Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.

Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing.

Double-strand breaks (DSBs) occur frequently during DNA replication. They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis. In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination. Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e. exonuclease resected) duplex DNA molecules. Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini. Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

Phase II study of weekly plitidepsin as second-line therapy for small cell lung cancer.

OBJECTIVE: To evaluate the antitumor activity and safety profile of plitidepsin administered as a 1h weekly intravenous (i.v.) infusion of 3.2mg/m(2) to patients with small cell lung cancer (SCLC) who relapsed or progressed after one line of chemotherapy. PATIENTS AND METHODS: This was a multicenter, open-label, single-arm, exploratory, phase II clinical trial. Treatment lasted until disease progression, unacceptable toxicity, patient refusal or treatment delay for >2 weeks. Objective response rate (primary efficacy endpoint) was evaluated according to response evaluation criteria in solid tumors (RECIST). The rate of stable disease (SD) lasting for at least 6 months and time-to-event variables were secondary endpoints of efficacy. Toxicity was assessed using National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 2.0. RESULTS: Twenty pretreated SCLC patients (median age, 60 years) with extensive (n=13) or limited-stage disease (n=7) received a total of 24 treatment cycles (median, one cycle per patient; range, 1-2). Objective tumor responses were not observed and only one of the 17 evaluable patients had SD. With a median follow-up of 11.8 months, the progression-free survival and the median overall survival were 1.3 months and 4.8 months, respectively. The most troubling or common toxicities were fatigue, muscle weakness, lymphopenia, anemia (no patients showed neutropenia), and asymptomatic, non-cumulative increase of transaminases levels and alkaline phosphatase. CONCLUSION: This clinical trial shows that a cycle of 1h weekly i.v. infusion of plitidepsin (3.2mg/m(2)) was generally well tolerated other than fatigue and muscle weakness in patients with pretreated SCLC. One patient died due to multi-organ failure. The absence of antitumor activity found here precludes further studies of this plitidepsin schedule as second-line single-agent treatment of SCLC.

