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The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “context of use” [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Measurable residual disease (MRD) evaluation may help to guide treatment duration in multiple myeloma (MM). Paradoxically, limited longitudinal data exist on MRD during maintenance. We investigated the prognostic value of MRD dynamics in 1280 transplant-eligible and -ineligible patients from the TOURMALINE-MM3 and -MM4 randomized, placebo-controlled, phase 3 studies of 2-year ixazomib maintenance. MRD status at randomization showed independent prognostic value (median progression-free survival [PFS]: 38.6 vs 15.6 months in MRD- vs MRD+ patients, HR 0.47). However, MRD dynamics during maintenance provided more detailed risk stratification. A 14-month landmark analysis showed prolonged PFS in patients converting from MRD+ to MRD- status vs those with persistent MRD+ status (76.8% vs 27.6% 2-year PFS rates). Prolonged PFS was observed in patients with sustained MRD- status vs those converting from MRD- to MRD+ status (75.0% vs 34.2% 2-year PFS rates). Similar results were observed at a 28-month landmark analysis. Ixazomib maintenance vs placebo improved PFS in patients who were MRD+ at randomization (median 18.8 vs 11.6 months, HR 0.65) or at the 14-month landmark (median 16.8 vs 10.6 months, HR 0.65); no difference was observed in patients who were MRD-. This is the largest MM population undergoing yearly MRD evaluation during maintenance reported to date. We demonstrate the limited prognostic value of a single timepoint MRD evaluation, because MRD dynamics over time substantially impact PFS risk. These findings support MRD- status as a relevant endpoint during maintenance and confirm the increased progression risk in patients converting to MRD+ from MRD- status.

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/OralMultispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, GeneCell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9CAR-T Immunogenicity; PCRVaccine Assay Performance; ADA Assay ComparabilityCut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Aims This investigation characterised tolerability, pharmacokinetics and pharmacodynamics of the anti-CD38 antibody TAK-079. Methods A randomised, double-blind, placebo-controlled trial of a single intravenous (i.v.) infusion or subcutaneous (s.c.) injection of TAK-079 at escalating doses in healthy subjects (n = 74), who were followed for 92 days postexposure. Results TAK-079 was well tolerated. All adverse events were mild or moderate. There were no withdrawals, infusion, or injection site reactions over the tested i.v. and s.c. doses up to 0.06 and 0.6 mg kg-1 , respectively. At higher doses, transient cytokine level increases, following i.v. administration, coincided with reduction in CD38-expressing cells; clinical symptoms included mild pyrexia, headache, and postural hypotension. Following an i.v. infusion of 0.06 mg kg-1 TAK-079, maximum observed serum concentration (Cmax ) was 100.4 (%CV: 52) ng mL-1 , time to Cmax was the end of infusion and natural killer (NK_ cells were reduced 93.8 (±8.5) % from baseline levels. Following a s.c. injection of 0.6 mg kg-1 TAK-079, Cmax was 23.0 (%CV: 67) ng mL-1 with time to Cmax of 24 (range 7.98-96.02) hours, and plasmablasts were subsequently reduced 93.4 (±8.8) % from predose levels. Serum immunoglobulin (Ig)M, IgA and IgG levels were reduced by 15-60% and had not returned to baseline levels within 78 days after administration at ≥0.3 mg kg-1 s.c. Reductions in NK cells at 0.6 mg kg-1 s.c. were approximately 2-3 times more durable than at 0.06 mg kg-1 i.v. Conclusions TAK-079 was well tolerated and s.c. administration elicited more durable reductions in plasmablasts and NK cells. This plasmacytolytic profile could be useful for treating disorders caused by plasma or NK cells, malignant counterparts, and/or pathogenic antibodies.

Measurable residual disease (MRD) evaluation may help to guide treatment duration in multiple myeloma (MM). Paradoxically, limited longitudinal data exist on MRD during maintenance. We investigated the prognostic value of MRD dynamics in 1280 transplant-eligible and -ineligible patients from the TOURMALINE-MM3 and -MM4 randomized placebo-controlled phase 3 studies of 2-year ixazomib maintenance. MRD status at randomization showed independent prognostic value (median progression-free survival [PFS], 38.6 vs 15.6 months in MRD− vs MRD+ patients; HR, 0.47). However, MRD dynamics during maintenance provided more detailed risk stratification. A 14-month landmark analysis showed prolonged PFS in patients converting from MRD+ to MRD− status vs those with persistent MRD+ status (76.8% vs 27.6% 2-year PFS rates). Prolonged PFS was observed in patients with sustained MRD− status vs those converting from MRD− to MRD+ status (75.0% vs 34.2% 2-year PFS rates). Similar results were observed at a 28-month landmark analysis. Ixazomib maintenance vs placebo improved PFS in patients who were MRD+ at randomization (median, 18.8 vs 11.6 months; HR, 0.65) or at the 14-month landmark (median, 16.8 vs 10.6 months; HR, 0.65); no difference was observed in patients who were MRD−. This is the largest MM population undergoing yearly MRD evaluation during maintenance reported to date. We demonstrate the limited prognostic value of a single–time point MRD evaluation, because MRD dynamics over time substantially impact PFS risk. These findings support MRD− status as a relevant end point during maintenance and confirm the increased progression risk in patients converting to MRD+ from MRD− status. These trials were registered at www.clinicaltrials.gov as #NCT02181413 and #NCT02312258.

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Summary Weekly ixazomib with lenalidomide‐dexamethasone (Rd) is feasible and has shown activity in newly diagnosed multiple myeloma (NDMM) patients. This phase 1/2 study (NCT01383928) evaluated the recommended phase 2 dose (RP2D), pharmacokinetics, safety and efficacy of twice‐weekly ixazomib plus Rd in NDMM; 64 patients were enrolled across both phases. Patients received twice‐weekly oral ixazomib 3·0 or 3·7 mg plus lenalidomide 25 mg and dexamethasone 20 mg (10 mg in cycles 9–16) for up to sixteen 21‐day cycles, followed by maintenance with twice‐weekly ixazomib alone. No dose‐limiting toxicities were reported in cycle 1; the RP2D was 3·0 mg based on overall tolerability across multiple cycles. In 62 evaluable patients, the confirmed overall response rate was 94% (68% ≥very good partial response; 24% complete response). Median progression‐free survival was 24·9 months. Responses (median duration 36·9 months for patients receiving the RP2D) deepened during treatment. Grade 3 drug‐related adverse events (AEs) occurred in 64% of patients, including: rash, 13%; peripheral neuropathy, 8%; hyperglycaemia, 8%. There were no grade 4 drug‐related AEs. Thirteen patients discontinued due to AEs. Twice‐weekly ixazomib‐Rd offers substantial activity with promising long‐term outcomes in NDMM patients but may be associated with greater toxicity compared with weekly ixazomib‐Rd in this setting.

