James Hu

Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial

BACKGROUND Patients with advanced sarcomas have a poor prognosis and few treatment options that improve overall survival. Chemotherapy and targeted therapies offer short-lived disease control. We assessed pembrolizumab, an anti-PD-1 antibody, for safety and activity in patients with advanced soft-tissue sarcoma or bone sarcoma. METHODS In this two-cohort, single-arm, open-label, phase 2 study, we enrolled patients with soft-tissue sarcoma or bone sarcoma from 12 academic centres in the USA that were members of the Sarcoma Alliance for Research through Collaboration (SARC). Patients with soft-tissue sarcoma had to be aged 18 years or older to enrol; patients with bone sarcoma could enrol if they were aged 12 years or older. Patients had histological evidence of metastatic or surgically unresectable locally advanced sarcoma, had received up to three previous lines of systemic anticancer therapy, had at least one measurable lesion according to the Response Evaluation Criteria In Solid Tumors version 1.1, and had at least one lesion accessible for biopsy. All patients were treated with 200 mg intravenous pembrolizumab every 3 weeks. The primary endpoint was investigator-assessed objective response. Patients who received at least one dose of pembrolizumab were included in the safety analysis and patients who progressed or reached at least one scan assessment were included in the activity analysis. Accrual is ongoing in some disease cohorts. This trial is registered with ClinicalTrials.gov, number NCT02301039. FINDINGS Between March 13, 2015, and Feb 18, 2016, we enrolled 86 patients, 84 of whom received pembrolizumab (42 in each disease cohort) and 80 of whom were evaluable for response (40 in each disease cohort). Median follow-up was 17·8 months (IQR 12·3-19·3). Seven (18%) of 40 patients with soft-tissue sarcoma had an objective response, including four (40%) of ten patients with undifferentiated pleomorphic sarcoma, two (20%) of ten patients with liposarcoma, and one (10%) of ten patients with synovial sarcoma. No patients with leiomyosarcoma (n=10) had an objective response. Two (5%) of 40 patients with bone sarcoma had an objective response, including one (5%) of 22 patients with osteosarcoma and one (20%) of five patients with chondrosarcoma. None of the 13 patients with Ewings sarcoma had an objective response. The most frequent grade 3 or worse adverse events were anaemia (six [14%]), decreased lymphocyte count (five [12%]), prolonged activated partial thromboplastin time (four [10%]), and decreased platelet count (three [7%]) in the bone sarcoma group, and anaemia, decreased lymphocyte count, and prolonged activated partial thromboplastin time in the soft-tissue sarcoma group (three [7%] each). Nine (11%) patients (five [12%] in the bone sarcoma group and four [10%] in the soft-tissue sarcoma group) had treatment-emergent serious adverse events (SAEs), five of whom had immune-related SAEs, including two with adrenal insufficiency, two with pneumonitis, and one with nephritis. INTERPRETATION The primary endpoint of overall response was not met for either cohort. However, pembrolizumab showed encouraging activity in patients with undifferentiated pleomorphic sarcoma or dedifferentiated liposarcoma. Enrolment to expanded cohorts of those subtypes is ongoing to confirm and characterise the activity of pembrolizumab. FUNDING Merck, SARC, Sarcoma Foundation of America, QuadW Foundation, Pittsburgh Cure Sarcoma, and Ewan McGregor.

Proteome analysis of Escherichia coli K-12 by two-dimensional native-state chromatography and MALDI-MS

To identify proteins expressed in Escherichia coli K‐12 MG1655 during exponential growth in defined medium, we separated soluble proteins of E. coli over two dimensions of native‐state high‐performance liquid chromatography, and examined the components of the protein mixtures in each of 380 fractions by peptide mass fingerprinting. To date, we have identified the products of 310 genes covering a wide range of cellular functions. Validation of protein assignments was made by comparing the assignments of proteins to specific first‐dimension fractions to proteins visualized by two‐dimensional gel electrophoresis. Co‐fractionation of proteins suggests the possible identities of components of multiprotein complexes. This approach yields high‐throughput gel‐independent identification of proteins. It can also be used to assign identities to spots visualized by two‐dimensional gels, and should be useful to evaluate differences in expressed proteome content and protein complexes among strains or between different physiological states.