Characterization of the effects of IL-17F on CHO cell line generation

CHO is the most commonly used mammalian host for the generation of cell lines allowing for the production of high quality therapeutic proteins. The generation of such cell lines is a lengthy and resource-intensive process requiring extensive screening in order to isolate candidates with optimal characteristics, such as growth, stability and productivity. For this reason, the biotechnology industry invests much effort in attempts to optimize CHO expression systems in order to streamline and shorten the cell line selection process. Based on preliminary observations of a facilitated selection of CHO-GS cell lines expressing members of the IL-17 cytokine family, this study investigates the use of IL-17F as a novel enhancing factor for CHO cell line generation. Using two different CHO expression systems (exploiting GS and DHFR-based selection), we demonstrated that IL-17F expression caused a significant increase in the occurrence of colonies during the selection process. All colonies selected produced substantial amounts of IL-17F, suggesting that benefits were conferred, during selection, to those cells expressing the cytokine. Furthermore, transgene expression levels were significantly increased when the selection pressure was raised to a level that would not normally be permissive for colony selection (i.e. 100 |o.M MSX for the CHO-GS expression system or 1000 nM MTX for the CHO-DHFR system). Finally, IL-17F expression was also found to enhance the rate of appearance of clones during single cell subcloning in the absence of selection pressure. Overall, these benefits have the potential to allow a substantial reduction in the length of cell line generation while significantly increasing cell line productivity. Nevertheless, we found that the high IL-17F expression levels required to convey enhancing effects was a limitation when attempting to co-express IL-17F and a recombinant soluble protein of therapeutic interest from independent CMV promoters within the same expression vector. In order to understand and overcome this limitation, studies were designed to characterize the IL-17F enhancing effect at the molecular and cellular level. Regular supplementation of recombinant biologically-active IL-17F into the culture medium during cell line selection was not able to reproduce the enhancing effects of endogenous IL-17F expression. In addition, increased IL-17F expression correlated with increased CHO-GS selection transgene expression at the single cell level. This data suggested a possible effect of IL-17F on viral promoter activity or transgene mRNA stability. It also provided direct evidence that the cells expressing the highest amounts of IL-17F obtained the most benefit. Overall data obtained from these study implied that IL-17F may act through an intracellular mechanism, possibly exerted during secretion. We therefore initiated experiments designed to determine the specific compartment(s) within which IL-17F triggers its effect. This work has identified IL-17F as a potentially powerful tool to optimize the CHO cell line generation process. The characterization of this enhancing effect at the molecular level has given us several insights into overcoming the current limitations, thus paving the way for the development of a viable technology that can be exploited within the biotechnology industry. - La CHO est la cellule hôte de mammifere la plus couramment utilisée dans la création de lignée cellulaire produisant des protéines thérapeutiques de haute qualité. La génération de ces lignées cellulaires est un processus long et exigeant l'utilisation de techniques de sélection robustes afin d'isoler des candidats possédants les caractéristiques optimales de croissance, de productivité et de stabilité d'expression. Les industries biopharmaceutiques ont investi beaucoup d'efforts afin d'optimiser les systèmes d'expression CHO dans le but raccourcir la longueur du procédé de sélection de lignées cellulaires et aussi d'en augmenter l'efficacité. A partir d'observations préliminaires obtenues lors de la génération de lignées cellulaires CHO- GS exprimant une cytokine appartenant à la famille des IL-17, nous avons réalisé une étude portant sur l'utilisation de l'IL-17F humaine (IL-17F) comme nouveau facteur d'optimisation pour la génération de lignées cellulaires CHO. Nous avons démontré, en utilisant les deux systèmes de sélection et d'expression CHO couramment utilisés (le premier exploitant la GS et l'autre basée sur la DHFR), que l'expression de l'IL-17F permet une augmentation significative de la fréquence d'apparition de colonies durant le processus de sélection de lignées cellulaires. Les différentes colonies sélectionnées expriment des quantités substantielles d'IL-17F, suggérant un effet bénéfique lors de la sélection qui serait exclusivement conféré aux cellules exprimant la cytokine. En outre, le niveau d'expression du transgene se trouve significativement augmenté lorsque la pression de sélection est portée à un niveau habituellement trop élevé pour permettre la sélection de colonies (soit 100 |JM MSX pour le système d'expression CHO-GS ou 1000 nM MTX pour le système CHO- DHFR). Enfin, l'expression d'IL-17F permet également d'améliorer la vitesse d'apparition de clones pendant une étape de sous-clonage en l'absence de pression de sélection. L'ensemble de ces effets bénéfiques permettent une réduction substantielle de la durée de génération de lignées cellulaires tout en augmentant considérablement la productivité des lignées obtenues. Néanmoins, nous avons constaté que la nécessité d'exprimer des niveaux élevés d'IL-17F afin obtenir l'ensemble de ses effets bénéfiques devient une contrainte lors de l'utilisation d'un vecteur d'expression composé de deux promoteurs CMV indépendants pour la co-expression de la cytokine et d'une protéine soluble présentant un intérêt thérapeutique. Afin de mieux comprendre et de surmonter cette limitation, plusieurs études ont été effectuées dans le but de mieux caractériser l'effet de IL-17F au niveau subcellulaire. L'apport régulier en IL-17F recombinante et biologiquement active dans le milieu de culture lors de la sélection de lignées cellulaires ne permet pas de reproduire les effets bénéfiques observés par l'expression endogène d'IL-17F. En outre, nous avons constaté que, lors de l'utilisation du système CHO- GS, l'augmentation d'expression de 1TL-17F est corrélée à un accroissement de l'expression du marqueur de sélection au niveau cellulaire. Ces résultats suggèrent un possible effet d'IL- 17F sur l'activité des promoteurs viraux et ainsi fournissent une preuve directe que les cellules exprimant de haut niveau d'IL-17F sont celles qui en profitent le plus. L'ensemble de ces observations mettrait en avant que l'effet d'IL-17F se ferait selon un mécanisme intracellulaire. Nous avons donc étudié le(s) compartiment(s) spécifique(s) dans lequel IL-17F pourrait exercer son effet. Ce travail a permis de définir IL-17F comme un puissant outil pour l'optimisation des procédés de génération de lignées cellulaires CHO. La caractérisation de cette amélioration de l'effet au niveau moléculaire nous a donné plusieurs indications sur la manière de dépasser les limitations actuelles, ouvrant ainsi la voie au développement d'une technologie viable qui peut être exploitée pars l'industrie biotechnologique.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Outcome of long-axis percutaneous sacroplasty as first-line treatment of sacral insufficiency fractures : P219