The advent of time-of-flight mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system to simultaneous interrogate a multitude of parameters and gain a better understanding of immunologic data from clinical trial samples. Here we describe the development and validation of a 33-marker mass cytometry workflow for measuring gastrointestinal (GI) trafficking peripheral blood mononuclear cells (PBMCs) in patients with celiac disease (CeD). This panel builds upon identification of well-characterized immune cells and expands to include markers modulated in response to gluten challenge in patients with CeD. The CeD panel was optimized and validated according to accepted industry practice for validation of flow cytometry assays and builds upon established sample processing workflows for mass cytometry studies. Several critical parameters were evaluated during the assay development phase of this study including optimization of the sample processing steps, antibody specificity, and ensuring the panel as a whole performed to expectation. The panel was then validated using a fit-for-purpose approach tailored to the intended use of the data in the clinical trial. Validation included assessment of analytical parameters essential to understanding the reliability and robustness of the CeD panel such as intra-assay precision, inter-assay precision, inter-operator precision and sample processing stability. Together, this validated mass cytometry workstream provides robust and reproducible high-dimensional analysis of human peripheral blood immune cells to characterize patient samples from clinical trials.

Background Mezagitamab is a fully human immunoglobulin (Ig) G1 monoclonal antibody with high affinity to CD38 that depletes tumor cells expressing CD38 by antibody- and complement-dependent cytotoxicity. CD38 is a cell surface molecule that is highly expressed on myeloma cells, plasma cells, plasmablasts, and natural killer (NK) cells, and is induced on activated T cells and other suppressor cells including regulatory T (Tregs) and B (Bregs) cells. Data suggest that immune landscape changes in cancer patients and this may correlate with disease stage and clinical outcome. Monitoring specific immune cell subsets could predict treatment responses since certain cell populations either enhance or attenuate the anti-tumor immune response. Method To monitor the immune landscape changes in RRMM patients we developed a mass cytometry panel that measures 39-biomarkers to identify multiple immune cell subsets, including T cells (naïve, memory, effector, regulatory), B cells (naïve, memory, precursors, plasmablasts, regulatory), NK cells, NKT cells, gamma delta T cells, monocytes (classical, non-classical and intermediate), dendritic cells (mDC; myeloid and pDC; plasmacytoid) and basophils. After a robust analytical method validation, we tested cryopreserved peripheral blood and bone marrow mononuclear cells from 19 RRMM patients who received ≥ 3 prior lines of therapy. Patients were administered 300 or 600 mg SC mezagitamab on a QWx8, Q2Wx8 and then Q4Wx until disease progression schedule (NCT03439280). We compared the percent change in immune cell subsets at baseline versus week 4 and week 16. Results CD38 is expressed at different levels on immune cells and sensitivity to depletion by mezagitamab generally correlates positively with the density of expression. CD38 is expressed at high densities on plasmablasts, Bregs, NK-cells, pDC and basophils at baseline and this was associated with reductions in peripheral blood and bone marrow (plasmablasts, 95%, Bregs, 90%, NK-cells, 50%, pDC, 55% and basophils, 40%) at week 4 post treatment. In contrast, no changes occurred in the level of total T-cells and B-cells, which is consistent with low expression of CD38 on most cells of these large populations. Among the insensitive cell types, remaining NK-cells acquired an activated, proliferative and effector phenotype. We observed 60-150% increase in activation (CD69, HLA-DR), 110-200% increase in proliferation (Ki-67), and 40-375% increase in effector (IFN-γ) markers in peripheral blood and bone marrow. Importantly, NK-cells which did not express detectable CD38, also exhibited a similar phenotype possibly by a mechanism independent of CD38. Consistent with these data, the remaining CD4 and CD8 T-cell populations exhibited an activated effector phenotype as observed by 40-200% increase in activation, 60-200% increase in proliferation and 40-90% increase in effector markers in peripheral blood. A potential explanation for this acquisition of activated effector phenotypes could be a reduction in suppressive regulatory lymphocytes. Next, we measured levels of Tregs and Bregs, and observed that Bregs which are CD24hiCD38hi were reduced to 60-90% in peripheral blood and bone marrow. In contrast, total Tregs were reduced by only 5-25% because CD38 expression in Tregs appears as a spectrum where only ~10-20% are CD38+, and thus CD38+ Tregs were reduced more significantly (45-75%), reflecting the selectively of mezagitamab to cells expressing high levels of CD38. CD38+ Tregs are induced in RRMM patients, thus we looked at the phenotype of CD38-, CD38mid, and CD38high -expressing Tregs. We observed higher level of markers that correlate with highly suppressive Tregs such as Granzyme B, Ki-67, CTLA-4 and PD-1 in CD38high Tregs. Accordingly, the total Treg population exhibited a less active phenotype after exposure to mezagitamab, which selectively depleted the highly suppressive CD38+ Tregs. Conclusions Chronic treatment with mezagitamab is immunomodulatory in patients with RRMM, which is associated with reductions in tumor burden, subpopulations of B and T regulatory cells, and characterized by conventional NK and T cells exhibiting an activated, proliferative and effector phenotype. The immune landscape changes observed is consistent with the immunologic concept of converting the tumor microenvironment from cold-to-hot and highlights a key mechanistic effect of mezagitamab. Disclosures Berg: Takeda Pharmaceuticals Inc: Current Employment.