EcoliWiki: a wiki-based community resource for Escherichia coli

EcoliWiki is the community annotation component of the PortEco (http://porteco.org; formerly EcoliHub) project, an online data resource that integrates information on laboratory strains of Escherichia coli, its phages, plasmids and mobile genetic elements. As one of the early adopters of the wiki approach to model organism databases, EcoliWiki was designed to not only facilitate community-driven sharing of biological knowledge about E. coli as a model organism, but also to be interoperable with other data resources. EcoliWiki content currently covers genes from five laboratory E. coli strains, 21 bacteriophage genomes, F plasmid and eight transposons. EcoliWiki integrates the Mediawiki wiki platform with other open-source software tools and in-house software development to extend how wikis can be used for model organism databases. EcoliWiki can be accessed online at http://ecoliwiki.net.

Resistance to Methotrexate Due to AcrAB-Dependent Export from Escherichia coli

ABSTRACT Many laboratory strains of Escherichia coli are resistant to methotrexate (MTX), a folate analogue that binds dihydrofolate reductase (DHFR). Mutations that inactivate eithertolC or acrA confer MTX sensitivity. Further, overexpression of a fusion protein with DHFR activity reverses this sensitivity by titrating out intracellular MTX. These results suggest that MTX accumulates in cells where mutations inacrA or tolC have inactivated the TolC-dependent AcrAB multidrug resistance efflux pump.

PortEco: a resource for exploring bacterial biology through high-throughput data and analysis tools

PortEco (http://porteco.org) aims to collect, curate and provide data and analysis tools to support basic biological research in Escherichia coli (and eventually other bacterial systems). PortEco is implemented as a ‘virtual’ model organism database that provides a single unified interface to the user, while integrating information from a variety of sources. The main focus of PortEco is to enable broad use of the growing number of high-throughput experiments available for E. coli, and to leverage community annotation through the EcoliWiki and GONUTS systems. Currently, PortEco includes curated data from hundreds of genome-wide RNA expression studies, from high-throughput phenotyping of single-gene knockouts under hundreds of annotated conditions, from chromatin immunoprecipitation experiments for tens of different DNA-binding factors and from ribosome profiling experiments that yield insights into protein expression. Conditions have been annotated with a consistent vocabulary, and data have been consistently normalized to enable users to find, compare and interpret relevant experiments. PortEco includes tools for data analysis, including clustering, enrichment analysis and exploration via genome browsers. PortEco search and data analysis tools are extensively linked to the curated gene, metabolic pathway and regulation content at its sister site, EcoCyc.

GONUTS: the Gene Ontology Normal Usage Tracking System

The Gene Ontology Normal Usage Tracking System (GONUTS) is a community-based browser and usage guide for Gene Ontology (GO) terms and a community system for general GO annotation of proteins. GONUTS uses wiki technology to allow registered users to share and edit notes on the use of each term in GO, and to contribute annotations for specific genes of interest. By providing a site for generation of third-party documentation at the granularity of individual terms, GONUTS complements the official documentation of the Gene Ontology Consortium. To provide examples for community users, GONUTS displays the complete GO annotations from seven model organisms: Saccharomyces cerevisiae, Dictyostelium discoideum, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus and Arabidopsis thaliana. To support community annotation, GONUTS allows automated creation of gene pages for gene products in UniProt. GONUTS will improve the consistency of annotation efforts across genome projects, and should be useful in training new annotators and consumers in the production of GO annotations and the use of GO terms. GONUTS can be accessed at http://gowiki.tamu.edu. The source code for generating the content of GONUTS is available upon request.

A randomised phase II trial of selumetinib vs selumetinib plus temsirolimus for soft-tissue sarcomas.

Background:The MEK inhibitor, selumetinib, suppresses soft-tissue sarcoma (STS) cell proliferation in vitro. Mammalian target of rapamycin inhibitors possess modest activity against STS; however, resistance develops via MAPK pathway feedback activation. The combination of selumetinib and temsirolimus synergistically inhibits STS cell line growth. Therefore, a randomized phase II trial of selumetinib vs selumetinib plus temsirolimus was conducted.Methods:Seventy-one adults with advanced STS who received ⩽2 prior chemotherapeutics were randomized to selumetinib 75 mg p.o. bid and allowed to crossover upon progression, or to selumetinib 50 mg p.o. bid plus temsirolimus 20 mg i.v. weekly, with primary endpoint of progression-free survival (PFS).Results:There was no difference in PFS between the two arms for the overall cohort (median 1.9 vs 2.1 months); an improved median PFS was observed in the combination arm (N=11) over single agent (N=10) in the prespecified leiomyosarcoma stratum (median 3.7 vs 1.8 months; P=0.01). Four-month PFS rate was 50% (95% confidence interval 0.19–0.81) with the combination vs 0% with selumetinib alone in the leiomyosarcoma cohort. Most common grade 3/4 adverse events with the combination were mucositis (29%), lymphopenia (26%), neutropenia and anaemia (20% each).Conclusions:While single-agent selumetinib has no significant activity in STS, the combination may be active for leiomyosarcomas.

