analysis scenarios: it is able to handle both single-end and paired-end reads, it does not rely on the presence of canonical splicing signals, and it uses organism-specific regression models to accurately identify junctions. Conclusions: PASTA is a highly efficient and sensitive tool to identify splicing junctions from RNA-Seq data. Compared to similar programs, it has the ability to identify a higher number of real splicing junctions, and provides highly annotated output files containing detailed information about their location and characteristics. Accurate junction data in turn facilitates the reconstruction of the splicing isoforms and the analysis of their expression levels, which will be performed by the remaining modules of the PASTA pipeline, still under development. Use of PASTA can therefore enable the large-scale investigation of transcription and alternative splicing.

Description: Background: Truck driving is the second most common occupation among Canadian men. Transportation of goods via roads is of crucial importance for the Canadian economy. The industry is responsible annually for $17 billion in GDP and is projected to increase by 28% over the next 10 years. Recruitment is an issue with 20% of drivers projected to retire or leave the profession in the next 10 years. Despite the reliance on transport truck drivers for the delivery of goods which affects Canada’s economy and daily living of residents, little is known about the health care needs of this large cohort of primarily male lone workers from a drivers’ perspective. Transport truck drivers are independent workers whose non traditional workplace is their tractor, the truck stops and the journey on the road.The objective of this study was to obtain a contextually informed description of lifestyle issues, health and disease risk factors experienced by drivers and perceived by their managers in the truck driving occupation. Methods: Using a grounded theory approach, 4 focus groups were conducted with drivers (n = 16) and managers (n = 10) from two trucking companies in Southwestern Ontario to identify the lived experience of the drivers as it relates to preventable risks to health and wellness. A semi structured guided interview was used to explore the lifestyle context of transport truck driving and organizational aspects of the occupation (workplace culture, working conditions and health and wellness promotion). Results: The predominant themes described stress, workplace, communication, lifestyle, driving culture, family, and fatigue concerns. In terms of the transportation work environment, drivers and managers were aware of the profession’s potential to foster lifestyle related chronic diseases but described challenges in making the profession more amenable to a healthy lifestyle. Conclusions: Workplace environmental determinants are significant in shaping health behaviours. Chronic disease health risks were the main health concerns identified. Health risks were exacerbated by working conditions (job demands, work hours, financial pressure and the sedentary nature of the job). Workplace health strategies will need to take into account the unique challenges of the occupation.

Description: Background: Truck driving is the second most common occupation among Canadian men. Transportation of goods via roads is of crucial importance for the Canadian economy. The industry is responsible annually for $17 billion in GDP and is projected to increase by 28% over the next 10 years. Recruitment is an issue with 20% of drivers projected to retire or leave the profession in the next 10 years. Despite the reliance on transport truck drivers for the delivery of goods which affects Canada's economy and daily living of residents, little is known about the health care needs of this large cohort of primarily male lone workers from a drivers' perspective. Transport truck drivers are independent workers whose non traditional workplace is their tractor, the truck stops and the journey on the road.The objective of this study was to obtain a contextually informed description of lifestyle issues, health and disease risk factors experienced by drivers and perceived by their managers in the truck driving occupation. Methods: Using a grounded theory approach, 4 focus groups were conducted with drivers (n = 16) and managers (n = 10) from two trucking companies in Southwestern Ontario to identify the lived experience of the drivers as it relates to preventable risks to health and wellness. A semi structured guided interview was used to explore the lifestyle context of transport truck driving and organizational aspects of the occupation (workplace culture, working conditions and health and wellness promotion). Results: The predominant themes described stress, workplace, communication, lifestyle, driving culture, family, and fatigue concerns. In terms of the transportation work environment, drivers and managers were aware of the profession's potential to foster lifestyle related chronic diseases but described challenges in making the profession more amenable to a healthy lifestyle. Conclusions: Workplace environmental determinants are significant in shaping health behaviours. Chronic disease health risks were the main health concerns identified. Health risks were exacerbated by working conditions (job demands, work hours, financial pressure and the sedentary nature of the job). Workplace health strategies will need to take into account the unique challenges of the occupation.