Background Vedolizumab is a monoclonal antibody approved for use in ulcerative colitis and Crohn's disease. By specifically binding to α4β7 integrin, vedolizumab prevents trafficking of lymphocytes to the gut, thereby interfering with disease pathology. During the clinical development program, the pharmacodynamic effect of vedolizumab was evaluated by 2 flow cytometry receptor occupancy assays: act-1 (ACT-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Here we describe the development and validation of these assays. Methods The ACT-1 assay is a receptor occupancy free-site assay that uses a monoclonal antibody with the same binding epitope as vedolizumab to detect free (unbound) sites on α4β7 integrin. The MAdCAM-1 assay used a soluble version of the natural ligand for α4β7 integrin to detect free sites. The assays were validated using a fit-for-purpose approach throughout the clinical development of vedolizumab. Results Both the ACT-1 assay and the MAdCAM-1 assay demonstrated acceptable reproducibility and repeatability. The assays were sufficiently stable to allow for clinical use. During clinical testing the assays demonstrated that vedolizumab was able to saturate peripheral cells at all doses tested. Conclusions Two pharmacodynamic receptor occupancy assays were developed and validated to assess the effect of vedolizumab on peripheral blood cells. The results of these assays demonstrated the practical use of flow cytometry to examine pharmacodynamic response in clinical trials. © 2015 International Clinical Cytometry Society

Guanylyl cyclase C (GCC) is a cell-surface protein that is expressed by normal intestinal epithelial cells, more than 95% of metastatic colorectal cancers (mCRC), and the majority of gastric and pancreatic cancers. Due to strict apical localization, systemically delivered GCC-targeting agents should not reach GCC in normal intestinal tissue, while accessing antigen in tumor. We generated an investigational antibody-drug conjugate (TAK-264, formerly MLN0264) comprising a fully human anti-GCC monoclonal antibody conjugated to monomethyl auristatin E via a protease-cleavable peptide linker. TAK-264 specifically bound, was internalized by, and killed GCC-expressing cells in vitro in an antigen-density-dependent manner. In GCC-expressing xenograft models with similar GCC expression levels/patterns observed in human mCRC samples, TAK-264 induced cell death, leading to tumor regressions and long-term tumor growth inhibition. TAK-264 antitumor activity was generally antigen-density-dependent, although some GCC-expressing tumors were refractory to TAK-264-targeted high local concentrations of payload. These data support further evaluation of TAK-264 in the treatment of GCC-expressing tumors.

Tooth erosion from an acidic insult may be exacerbated by toothbrushing. The purposes of this study were to develop an in vitro methodology to measure enamel loss after brushing immediately following an acidic episode and to investigate the effect of brushing with an anti-erosive toothpaste. The null hypotheses tested were that tooth erosion after brushing with the toothpaste would not be different from brushing with water and that a 1-hour delay before brushing would not reduce tooth erosion. Forty bovine enamel slabs were embedded, polished, and subjected to baseline profilometry. Specimens were bathed in hydrochloric acid for 10 minutes to simulate stomach acid exposure before post-acid profilometry. Toothbrushing was then simulated with a cross-brushing machine and followed by postbrushing profilometry. Group 1 was brushed with water; group 2 was brushed with a 50:50 toothpaste-water slurry; and groups 3 and 4 were immersed in artificial saliva for 1 hour before brushing with water or the toothpaste slurry, respectively. The depth of enamel loss was analyzed and compared using 1-way analysis of variance and post hoc testing (α = 0.05). Greater enamel loss was measured in groups brushed with toothpaste than in groups brushed with water. One-hour immersion in artificial saliva significantly reduced enamel loss when teeth were brushed with water (group 3; P0.05) but not with toothpaste (group 4). This study established a protocol for measuring enamel loss resulting from erosion followed by toothbrush abrasion. The results confirmed the abrasive action of toothpaste on acid-softened enamel.

Introduction: The CD38 antigen is a target for treating multiple myeloma and is being investigated for disorders caused by plasma cells. Therapeutics bind to CD38 on red blood cells and platelets in the vasculature, prior to myeloma and plasma cells in the bone marrow and this initial activity could compromise downstream binding to target cells. Methods: We hypothesized that selectively targeting myeloma and plasma cells by circumventing CD38 expressed on RBCs and platelets could improve the potency of next generation therapeutics. We selected the human antibody TAK-079 (IgG1) because it demonstrated relatively low binding to CD38 on RBCs and platelets, in comparison to the first-generation antibody daratumumab (IgG1) in vitro, and characterized the pharmacokinetics and pharmacodynamics in relapsed/refractory multiple myeloma (RRMM) patients (NCT03439280). Results: TAK-079 and daratumumab bind to distinct amino acids of CD38 and exhibit similar affinities to recombinant CD38 protein, immobilized on a silicon chip (e.g. KD of 0.45 & 0.69 nM, respectively) and to endogenously expressed CD38 on a clonal cell line (e.g. EC50s = 0.30 & 0.34 nM, respectively). In contrast, these antibodies bound differently to components of human blood. Daratumumab bound 610% more intensively to a subpopulation of RBCs than TAK-079. Similarly, daratumumab bound 622% more intensively to a subpopulation of platelets than TAK-079. Conversely, TAK-079 bound 181% more potently than daratumumab to CD38 expressed on blood B lymphocytes (e.g. MFI mean EC50 =3.6 & 6.6 nM, respectively) and 259% more potently to blood T lymphocytes (e.g. MFI mean EC50 = 9.3 & 24.2 nM, respectively). Consistent with these data, trough exposures of TAK-079 were approximately 200% higher than those reported for daratumumab (NCT02519452) in RRMM patients, when compared at equivalent subcutaneous doses. Furthermore, bone marrow myeloma and plasma cells were completely saturated by TAK-079 and reduced at subcutaneous doses ≥ 300mg administered weekly. Conclusion: An effective strategy for developing more potent therapeutics may be to circumvent binding to CD38 on circulating RBCs and PLTs because the magnitude of CD38 antibody binding to leukocytes is inversely related to binding of circulating RBCs and PLTs in vitro. In addition, lower binding to RBCs and PLTs is associated with higher availability of CD38 antibody for binding target cells in RRMM patients, as well as target saturation and depletion by TAK-079 when administered subcutaneously at relatively low doses (eg, 300mg). Disclosures Fedyk: Millennium, a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd: Employment, Patents & Royalties. Idamakanti:Takeda: Employment. Chen:Takeda: Employment. Estevam:Takeda: Employment. Hernandez:Takeda: Employment. Wagoner:Takeda: Employment. Carsillo:Takeda: Employment. Berg:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd: Employment, Patents & Royalties. Allikmets:Takeda: Employment, Patents & Royalties. Lahu:Think AQ: Consultancy. Palumbo:Millennium, a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd: Employment, Patents & Royalties.