Paclitaxel, Ifosfamide, and Cisplatin Efficacy for First-Line Treatment of Patients With Intermediate- or Poor-Risk Germ Cell Tumors

PURPOSE Paclitaxel, ifosfamide, and cisplatin (TIP) achieved complete responses (CRs) in two thirds of patients with advanced germ cell tumors (GCTs) who relapsed after first-line chemotherapy with cisplatin and etoposide with or without bleomycin. We tested the efficacy of first-line TIP in patients with intermediate- or poor-risk disease. PATIENTS AND METHODS In this prospective, multicenter, single-arm phase II trial, previously untreated patients with International Germ Cell Cancer Collaborative Group poor-risk or modified intermediate-risk GCTs received four cycles of TIP (paclitaxel 240 mg/m(2) over 2 days, ifosfamide 6 g/m(2) over 5 days with mesna support, and cisplatin 100 mg/m(2) over 5 days) once every 3 weeks with granulocyte colony-stimulating factor support. The primary end point was the CR rate. RESULTS Of the first 41 evaluable patients, 28 (68%) achieved a CR, meeting the primary efficacy end point. After additional accrual on an extension phase, total enrollment was 60 patients, including 40 (67%) with poor risk and 20 (33%) with intermediate risk. Thirty-eight (68%) of 56 evaluable patients achieved a CR and seven (13%) achieved partial responses with negative markers (PR-negative) for a favorable response rate of 80%. Five of seven achieving PR-negative status had seminoma and therefore did not undergo postchemotherapy resection of residual masses. Estimated 3-year progression-free survival and overall survival rates were 72% (poor risk, 63%; intermediate risk, 90%) and 91% (poor risk, 87%; intermediate risk, 100%), respectively. Grade 3 to 4 toxicities consisted primarily of reversible hematologic or electrolyte abnormalities, including neutropenic fever in 18%. CONCLUSION TIP demonstrated efficacy as first-line therapy for intermediate- and poor-risk GCTs with an acceptable safety profile. Given higher rates of favorable response, progression-free survival, and overall survival compared with prior first-line studies, TIP warrants further study in this population.

An ontology for microbial phenotypes

BackgroundPhenotypic data are routinely used to elucidate gene function in organisms amenable to genetic manipulation. However, previous to this work, there was no generalizable system in place for the structured storage and retrieval of phenotypic information for bacteria.ResultsThe Ontology of Microbial Phenotypes (OMP) has been created to standardize the capture of such phenotypic information from microbes. OMP has been built on the foundations of the Basic Formal Ontology and the Phenotype and Trait Ontology. Terms have logical definitions that can facilitate computational searching of phenotypes and their associated genes. OMP can be accessed via a wiki page as well as downloaded from SourceForge. Initial annotations with OMP are being made for Escherichia coli using a wiki-based annotation capture system. New OMP terms are being concurrently developed as annotation proceeds.ConclusionsWe anticipate that diverse groups studying microbial genetics and associated phenotypes will employ OMP for standardizing microbial phenotype annotation, much as the Gene Ontology has standardized gene product annotation. The resulting OMP resource and associated annotations will facilitate prediction of phenotypes for unknown genes and result in new experimental characterization of phenotypes and functions.

[18] Molecular applications of fusions to leucine zippers

Publisher Summary The use of chimeric proteins to study protein function dates back as far as the use of illegitimate recombination events among lambdoid phage to generate interspecies variants containing different amounts of polypeptide derived from each parent. Recombinant deoxyribonucleic acid (DNA) methods made the use of fusion proteins more easily generalizable and allowed fundamentally important insights to be derived for many biological systems. For example, understanding of transcriptional activation was fundamentally altered by the experiments of Brent and Ptashne, who showed that targeting of an activation domain to a particular chromosomal location through a fused bacterial LexA DNA-binding protein was sufficient to activate transcription. There are now many ways to use fused motifs to hold proteins together. This chapter briefly discusses the use of natural and mutant leucine zippers as molecular damps.