Purpose: To assess the clinical outcome of patients who were subjected to long-axis sacroplasty as first line treatment for sacral insufficiency fractures. Methods and materials: Nineteen patients with unilateral (n = 3) or bilateral (n = 16) sacral fractures were involved. Under local anaesthesia, each patient was subjected to CT guided sacroplasty using the long-axis approach through a single entry point. An average of 6 ml of PMMA was delivered along the path of each sacral fracture. For each individual patient, the VAS pain score before sacroplasty and at 1, 4, 24, and 48 weeks after the procedure was obtained. Furthermore, the use of analgesics (narcotic/non-narcotic) along with the evolution of post interventional patient mobility before and after sacroplasty was also recorded. Results: The mean pre-procedure VAS score was 8 ± 1.9. This has rapidly declined in the first week after the procedure (mean 4 ± 1.5) followed by gradual decrease along the rest of follow-up period at 4 weeks (mean 3 ± 1.2), 24 weeks (mean 2 ± 1.3), and 48 weeks (mean 1.3 ± 1.4), respectively. Eleven (58%) patients were under narcotic analgesia before sacroplasty, whereas, 8 (42%) patients were using non-narcotics. Corresponding values after the procedure were 2/19 (10%) (narcotic) and 10/19 53% (non-narcotic). Seven (37%) patients did not address post-procedure analgesic use. The evolution of post interventional mobility was favourable in the study group since they revealed a significant improvement in their mobility point scale. Conclusion: Long-axis percutaneous sacroplasty is a suitable minimally invasive treatment option for patients who present with sacral insufficiency fractures. Future studies with larger patient number are warranted to grasp any potential limitations of this therapeutic approach.

Identification of a novel 45-kDa cell surface molecule involved in activation of the human Jurkat T cell line.

This report describes a surface molecule, Tp45, which appears to be involved in interleukin 2 production and Ca2+ mobilization by Jurkat cells. The Tp45 molecule was identified by a monoclonal antibody, MX13, on the surface of either T3/TCR+ or T3/TCR- human T cell lines. Biochemical data showed that mAb MX13 precipitated a single polypeptide chain of 45 kDa both under reduced and nonreduced conditions from lysates of 125I-surface-labeled cells. Sequential immunodepletion experiments using lysates of 125I-labeled T3/TCR+ cells showed that Tp45 was distinct from the alpha chain of the TCR complex. However, incubation of such cells with either anti-T3 or anti-TCR monoclonal antibody induced complete modulation of both the T3/TCR complex and Tp45. Conversely, complete modulation of both Tp45 and the T3/TCR complex was observed after incubation with anti-Tp45 antibody. Functional studies showed that anti-Tp45 antibody induced high levels of interleukin 2 production in Jurkat cells. In addition, incubation of these cells with the antibody resulted in Ca2+ mobilization from internal stores. Anti-Tp45 antibody reacted with 3-19% peripheral blood (E-rosette-positive) T cells in individual donors. The magnitude of the proliferative response elicited by anti-Tp45 antibody for peripheral blood T cells was lower than that induced by an anti-T3 antibody. This observation is compatible with the idea that only a subpopulation of T cells is reactive with anti-Tp45. Multicolor flow cytometry analysis showed that the Tp45+ cells belong preferentially to the T8 subset.