Pharmacodynamic assays are important aspects for understanding molecularly targeted anticancer agents to investigate the relationship between drug concentration (pharmacokinetics) and drug "effect" or biological activity. As new drug entities are developed that affect DNA cell cycle, a pharmacodynamic assay which measures cell cycle perturbation would be a valuable clinical trial tool. During recent years, flow cytometry has established itself as a useful method to determine the relative nuclear DNA content and percentage of cycling cells of biological specimens. However to date, the analytical validation of cytometry based assays is limited and there is no suitable guidance for method validation of flow cytometry based cell cycle assays. Here we report the validation of a flow cytometry based cell cycle G(2)/M delay assay for use in evaluating the effect of investigational drug MLN8237, a small molecule inhibitor of a mitotic kinase Aurora A, for clinical trial use. The assay method was validated by examining assay robustness, repeatability, reproducibility, precision, and determining the cutoff for a true drug effect based on biostatistical analysis models. Experimental results show that the intra-assay repeatability was less than 20% with an intra-donor variability of less than 40%. The robustness of the assay was less than 30%. Since this is an ex-vivo stimulation assay, variability parameters were expected to be higher. Based on biostatistical modeling, an absolute change in %G(2)M of 5.2% (95% CI) was needed in order to detect a true drug effect. Overall, the assay demonstrated acceptable variability to warrant further in vivo testing.

Background: A study investigating the safety, efficacy, pharmacokinetics (PK) and pharmacodynamics (PD) of TAK-079 administered subcutaneously as a single agent in participants with relapsed/refractory multiple myeloma (RRMM) is ongoing because this investigational agent demonstrated distinct pharmacologic properties in a first in human study involving healthy volunteers. CD38 is expressed at high levels on subtypes of leukocytes which mediate immune disorders (e.g. plasma cells, activated B and T lymphocytes) and the selective removal of these cells could also restore immune homeostasis in these patients. TAK-079 is a fully human monoclonal antibody which binds to human CD38 protein with high affinity (KD = 0.7 nM) and lyses bound cells by CDC and ADCC in vitro. Methods: A randomized, double-blind, placebo-controlled study was conducted to assess the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of single intravenous (IV) infusion or subcutaneous (SC) injection of TAK-079 in escalating dose cohorts of healthy subjects. A starting single dose of 0.0003 mg/kg IV was selected, based upon modeling of responses in human and monkey model systems. Dose escalation ceased once a prespecified PD objective was achieved (i.e. ≥ 50% reduction in a leukocyte subset within peripheral blood which was sustained for ≥ 1 month). Six subjects were enrolled per dose cohort (4 receiving TAK-079; 2 receiving matching placebo). Subjects were monitored for at least 92 days post-dosing. Results: After IV (n=30) or SC (n=24) administration at all dose levels, TAK-079 was well tolerated and no safety concerns were identified. The maximum doses given were 0.06 mg/kg IV and 0.6 mg/kg SC. There were no serious adverse events (AE), severe AEs, withdrawals due to AEs, or infusion reactions. All AEs were mild or moderate. At the higher doses of TAK-079 administration, transient, mild to moderate increases in cytokine levels coincided with anticipated cell depletion; clinical symptoms primarily included mild pyrexia, headache, and postural hypotension. No remarkable findings for laboratory tests, ECGs, vital signs, or physical examinations were reported. Following a 2-hour IV infusion of 0.06 mg/kg, Cmax was observed at 5 minutes post EOI with a mean Cmax of 100.4 ng/mL. After Cmax was reached, serum concentrations decreased below the limit of quantification within 1 to 4 hours after the end of infusion. Following a single SC injection of 0.6 mg/kg TAK-079, the mean Cmax of 23.0 ng/mL was observed at approximately 24 hours post-dose and decreased gradually to below the limit of quantification by a median of 8 days (range 3-14). The mean AUClast and AUC∞ values were 90.4 and 212 ng*day/mL, respectively. Pharmacodynamic effects included a transient, dose-dependent reduction in NK cells at doses ≥0.003 mg/kg IV, approximately 167-fold less than the lowest dose reported for NK cell reduction by daratumumab administered to RRMM patients IV (≥0.5 mg/kg1). TAK-079 administered IV or SC reduced NK cells to a comparable extent (EC75 = 21 and 23 ng/mL, respectively). Greater than 90% reduction of plasmablasts was also observed in each subject dosed at 0.6 mg/kg SC, which generally peaked 2 days after injection and returned to baseline levels by 29 days. Neutrophil, lymphocyte, monocyte, red blood cell and platelet counts remained within normal ranges for all dose cohorts. Conclusion: TAK-079 may be a potent and convenient second generation anti-CD38 therapeutic, which warrants development for treatment of hematologic malignancies. Therefore, the safety, efficacy, pharmacokinetics, and pharmacodynamics of subcutaneous TAK-079 is being investigated in patients with RRMM in an ongoing study (NCT03439280; Figure 1) The starting dose in the RRMM study is fixed at 45 mg, based on the profile of the equivalent 0.6 mg/kg in healthy subjects. References Blood Adv. 2017 Oct 24;1(23):2105-2114. Disclosures Fedyk: Takeda Pharmaceuticals Inc.: Employment. Berg:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Smithson:Takeda Pharmaceuticals Inc.: Employment. Estevam:Takeda Pharmaceuticals Inc.: Employment. Mclean:Takeda Pharmaceuticals Inc.: Employment. Allikmets:Takeda Pharmaceuticals Inc.: Employment. Palumbo:Takeda Pharmaceuticals Inc.: Employment.

