ALBERT

Description: Introduction Lenalidomide (LEN) is a weak substrate of P-glycoprotein (P-gp) in vitro and renal excretion of LEN is the primary elimination route following oral administration.  A P-gp inhibitor may have the potential to increase systemic exposure to LEN by reducing renal elimination at the tubular level and enhancing oral absorption at the gut level. Recently, a single uncontrolled phase 1 study (Hofmeister, 2011) and a case report (Takahashi, 2012) described that plasma exposure to LEN and temsirolimus was increased in multiple myeloma patients when lenalidomide was co-administered with a P-gp inhibitor (temsirolimus and intraconazole, respectively).  This clinical study assessed LEN-drug interactions via P-gp using two probe drugs under controlled conditions.  Quinidine, a P-gp inhibitor with high in vivo inhibition potential and proven effect on plasma exposure of the prototype P-gp substrate digoxin in humans, was used to maximize the likelihood of detecting drug-drug interactions via P-gp.  Temsirolimus, a P-gp inhibitor/substrate, was used to evaluate P-gp mediated interactions on either drug in comparison with the results reported in literature. Methods This was a phase 1, single-center, open-label, 2-part, fixed-sequence crossover study conducted in healthy men. Part 1 comprised of 2 treatment periods with LEN alone (25 mg single dose on Day 1) in period 1, followed by LEN (on Day 4) plus quinidine (300 mg twice daily [BID] on Day 1 and 600 mg BID on Days 2–5) in period 2.  Part 2 consisted of three treatment periods with LEN (25 mg single dose on Day 1) alone in period 1, temsirolimus (25 mg single dose intravenously [IV] on Day 1) alone in period 2, and LEN plus temsirolimus in period 3 (Day 1).  Treatment periods were separated by a washout of 7–10 days. Serial samples were collected to determine the plasma, whole blood or urine concentrations of LEN, quinidine, and temsirolimus (and its active metabolite, sirolimus [also a P-gp inhibitor/substrate]).  Safety was monitored throughout the study. Results A total of 31 healthy men, aged 22 to 62 years; were enrolled (14 in Part 1 and 17 in Part 2). Renal excretion of LEN was almost complete at 12 h post dose for all treatments (Figure 1). In the absence or presence of a P-gp inhibitor, the mean percentage LEN dose excreted in the urine (74% vs 70% in Part 1, respectively; 81% vs 80% in Part 2) and renal clearance (227 vs 245 mL/min in Part 1; 251 vs 229 mL/min in Part 2) were similar, demonstrating that the rate and capacity of LEN renal excretion were not reduced by P-gp inhibition. Both the median time (1 h) to reach the maximum concentration (Cmax) and the oral bioavailability (70–80% of the administrated dose, as indicated by the renal excretion of unchanged drug) of LEN, were comparable in the absence or presence of a P-gp inhibitor (0.5–1 h and 74–81% of the dose, respectively); therefore, the rate and extent of LEN oral absorption were also not altered by P-gp inhibition. Consequently, there was no significant change in the plasma exposure to LEN in the presence of a P-gp inhibitor (Figure 1). The 90% confidence intervals (CIs) for the ratio of geometric means between LEN alone and LEN plus a P-gp inhibitor were completely contained within the equivalence limits of 80–125% for Cmaxand area under the concentration-time curve (AUC) of LEN. In addition, LEN had no effect on blood exposure to temsirolimus and sirolimus, with the 90% CIs for the ratio of their geometric mean Cmax and AUC between temsirolimus alone and temsirolimus plus LEN between 80–125%. No significant safety findings were reported when LEN was given with quinidine or temsirolimus compared to LEN alone. Conclusions Co-administration of either the P-gp inhibitor quinidine or temsirolimus had no clinically relevant effect on the systemic exposure of LEN.  Similarly, co-administration of LEN had no clinically relevant effect on the systemic exposure of the P-gp substrates temsirolimus and sirolimus.  A single dose of LEN was well tolerated when co-administered with quinidine or temsirolimus in healthy men. Disclosures: Chen: Celgene Corporation: Employment, Equity Ownership. Weiss:Celegene Corporation: Employment, Equity Ownership. Reyes:Celgene Corporation: Employment, Equity Ownership. Liu:Celgene Corporation: Employment, Equity Ownership. Kasserra:Celgene: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Zhou:Celgene Corporation: Employment, Equity Ownership. Kumar:Celgene Corporation: Employment, Equity Ownership. Weiss:Celgene Corporation: Employment, Equity Ownership. Palmisano:Celgene Corporation: Employment, Equity Ownership.