Role of Kras Status in Patients with Metastatic Colorectal Cancer Receiving First-Line Chemotherapy plus Bevacizumab: A TTD Group Cooperative Study

BACKGROUND In the MACRO study, patients with metastatic colorectal cancer (mCRC) were randomised to first-line treatment with 6 cycles of capecitabine and oxaliplatin (XELOX) plus bevacizumab followed by either single-agent bevacizumab or XELOX plus bevacizumab until disease progression. An additional retrospective analysis was performed to define the prognostic value of tumour KRAS status on progression-free survival (PFS), overall survival (OS) and response rates. METHODOLOGY/PRINCIPAL FINDINGS KRAS data (tumour KRAS status and type of mutation) were collected by questionnaire from participating centres that performed KRAS analyses. These data were then cross-referenced with efficacy data for relevant patients in the MACRO study database. KRAS status was analysed in 394 of the 480 patients (82.1%) in the MACRO study. Wild-type (WT) KRAS tumours were found in 219 patients (56%) and mutant (MT) KRAS in 175 patients (44%). Median PFS was 10.9 months for patients with WT KRAS and 9.4 months for patients with MT KRAS tumours (p=0.0038; HR: 1.40; 95% CI:1.12-1.77). The difference in OS was also significant: 26.7 months versus 18.0 months for WT versus MT KRAS, respectively (p=0.0002; HR: 1.55; 95% CI: 1.23-1.96). Univariate and multivariate analyses showed that KRAS was an independent variable for both PFS and OS. Responses were observed in 126 patients (57.5%) with WT KRAS tumours and 76 patients (43.4%) with MT KRAS tumours (p=0.0054; OR: 1.77; 95% CI: 1.18-2.64). CONCLUSIONS/SIGNIFICANCE This analysis of the MACRO study suggests a prognostic role for tumour KRAS status in patients with mCRC treated with XELOX plus bevacizumab. For both PFS and OS, KRAS status was an independent factor in univariate and multivariate analyses.

Plerixafor and granulocyte colony-stimulating factor for first-line steady-state autologous peripheral blood stem cell mobilization in lymphoma and multiple myeloma: results of the prospective PREDICT trial.

In Europe, the combination of plerixafor + granulocyte colony-stimulating factor is approved for the mobilization of hematopoietic stem cells for autologous transplantation in patients with lymphoma and myeloma whose cells mobilize poorly. The purpose of this study was to further assess the safety and efficacy of plerixafor + granulocyte colony-stimulating factor for front-line mobilization in European patients with lymphoma or myeloma. In this multicenter, open label, single-arm study, patients received granulocyte colony-stimulating factor (10 μg/kg/day) subcutaneously for 4 days; on the evening of day 4 they were given plerixafor (0.24 mg/kg) subcutaneously. Patients underwent apheresis on day 5 after a morning dose of granulocyte colony-stimulating factor. The primary study objective was to confirm the safety of mobilization with plerixafor. Secondary objectives included assessment of efficacy (apheresis yield, time to engraftment). The combination of plerixafor + granulocyte colony-stimulating factor was used to mobilize hematopoietic stem cells in 118 patients (90 with myeloma, 25 with non-Hodgkin's lymphoma, 3 with Hodgkin's disease). Treatment-emergent plerixafor-related adverse events were reported in 24 patients. Most adverse events occurred within 1 hour after injection, were grade 1 or 2 in severity and included gastrointestinal disorders or injection-site reactions. The minimum cell yield (≥ 2 × 10(6) CD34(+) cells/kg) was harvested in 98% of patients with myeloma and in 80% of those with non-Hodgkin's lymphoma in a median of one apheresis. The optimum cell dose (≥ 5 × 10(6) CD34(+) cells/kg for non-Hodgkin's lymphoma or ≥ 6 × 10(6) CD34(+) cells/kg for myeloma) was harvested in 89% of myeloma patients and 48% of non-Hodgkin's lymphoma patients. In this prospective, multicenter European study, mobilization with plerixafor + granulocyte colony-stimulating factor allowed the majority of patients with myeloma or non-Hodgkin's lymphoma to undergo transplantation with minimal toxicity, providing further data supporting the safety and efficacy of plerixafor + granulocyte colony-stimulating factor for front-line mobilization of hematopoietic stem cells in patients with non-Hodgkin's lymphoma or myeloma.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia.

Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

A Multi-Center Phase II Study (SAKK 36/06) of Single Agent Everolimus (RAD001) In Patients with Relapsed or Refractory Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) accounts for 6% of all B-cell lymphomas and remains incurable for most patients. Those who relapse after first line therapy or hematopoietic stem cell transplantation have a dismal prognosis with short response duration after salvage therapy. On a molecular level, MCL is characterised by the translocation t[11;14] leading to Cyclin D1 overexpression. Cyclin D1 is downstream of the mammalian target of rapamycin (mTOR) kinase and can be effectively blocked by mTOR inhibitors such as temsirolimus. We set out to define the single agent activity of the orally available mTOR inhibitor everolimus (RAD001) in a prospective, multi-centre trial in patients with relapsed or refractory MCL (NCT00516412). The study was performed in collaboration with the EU-MCL network. Methods: Eligible patients with histologically/cytologically confirmed relapsed (not more than 3 prior lines of systemic treatment) or refractory MCL received everolimus 10 mg orally daily on day 1 - 28 of each cycle (4 weeks) for 6 cycles or until disease progression. The primary endpoint was the best objective response with adverse reactions, time to progression (TTP), time to treatment failure, response duration and molecular response as secondary endpoints. A response rate of 10% was considered uninteresting and, conversely, promising if 30%. The required sample size was 35 pts using the Simon's optimal two-stage design with 90% power and 5% significance. Results: A total of 36 patients with 35 evaluable patients from 19 centers were enrolled between August 2007 and January 2010. The median age was 69.4 years (range 40.1 to 84.9 years), with 22 males and 13 females. Thirty patients presented with relapsed and 5 with refractory MCL with a median of two prior therapies. Treatment was generally well tolerated with anemia (11%), thrombocytopenia (11%), neutropenia (8%), diarrhea (3%) and fatigue (3%) being the most frequent complications of CTC grade III or higher. Eighteen patients received 6 or more cycles of everolimus treatment. The objective response rate was 20% (95% CI: 8-37%) with 2 CR, 5 PR, 17 SD, and 11 PD. At a median follow-up of 6 months, TTP was 5.45 months (95% CI: 2.8-8.2 months) for the entire population and 10.6 months for the 18 patients receiving 6 or more cycles of treatment. Conclusion: This study demonstrates that single agent everolimus 10 mg once daily orally is well tolerated. The null hypothesis of inactivity could be rejected indicating a moderate anti-lymphoma activity in relapsed/refractory MCL. Further studies of either everolimus in combination with chemotherapy or as single agent for maintenance treatment are warranted in MCL.

Advantages of extended bottom-up proteomics using sap9 for analysis of monoclonal antibodies.

Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence coverage per liquid chromatography (LC) MS experiment, and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixtures. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for analysis of monoclonal antibodies. Key findings of the Sap9-based proteomics analysis of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on average) containing up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The analysis of a mixture of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixtures compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.

Evaluation of the chicken transcriptome by SAGE of B cells and the DT40 cell line