Summary Background The combination of bortezomib, lenalidomide, and dexamethasone is a highly effective therapy for newly diagnosed multiple myeloma. Ixazomib is an investigational, oral, proteasome inhibitor with promising anti-myeloma effects and low rates of peripheral neuropathy. In a phase 1/2 trial we aimed to assess the safety, tolerability, and activity of ixazomib in combination with lenalidomide and dexamethasone in newly diagnosed multiple myeloma. Methods We enrolled patients newly diagnosed with multiple myeloma aged 18 years or older with measurable disease, Eastern Cooperative Oncology Group performance status 0–2, and no grade 2 or higher peripheral neuropathy, and treated them with oral ixazomib (days 1, 8, 15) plus lenalidomide 25 mg (days 1–21) and dexamethasone 40 mg (days 1, 8, 15, 22) for up to 12 28-day cycles, followed by maintenance therapy with ixazomib alone. In phase 1, we gave patients escalating doses of ixazomib (1·68–3·95 mg/m 2 ) to establish the recommended dose for phase 2. The primary endpoints were maximum tolerated dose for phase 1, and the rate of very good partial response or better for phase 2. Safety analyses were done in all patients who received at least one dose of study drug; efficacy analyses were done in all patients who received at least one dose of study drug at the phase 2 dose, had measurable disease at baseline, and had at least one post-baseline response assessment. This study is registered at ClinicalTrials.gov, number NCT01217957. Findings Between Nov 22, 2010, and Feb 28, 2012, we enrolled 65 patients (15 to phase 1 and 50 to phase 2). Four dose-limiting toxic events were noted in phase 1: one at a dose of ixazomib of 2·97 mg/m 2 and three at 3·95 mg/m 2 . The maximum tolerated dose of ixazomib was established as 2·97 mg/m 2 and the recommended phase 2 dose was 2·23 mg/m 2 , which was converted to a 4·0 mg fixed dose based on population pharmacokinetic results. Grade 3 or higher adverse events related to any drug were reported in 41 (63%) patients, including skin and subcutaneous tissue disorders (11 patients, 17%), neutropenia (eight patients, 12%), and thrombocytopenia (five patients, 8%); drug-related peripheral neuropathy of grade 3 or higher occurred in four (6%) patients. Five patients discontinued because of adverse events. In 64 response-evaluable patients, 37 (58%, 95% CI 45–70) had a very good partial response or better. Interpretation The all-oral combination of weekly ixazomib plus lenalidomide and dexamethasone was generally well tolerated and appeared active in newly diagnosed multiple myeloma. These results support the phase 3 trial development of this combination for multiple myeloma. Funding Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda Pharmaceutical International Company.

Vedolizumab is a monoclonal antibody approved for use in ulcerative colitis and Crohn's disease. By specifically binding to α4 β7 integrin, vedolizumab prevents trafficking of lymphocytes to the gut, thereby interfering with disease pathology. During the clinical development program, the pharmacodynamic effect of vedolizumab was evaluated by 2 flow cytometry receptor occupancy assays: act-1 (ACT-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Here we describe the development and validation of these assays.The ACT-1 assay is a receptor occupancy free-site assay that uses a monoclonal antibody with the same binding epitope as vedolizumab to detect free (unbound) sites on α4 β7 integrin. The MAdCAM-1 assay used a soluble version of the natural ligand for α4 β7 integrin to detect free sites. The assays were validated using a fit-for-purpose approach throughout the clinical development of vedolizumab.Both the ACT-1 assay and the MAdCAM-1 assay demonstrated acceptable reproducibility and repeatability. The assays were sufficiently stable to allow for clinical use. During clinical testing the assays demonstrated that vedolizumab was able to saturate peripheral cells at all doses tested.Two pharmacodynamic receptor occupancy assays were developed and validated to assess the effect of vedolizumab on peripheral blood cells. The results of these assays demonstrated the practical use of flow cytometry to examine pharmacodynamic response in clinical trials.

Made available in DSpace on 2022-04-28T18:57:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-01 Purpose: The purpose of this study was to evaluate the histological features of the pulp tissue in primary pig teeth submitted to pulpotomy and capped with calcium hydroxide-based and standardized propolis extract-based pastes, and the combination of these pastes. Methods: Nine 4-month-old male pigs were used in this study, which were distributed into 4 groups, according to the studied pastes: Group 1-calcium hydroxide; Group 2-standardized propolis extract; Group 3-combination of pastes 1 and 2 in the proportion 1:1; and Group 4-control. The teeth used for the pulpotomy were the 4 mandibular primary incisors. Results: After 7, 21, and 42 days, the animals were killed and the teeth were removed for histological analysis. At 42 days, all teeth in Groups 1 to 3 presented a complete hard tissue barrier and the pulp tissue beneath was without inflammation. Conclusion: According to these findings, calcium hydroxide and standardized propolis extract favored the formation of a hard tissue barrier in primary pig teeth submitted to pulpotomy. School of Dentistry University of Ribeirao Preto School of Dentistry University of Sao Paulo Ribeirao Preto, Sao Paulo School of Agrarian and Veterinary Sciences of Jaboticabal State University of Sao Paulo, Jaboticabal, Sao Paulo Federal University of Pernambuco Oral Pathology Section, Recife, Pernambuco