Description: Pro-inflammatory IL-17-producing T cells termed Th17 are actively involved in the pathogenesis of GVHD. The development and function of Th17 cells is dependent on activation of STAT3, RORgt and IRF4 transcription factors. Aberrant activation of Rho-associated kinase 2 (ROCK2) leads to induction of IL-17 and IL-21 secretion via IRF4-dependent mechanism. KD025, is a potent and selective ROCK2 inhibitor, which when given to healthy human subjects down-regulated the ability of T cells to secrete IL-21 and IL-17, but not interferon (IFN)-g, in response to TCR stimulation in vitro (Figure 1). KD025 inhibits STAT3 phosphorylation which supports RORgt, Th17 generation, and IL-21 production. Concurrently, KD025 increases STAT5 phosphorylation and Treg suppressor function in a dose-responsive fashion. KD025 treatment therefore shifts Th17/Treg balance. Th17 cells have been linked to in vivo pro-inflammatory responses, antibody production, and fibrosis. Conversely, Tregs can offset these pathogenic responses. Given the profile of KD025, we tested the effects of this inhibitor on cGVHD pathogenesis in a multi-organ system rodent model of disease that is driven by IL-21 responses and is associated with lung, liver and intestinal fibrosis. We observed that Th17/Rorc deficient T cells are unable to mediate cGVHD pathogenesis. In mice with established cGVHD, therapy was initiated with 30, 100, or 150 mg/kg/dose of KD025 daily from d28-56. Treated mice had a dose dependent decrease in the development of pathogenic pulmonary function as determined by whole body plethysmography (Figure 2) which correlated with a marked reduction of antibody deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. KD025 administration also resulted in a 2-fold decrease in collagen deposition in the lungs of mice treated with the highest dose of KD025. The spleens of mice treated with 150 mg/kg dose of KD025 had a decrease in the frequency of germinal centers compared to the vehicle treated mice. To determine the selective role of STAT3 on T cells, mice were transplanted with wildtype (WT) bone marrow (BM) and WT or inducible STAT3 deficient T cells. In parallel cohorts, the role of STAT3 in BM-derived B cells, precursors of germinal center B cells, was examined using WT vs inducible STAT3 deficient BM cells + WT T cells. We demonstrate here that mice transplanted with inducible STAT3 deficient T cells or BM cells had pulmonary function comparable to the healthy negative controls, suggesting that STAT3 is a potential therapeutic target in both T and B cells is necessary for the development of cGVHD and providing mechanistic insight into how KD025 may ameliorate active cGVHD. Studies are in progress to test KD025 administration in a murine scleroderma model using a minor histocompatibility antigen disparate donor-recipient strain that we have shown to be dependent upon STAT3 expressing donor T cells and a STAT3 inhibitor in both cGVHD models described here. Together, these data demonstrate that KD025 is effective at decreasing STAT3-dependent production of IL-21 and IL-17 and the use of KD025 is a potentially novel therapeutic intervention for the treatment of cGVHD. Fig 1 Oral administration of KD025 down-regulates the IL-17 and IL-21 secretion in human PBMCs upon stimulation ex vivo. Human PBMCs were purified from healthy human subjects before and after oral administration of KD025 at doses 40, 120, 240, 320 and stimulated ex vivo. Cytokine secretion was determined after 48 hours by ELISA. Fig 1. Oral administration of KD025 down-regulates the IL-17 and IL-21 secretion in human PBMCs upon stimulation ex vivo. Human PBMCs were purified from healthy human subjects before and after oral administration of KD025 at doses 40, 120, 240, 320 and stimulated ex vivo. Cytokine secretion was determined after 48 hours by ELISA. Fig 2 KD025 is an effective therapy for established murine cGVHD. Mice were given KD025 (150 mg/kg) d.28-56. PFTs indicate normal resistance, elastance and better compliance. Lung Ig deposition and fibrosis were comparable to BM controls. Fig 2. KD025 is an effective therapy for established murine cGVHD. Mice were given KD025 (150 mg/kg) d.28-56. PFTs indicate normal resistance, elastance and better compliance. Lung Ig deposition and fibrosis were comparable to BM controls. Disclosures No relevant conflicts of interest to declare.