Background: The understanding of whole genome sequences in higher eukaryotes depends to a large degree on the reliable definition of transcription units including exon/intron structures, translated open reading frames (ORFs) and flanking untranslated regions. The best currently available chicken transcript catalog is the Ensembl build based on the mappings of a relatively small number of full length cDNAs and ESTs to the genome as well as genome sequence derived in silico gene predictions.Results: We use Long Serial Analysis of Gene Expression (LongSAGE) in bursal lymphocytes and the DT40 cell line to verify the quality and completeness of the annotated transcripts. 53.6% of the more than 38,000 unique SAGE tags (unitags) match to full length bursal cDNAs, the Ensembl transcript build or the genome sequence. The majority of all matching unitags show single matches to the genome, but no matches to the genome derived Ensembl transcript build. Nevertheless, most of these tags map close to the 3' boundaries of annotated Ensembl transcripts.Conclusions: These results suggests that rather few genes are missing in the current Ensembl chicken transcript build, but that the 3' ends of many transcripts may not have been accurately predicted. The tags with no match in the transcript sequences can now be used to improve gene predictions, pinpoint the genomic location of entirely missed transcripts and optimize the accuracy of gene finder software.

Event-related potential analyses via single-subject topographic classification.

Introduction: Responses to external stimuli are typically investigated by averaging peri-stimulus electroencephalography (EEG) epochs in order to derive event-related potentials (ERPs) across the electrode montage, under the assumption that signals that are related to the external stimulus are fixed in time across trials. We demonstrate the applicability of a single-trial model based on patterns of scalp topographies (De Lucia et al, 2007) that can be used for ERP analysis at the single-subject level. The model is able to classify new trials (or groups of trials) with minimal a priori hypotheses, using information derived from a training dataset. The features used for the classification (the topography of responses and their latency) can be neurophysiologically interpreted, because a difference in scalp topography indicates a different configuration of brain generators. An above chance classification accuracy on test datasets implicitly demonstrates the suitability of this model for EEG data. Methods: The data analyzed in this study were acquired from two separate visual evoked potential (VEP) experiments. The first entailed passive presentation of checkerboard stimuli to each of the four visual quadrants (hereafter, "Checkerboard Experiment") (Plomp et al, submitted). The second entailed active discrimination of novel versus repeated line drawings of common objects (hereafter, "Priming Experiment") (Murray et al, 2004). Four subjects per experiment were analyzed, using approx. 200 trials per experimental condition. These trials were randomly separated in training (90%) and testing (10%) datasets in 10 independent shuffles. In order to perform the ERP analysis we estimated the statistical distribution of voltage topographies by a Mixture of Gaussians (MofGs), which reduces our original dataset to a small number of representative voltage topographies. We then evaluated statistically the degree of presence of these template maps across trials and whether and when this was different across experimental conditions. Based on these differences, single-trials or sets of a few single-trials were classified as belonging to one or the other experimental condition. Classification performance was assessed using the Receiver Operating Characteristic (ROC) curve. Results: For the Checkerboard Experiment contrasts entailed left vs. right visual field presentations for upper and lower quadrants, separately. The average posterior probabilities, indicating the presence of the computed template maps in time and across trials revealed significant differences starting at ~60-70 ms post-stimulus. The average ROC curve area across all four subjects was 0.80 and 0.85 for upper and lower quadrants, respectively and was in all cases significantly higher than chance (unpaired t-test, p<0.0001). In the Priming Experiment, we contrasted initial versus repeated presentations of visual object stimuli. Their posterior probabilities revealed significant differences, which started at 250ms post-stimulus onset. The classification accuracy rates with single-trial test data were at chance level. We therefore considered sub-averages based on five single trials. We found that for three out of four subjects' classification rates were significantly above chance level (unpaired t-test, p<0.0001). Conclusions: The main advantage of the present approach is that it is based on topographic features that are readily interpretable along neurophysiologic lines. As these maps were previously normalized by the overall strength of the field potential on the scalp, a change in their presence across trials and between conditions forcibly reflects a change in the underlying generator configurations. The temporal periods of statistical difference between conditions were estimated for each training dataset for ten shuffles of the data. Across the ten shuffles and in both experiments, we observed a high level of consistency in the temporal periods over which the two conditions differed. With this method we are able to analyze ERPs at the single-subject level providing a novel tool to compare normal electrophysiological responses versus single cases that cannot be considered part of any cohort of subjects. This aspect promises to have a strong impact on both basic and clinical research.