Analisou-se, por meio de espectrofotometria, a liberação de formaldeído de cimentos endodônticos à base de óxido de zinco e eugenol, resinas epóxica e polimetacrilato, hidróxido de cálcio e biocerâmico, estudados em diferentes oportunidades: durante a espatulação; decorrido três vezes o tempo de endurecimento e, por fim, na extração de sua massa endurecida. As amostras foram adquiridas pela coleta do volume da água utilizada em cada período e, determinada a curva de calibração da liberação do formaldeído e análise controle com água, foram submetidas à análise colorimétrica com adição dos reagentes: 5,0 mL de solução tampão, 0,5 mL de solução de parafenilenodiamina e 2,5 mL de peróxido de hidrogênio em balões volumétricos de 50,0 mL mantendo-os em repouso por 20 minutos. Após, foi realizada leitura em espectrofotômetro duplo feixe nos comprimentos de onda 326, 334 e 462 nm. Detectou-se formaldeído em todos os cimentos à base de resina epóxica, no EndoREZ® e no Endomèthasone N, quantificado apenas no Sealer 26 (7.40 mg.L-1) e Endomèthasone N (9.13 mg.L-1) durante a espatulação; no AH Plus® (4.44 mg.L-1) e no Endomèthasone N (18.14 mg.L-1) durante o endurecimento, e somente no Endomèthasone N depois de endurecido (9.43 mg.L- 1). Concluiu-se que, durante a espatulação, todos os cimentos de resina epóxica liberaram formaldeído, com quantificação apenas para o Sealer 26, o Endomèthasone N apresentou a maior quantidade, e o Endorez liberou a substância em quantidade insuficiente para quantificação. Após a espatulação, todos os de resina epóxica liberaram formaldeído, com quantificação no AH Plus e o Endomèthasone N apresentou a maior quantidade da substância, que também foi encontrada no Endorez, sem quantificação. Endurecidos, todos os cimentos de resina epóxica e o Endorez liberaram formaldeído em quantidade insuficiente para quantificação, e o cimento Endomèthasone N liberou a maior quantidade dessa substância. This study evaluated, by means of spectrophotometry, the release of formaldehyde in zinc oxide, epoxy resins and polymethylmethacrylate, calcium hydroxide and bioceramic based endodontic sealers in different situations: during mixing; after 3 time the setting time and, at extraction of its hardened mass. The samples were acquired by means of collection of the volume of water used in each period and - after determining the calibration curve of formaldehyde release and analyzing the control with water - were submitted to colorimetric analysis with the addition of the following reagents: 5.0mL of buffered solution, 0.5mL of paraphenylenediamine and 2.5mL of hydrogen peroxide to a 50.0mL volumetric flask, which was kept at rest for 20 minutes. Double-beam spectrophotometer readings were performed at 326, 334 and 462nm wavelengths. Formaldehyde was detected in all epoxy resin based sealers, in EndoREZ and in Endomèthasone N, quantified only in Sealer 26 (7.40 mg.L-1) and Endomèthasone N (9.13 mg.L-1) during mixing, and in AH Plus (4.44 mg.L-1) and Endomèthasone N (18.14 mg.L-1) during the setting time, and only in Endomèthasone N after setting (9.43 mg.L-1). It can be concluded that during mixing all epoxy resin based sealers released formaldehyde, with quantification being possible only for Sealer 26, Endomèthasone N showed higher quantities, and Endorez released the substance in insufficient quantity for detection. After mixing, all epoxy resin based sealers released formaldehyde, whereas quantification was possible for AH Plus and Endomèthasone N presented the highest quantity of the substance, that was also found in Endorez, but could not be quantified. When set, all epoxy resin sealers and Endorez released formaldehyde in insufficient quantity for quantification, and Endomèthasone N released the greater amount of this substance.

Combinations of bortezomib (V) and dexamethasone (D) with either lenalidomide (R) or cyclophosphamide (C) have shown significant efficacy. This randomized phase 2 trial evaluated VDC, VDR, and VDCR in previously untreated multiple myeloma (MM). Patients received V 1.3 mg/m2 (days 1, 4, 8, 11) and D 40 mg (days 1, 8, 15), with either C 500 mg/m2 (days 1, 8) and R 15 mg (days 1-14; VDCR), R 25 mg (days 1-14; VDR), C 500 mg/m2 (days 1, 8; VDC) or C 500 mg/m2 (days 1, 8, 15; VDC-mod) in 3-week cycles (maximum 8 cycles), followed by maintenance with V 1.3 mg/m2 (days 1, 8, 15, 22) for four 6-week cycles (all arms) ≥ very good partial response was seen in 58%, 51%, 41%, and 53% (complete response rate of 25%, 24%, 22%, and 47%) of patients (VDCR, VDR, VCD, and VCD-mod, respectively); the corresponding 1-year progression-free survival was 86%, 83%, 93%, and 100%, respectively. Common adverse events included hematologic toxicities, peripheral neuropathy, fatigue, and gastrointestinal disturbances. All regimens were highly active and well tolerated in previously untreated MM, and, based on this trial, VDR and VCD-mod are preferred for clinical practice and further comparative testing. No substantial advantage was noted with VDCR over the 3-drug combinations. This trial is registered at www.clinicaltrials.gov (NCT00507442).