Description: Background: A cost effective and comprehensive genomic profiling (CGP) approach for diagnosis, risk stratification and therapy would be useful for the evaluation of oncologic specimens. Available approaches involving additive testing for DNA and RNA abnormalities through traditional methods (e.g. Sanger, FISH, cytogenetics, qRT-PCR) are not comprehensive, require multiple different workflows and are sample consuming, often resulting in incomplete testing. While there are next generation sequencing (NGS) assays designed for detecting DNA and RNA abnormalities, they have separate workflows that require twice the amount of sample and effort. To address this, we developed a novel total nucleic acid (TNA) extraction method and single tube workflow utilizing TNA and a custom multimodal chemistry designed for hematologic malignancies. This consolidated workflow enables an efficient discovery based approach for both DNA/RNA abnormalities including single nucleotide variants (SNVs), InDels, copy number variants (CNVs), large structural changes from DNA and gene fusions and gene expression levels from RNA. This method maximizes data derived from valuable samples while delivering a comprehensive profile of the patient's tumor which can help guide therapeutic and clinical decisions. Methods: Total nucleic acid (TNA) was extracted from bone marrow and peripheral blood of 95 patients (CML, CMML, CLL, AML and myeloid disorders). 297 genes that have DNA mutations specific to hematological cancers were targeted, along with 213 genes that were targeted for clinically significant RNA abnormalities. Enriched genomic and transcriptomic regions of interest from 85 patients were successfully sequenced with unique dual indices on an Illumina NovaSeq 6000. DNA variant detection as well as fusion detection from RNA were compared to traditional orthogonal NGS assays that use DNA input or compared to qRT-PCR and Sanger sequencing assays that use RNA as input. Results: In this study, we developed an efficient and high-quality TNA extraction method that can purify enough total nucleic acid from bone marrow, peripheral blood, cytogenetic pellets, flow suspension, and FFPE samples for the downstream NGS assay. The average OD 260/280 value was 1.9 and the OD 260/230 was 2.18. After sequencing, 256/262 (97.7% accuracy) SNV and Indel variants that were candidate pathogenic mutations were concordant from 38 patients. Meanwhile, 100% (7/7) of all BCR/ABL1 gene fusions which had an international scale (IS) value above 6.4% were concordant. In addition, 69 fusion positive samples containing 20 unique gene fusions which had been previously reported by an independent ArcherDX assay designed specifically for gene fusions were also evaluated with this chemistry. Analysis revealed a 92.5% (64/69) concordance. More importantly, the QIAseq multimodal TNA NGS assay detected both DNA and RNA abnormalities in a single tube. For example, in one myeloid leukemia patient, we not only identified pathogenic variants of ASXL1 and JAK2 which had been previously detected by a DNA NGS assay, but also detected a concurrent BCR-FGFR1 fusion which had been previously reported by a FISH assay. Moreover, we were able to provide more comprehensive genomic profiling by investigating many DNA and RNA abnormalities simultaneously. In our study, for 5 patients that previously been tested for BCR-ABL1 fusion only, we are able to assess BCR-ABL1 fusion status from RNA as well as identify pathogenic DNA variants at the same time, including JAK2 p.V617F, U2AF1 p.S34F, ASXL1 p.E635Rfs*15, BRCA p.S1982Rfs*22, and DNMT3A p.S708Vfs*71, which provides valuable information to assist diagnosis and treatment in a cost effective and efficient way. Conclusions: We developed a single tube TNA based workflow with a custom multimodal chemistry that simultaneously detects many DNA and RNA abnormalities in a cost effective and efficient way while reducing sample requirements. This unique TNA NGS assay provides comprehensive genomic profiling for hematologic malignancies and improves the diagnostic testing options for precise patient care. Disclosures Yu: NeoGenomics: Current Employment. Alarcon:NeoGenomics: Current Employment. Mou:NeoGenomics: Current Employment. Jung:NeoGenomics: Current Employment. Nam:NeoGenomics: Current Employment. Thomas:NeoGenomics: Current Employment. Keeler:NeoGenomics: Current Employment. Shinbrot:NeoGenomics: Current Employment. Magnan:NeoGenomics: Current Employment. Bender:NeoGenomics: Current Employment. Jiang:NeoGenomics: Current Employment. Agersborg:NeoGenomics: Current Employment. Weiss:Bayer: Other: speaker; Genentech: Other: Speaker; Merck: Other: Speaker; NeoGenomics: Current Employment. Ye:NeoGenomics: Current Employment. Funari:NeoGenomics: Current Employment.

Description: Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.

Description: © The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nature Communications 8 (2017): 1870, doi:10.1038/s41467-017-01610-4.

Description: Peridotite carbonation represents a critical step within the long-term carbon cycle by sequestering volatile CO2 in solid carbonate. This has been proposed as one potential pathway to mitigate the effects of greenhouse gas release. Most of our current understanding of reaction mechanisms is based on hand specimen and laboratory-scale analyses. Linking laboratory-scale observations to field scale processes remains challenging. Here we present the first geophysical characterization of serpentinite carbonation across scales ranging from km to sub-mm by combining aeromagnetic observations, outcrop- and thin section-scale magnetic mapping. At all scales, magnetic anomalies coherently change across reaction fronts separating assemblages indicative of incipient, intermittent, and final reaction progress. The abundance of magnetic minerals correlates with reaction progress, causing amplitude and wavelength variations in associated magnetic anomalies. This correlation represents a foundation for characterizing the extent and degree of in situ ultramafic rock carbonation in space and time.

Description: This project was supported by the Woods Hole Oceanographic Institution Independent Study Award (Tivey and Tominaga) and by NASA Astrobiology Institute NNA15BB02A (Tominaga). M.T. and A.B. are grateful to B. Jamtveit and H. Austrheim (University of Oslo) for their support during the 2011 and 2013 field campaigns. B.W. and E.A.L. thank the National Science Foundation grant DMS-1521765 and Thomas F. Peterson, Jr for generous support.