Description: The combination of fludarabine, cyclophosphamide and rituximab (FCR) is still currently regarded as the standard regimen for treatment of physically fit patients with chronic lymphocytic leukemia (CLL). This therapy can be associated with significant toxicity, and patient adherence to the protocol may often be difficult outside of clinical trials. This retrospective study aimed to evaluate the efficacy and safety of FCR therapy in the real life setting, with particular focus on the influence of dose reduction on treatment outcome. A total of 132 CLL patients (≤70 years of age) treated with FCR as frontline therapy from 10 medical centers, were reviewed. The majority of patients were males (73.5%, n=97) and younger than 60 years (78%, n=103). Eleven patients had Binet stage A (8.3%), 72 (54.5%) were stage B and 49 (37.1%) had Binet stage C. Results of FISH analysis were available for 99 patients, with high risk cytogenetics of del(11q) in 21 patients (21.2%) and del(17p) in 9 cases (9.1%). The majority (56.5%, n=74) received rituximab at a dose of 500mg/m2 and the rest 375mg/m2. Almost half of the patients (49.2%, n=65) were given a reduced dose of chemotherapy (

Description: Background: Severe thrombocytopenia is an uncommon event in lower risk MDS patients, but it may significantly influence the prognosis. In fact, when it occurs, major bleeding may be a life-threatening complication. No licensed pharmacologic approach is nowadays available yet for these patients. Eltrombopag seems to be a very interesting product, but its efficacy and safeness are still to be better demonstrated. Romiplostim could be suitable too, but, at present, its safety is uncertain in MDS patients. Also danazol, an attenuated androgen, seems to have some ability to increase the platelet count in this context. Patients and methods: We retrospectively reviewed 17 thrombocytopenic patients affected by MDS, treated with danazol and observed for at least 6 months. Three patients of these had a therapy-related MDS. At the starting time of danazol therapy, the IPSS was “low” or “intermediate-1” in 16 cases; “intermediate-2” in 1 case. The IPSS-R was “very low”, “low” or “intermediate” in 16 cases; “very high” in 1 case. In 14 patients the platelet count was lower than 25x109/L, in the other 3 lower than 40x109/L, but with spontaneous bleeding. The initial dose was 600 mg/day for all the patients. The IWG criteria of response (Cheson 2006) were adopted. The outcomes were observed after 3 and 6 months from the beginning of therapy. Only descriptive statistical analysis was used. Results: At the beginning of therapy, the average platelet count of the 17 patients was 22.6 x109/L (S.D. 8.8, range 6-38). After 3 months, the therapy with danazol was ongoing in 16 patients (in 1 case the drug was discontinued due to renal failure). Platelet improvement, according to IWG criteria, was observed in 8 cases (47%). The average platelet count was 45.3x109/L (S.D. 32.9, range 4-133). The only one “high risk” patient did not show response. After 6 months danazol was still ongoing in 11 patients (in 5 cases the drug was stopped for inefficacy). The response according to IWG criteria was evident in 9 patients (52% of the initial 17 patients). The average platelet count was 66x109/L (S.D. 63.9, range 11-218). Adverse events recorded were as follows: increase in transaminases in 3 cases (in 2 of these the dose was reduced to 400 mg/day); severe but reversible renal failure in 1 case (the drug was stopped); moderate increasing of serum creatinine in 1 case (the drug was reduced to 400 mg/day); reversible cutaneous rush (the drug was reduced to 400mg/day); amenorrhea in 1 case (the only fertile woman in the series); weight loss and loss of appetite in 1 case, weight gain in 1 case. Conclusions This series confirms the efficacy of danazol to improve platelet count in approximately half of patients with severe thrombocytopenia due to “low-risk” MDS. In all patients with increased platelet count, the response was clinically significant. The response may not be immediate. Actually, there was an improvement of platelet count even after three months of therapy. The toxicity profile of this drug is low. The mechanism of action of danazol in MDS patients remains unclear. Waiting for more information on the efficacy and safety of eltrombopag from the clinical trials in progress, danazol may be a good therapeutic option for these patients. Disclosures Off Label Use: Danazol in MDS patients with severe trhombocytopenia.

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.