Background MLN9708 (ixazomib citrate) is an oral proteasome inhibitor that is being investigated in phase 3 trials. MLN9708 rapidly hydrolyzes to the biologically active form, MLN2238 (ixazomib). Preliminary findings from studies using weekly and twice-weekly schedules in relapsed/refractory MM have suggested evidence of single-agent activity (Kumar et al, ASCO 2013; Lonial et al, ASCO 2012), and a phase 1/2 study has suggested the feasibility and activity of weekly oral MLN9708 plus len-dex in newly diagnosed MM (Kumar et al, ASH 2012). Here we report the results of a phase 1/2 study, conducted in collaboration with the Multiple Myeloma Research Consortium, of twice-weekly oral MLN9708 plus len-dex (NCT01383928). Methods Phase 1 primary objectives were to determine safety, tolerability, the MTD, and the recommended phase 2 dose (RP2D); secondary objectives included characterizing MLN2238 pharmacokinetics (PK). Phase 2 primary objectives were to determine the combined CR+VGPR rate and further evaluate safety and tolerability; secondary objectives included overall response rate (≥PR), time to response, and duration of response (DOR). Pts aged ≥18 yrs who had no grade ≥2 peripheral neuropathy (PN) and no prior/concurrent DVT/pulmonary embolism received MLN9708 3.0 or 3.7 mg (d 1, 4, 8, 11), len 25 mg (d 1–14), and dex 20/10 mg (cycles 1–8/9–16; d 1, 2, 4, 5, 8, 9, 11, 12) for up to 16 21-day cycles, followed by MLN9708 maintenance (same schedule) until progression or unacceptable toxicity. Transplant-eligible pts could undergo stem cell collection after ≥4 cycles and discontinue for ASCT after ≥8 cycles. Responses were assessed per IMWG uniform response criteria. Adverse events (AEs) were graded per NCI-CTCAE v4.03. Blood samples for PK analysis were taken at multiple time points during cycles 1 and 2. Results 64 pts were enrolled; median age was 64 yrs (range 34–82), 63% were male, and 31%/16% had ISS stage II/III MM. In phase 1, 14 pts received MLN9708 3.0 mg (n=7) and 3.7 mg (n=7). No DLTs were seen in cycle 1; based on overall tolerability and incidence of rash at 3.7 mg, the RP2D was chosen as 3.0 mg. 50 pts were enrolled at this dose in phase 2. At data cut-off (July 1, 2013), the median follow-up was 6.9 months and median number of cycles received was 8 (range 1–26); 73% had received ≥8 cycles and 9% had received ≥16 cycles. At data cut-off, 22% of pts had discontinued to undergo ASCT (median CD34+ stem cell yield 14.9 x 106/kg [range 7–52 x 106]), a further 14%, 5%, and 19% had discontinued due to AEs, progressive disease, and other reasons, respectively, and the other 41% remained on treatment. In 58 response-evaluable pts, ≥PR rate to date was 93%, including 67% ≥VGPR (24% CR, including 14% sCR). 54% of pts had 100% decreases in M-protein or serum free light chain from baseline. Analysis of minimal residual disease is ongoing; data will be presented. Depth of response increased over the course of treatment; median time to first response (≥PR) was 0.69 mos and to best response to date was 2.07 mos. Median DOR to date was 5.9+ mos, ranging up to 18+ mos. Most common AEs were rash (61%; pooled high-level terms), fatigue, peripheral edema (each 50%), diarrhea (41%), and neuropathy peripheral (36%). Drug-related (to any drug in the regimen) grade 3 AEs were seen in 56% of pts, including rash (16%), hyperglycemia (8%), pneumonia (6%), and PN (5%; high-level term). No drug-related grade 4 AEs were seen; 58% of pts required dose reductions of at least one drug due to AEs including rash (16%), anxiety (11%), and PN (8%). AEs resulting in discontinuation were seen in 11%, with the majority reported as not related to therapy. There was 1 on-study death due to cardio-respiratory arrest, likely a pulmonary embolism, considered by the investigator to be unrelated to MLN9708 or dex, but probably len. Based on phase 1 preliminary PK data, MLN2238 was absorbed quickly with a Tmaxof 0.5–4 hours. Terminal half-life was 2–8 days. PK data were similar to single-agent twice-weekly dosing studies, suggesting no MLN2238 PK interaction with len or dex. Conclusions These data suggest that twice-weekly oral MLN9708 plus len-dex is feasible and active in pts with newly diagnosed MM. However, rates of rash, PN, and dose reductions appear higher than in the parallel study using weekly MLN9708, with similar response rates and better convenience, supporting use of weekly dosing in ongoing phase 3 trials. Disclosures: Richardson: Millennium: The Takeda Oncology Company: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees. Off Label Use: Investigational agent MLN9708 in combination with lenalidomide and dexamethasone for the first-line treatment of patients with multiple myeloma. Hofmeister:Celgene: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Rosenbaum:Janssen: Membership on an entity’s Board of Directors or advisory committees; Celgene: Speakers Bureau. Vesole:Millennium: The Takeda Oncology Company: Speakers Bureau; Celgene: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Liedtke:Celgene: Research Funding; Millennium: The Takeda Oncology Company: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Chari:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Millennium: The Takeda Oncology Company: Membership on an entity’s Board of Directors or advisory committees; Onyx Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees. Lebovic:Celgene: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees. Berg:Millennium: The Takeda Oncology Company: Employment. Liao:Millennium: The Takeda Oncology Company: Employment. Gupta:Millennium: The Takeda Oncology Company: Employment. Di Bacco:Millennium: The Takeda Oncology Company: Employment. Estevam:Millennium: The Takeda Oncology Company: Employment. Hui:Millennium: The Takeda Oncology Company: Employment. Baz:Celgene: Research Funding; Millennium: The Takeda Oncology Company: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding.

Abstract 621 Background: Two- and three-drug regimens incorporating bortezomib (Velcade®, V), lenalidomide (Revlimid®, R), dexamethasone (D), or cyclophosphamide (C) have been shown to be effective and well tolerated in previously untreated multiple myeloma (MM). Combining all four drugs in a single regimen (VDCR) may further enhance efficacy. Published results from the phase 1 dose-escalation portion of the non-comparative, multi-center EVOLUTION study showed that the VDCR regimen was highly active and generally well tolerated (Kumar et al Leukemia 2010). Here we present updated results from the phase 2 portion of the trial, focusing on efficacy and safety of the VDCR, VDR, and VDC regimens. Methods: Previously untreated patients with measurable disease were randomized to one of four treatment groups receiving up to eight 21-d cycles of VDCR (V 1.3 mg/m2 d 1, 4, 8, 11; D 40 mg d 1, 8, 15; R 15 mg d 1–14; C 500 mg/m2 d 1, 8), VDR (VD as in VDCR but with R 25 mg d 1–14), VDC (VDC as in VDCR), or VDC-mod (as for VDC but with an additional dose of C on d 15) as induction therapy, followed by four 42-d maintenance cycles of V 1.3 mg/m2 (d 1, 8, 15, 22) (all treatment arms). Patients eligible for autologous stem cell transplant (SCT) could undergo stem cell mobilization any time after cycle 2 and SCT any time after cycle 4. The primary endpoint was the combined complete response (CR) + very good partial response (VGPR) rate; secondary endpoints included safety/tolerability, time to response, duration of response, progression-free survival, and rate of minimal residual disease (MRD) negativity. Responses were assessed according to International Myeloma Working Group (IMWG) uniform criteria using an automated computer algorithm. Adverse events (AEs) were graded using the CTCAE v3.0. Results: Patient characteristics were similar among the groups with respect to age, performance status, ISS stage, and proportion of patients with high-risk cytogenetic features. Patients received a median of 5, 6, 6, and 6 treatment cycles in the VDCR, VDR, VDC, and VDC-mod arms, respectively; 65%, 60%, 52%, and 47% of patients had dose reductions of any drug. In the VDCR, VDR, VDC, and VDC-mod arms, respectively, 52%, 62%, 58%, and 65% of patients completed treatment; 31%, 43%, 24%, and 41%, respectively, underwent SCT. In the phase 2 response-evaluable patients (n=132), all treatment regimens showed substantial efficacy, with CR+VGPR rates of 59% (VDCR), 50% (VDR), 41% (VDC), and 59% (VDC-mod) (Table) (includes pre-transplant responses only in SCT patients). Of MRD-assessed patients, 46% (21/46) of those who achieved CR (including sCR) or nCR were MRD-negative; 48% (10/21), 75% (9/12), 0% (0/7) and 33% (2/6) in the VDCR, VDR, VDC and VDC-mod arms, respectively. Median time to first response was similar across arms (range 1.6–1.8 months); median time to best response of CR+VGPR was 4.0 months (VDCR), 3.4 months (VDR), 5.1 months (VDC), and 3.1 months (VDC-mod). Median duration of response has not been reached in any arm to date. All treatment regimens were generally well tolerated. In the VDCR, VDR, VDC, and VDC-mod arms, at least one grade ≥3 AE was observed in 81%, 76%, 79%, and 88% of enrolled patients, respectively; serious AEs were experienced by 42%, 40%, 21%, and 41% of patients, and AEs resulting in study discontinuation were reported for 19%, 17%, 12%, and 6% of patients. The five most common all-grade AEs across all treatment groups were fatigue (range 47–67%), nausea (36–67%), constipation (40–62%), diarrhea NOS (42–65%), and neutropenia (19–52%). The incidence of grade ≥3 (grade ≥2) peripheral neuropathy (PN) was 13% (40%) in the VDCR arm, 14% (45%) VDR, 9% (48%) VDC, and 18% (41%) VDC-mod; there was no grade 4 PN. Rates of grade ≥3 neutropenia/thrombocytopenia were 42%/10% for VDCR, 7%/7% VDR, 36%/12% VDC, and 65%/18% VDC-mod. Conclusions: All regimens appear highly active and generally well tolerated in previously untreated MM patients. The four-drug combination did not result in a substantial increase in response rate and was associated with a modest increase in the incidence of hematologic toxicities. Continuous weekly C in the VDC regimen was associated with high response rates and rapid responses, comparable to the VDR and VDCR arms. Outcome data will be presented following longer follow-up. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding; Bayer: Research Funding. Off Label Use: Lenalidomide for treatment of newly diagnosed myeloma. Flinn:Millennium Pharmaceuticals, Inc.: Research Funding. Richardson:Celgene, Millennium, Novartis, Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Hari:Celgene: Research Funding. Callander:Millennium Pharmaceuticals, Inc.: Research Funding. Noga:Amgen: Honoraria, Research Funding, Speakers Bureau; Millennium: Consultancy, Honoraria, Research Funding, Speakers Bureau; Ortho-Centicor: Honoraria, Speakers Bureau; Cephalon: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau. Stewart:Millennium Pharmaceuticals, Inc.: Honoraria, Research Funding; Celgene: Honoraria. Rifkin:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; Amgen: Speakers Bureau; Cephalon: Speakers Bureau; Dendreon: Speakers Bureau. Wolf:Millennium Pharmaceuticals: Consultancy, Honoraria, Speakers Bureau; Genentech and Multiple Myeloma Research: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Speakers Bureau; OrthoBiotech: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau. Estevam:Millennium Pharmaceuticals: Employment. Mulligan:Millennium Pharmaceuticals, Inc.: Employment. Shi:Millennium Pharmaceuticals: Employment. Webb:Millennium Pharmaceuticals: Employment.

A prospective study since the month 09/1991 to 09/1999 12113 endoscopic biopsies of gastric antrum in Santos - Sao Paulo - Brazil, accomplished redfaced anatomopathologic by Hematoxilin-eozina and observed for experienced pathologist, where we observed 663 cases of metaplasia intestinal type (Mp) (in the form enteric or colonic) of these 142 (21.41%) in the form enteric (Mpe) and 521 (78.58%) in the form colonic (Mpc), we correlated the presence of Helicobacter pylori to the two forms. The association, Metaplasia and Helicobacter pylori were present in 524 biopsies (79.03%) and the absence of Helicobacter pylori and Metaplasia were present in 139 biopsies (20.97%), being Metaplasia enteric (Mpe) (Hp+) 92 biopsies and (Hp-) 50 biopsies, and Metaplasia colonic (Mpc) (Hp+) 471 biopsies and (Mpc) (Hp-) 50 biopsies.With this study we can observe the great incidence of Helicobacter pylori (Hp) in association with the intestinal metaplasia (Mp), being your important association sees that the presence of intestinal metaplasia (Mp) and the Helicobacter pylori can be correlated the genesis of Gastric Cancer

Esta pesquisa tem por princípio investigar o campo de possibilidades de desvelamento criativo do ator, por meio de um campo-espaço de acontecimentos, sensações, ações, comportamentos, sentimentos, projetados para e entre atores. A partir da relação estabelecida entre o Campo Mórfico, do biólogo Rupert Sheldrake, e do Espaço Vazio, do encenador Peter Brook, objetiva-se potencializar o surgimento de elementos inovadores e criativos para a composição das cenas e das personagens. Esse estudo propõe uma forma de conhecimento pautado na transitoriedade, na impermanência, na efemeridade e em como elas se relacionam com a razão durante o processo criativo. E ainda, o aprofundamento e experimentação que não prevê ou cria expectativas sobre algo combinado antecipadamente acerca do fazer artístico, mas acentua o instante presente do esvaziar-se para preencher-se. This research has by premise the investigation of the field of possibilities of the actor\'s creative unveiling through a field-space of happenings, sensations, actions, behaviour, feelings, which is projected by actors and among actors. From the relation set between the Morphic Field of the biologist Rupert Sheldrake and the Void Space of scenemaker Peter Brook aims to enhancing the appearence of inovative and creative elements for the composition of scenes and characters. This study proposes a form of knowledge based on transience, impermanence, the ephemeral and how they relate to the reason during the creative process. And yet, the deepening and experimentation that does not foresee or create expectations about something combined in advance about art making, but emphasizes the present moment of hollowing out to be filled.