Stephen D. Mastrian

Niche adaptation and genome expansion in the chlorophyll d-producing cyanobacterium Acaryochloris marina

Acaryochloris marina is a unique cyanobacterium that is able to produce chlorophyll d as its primary photosynthetic pigment and thus efficiently use far-red light for photosynthesis. Acaryochloris species have been isolated from marine environments in association with other oxygenic phototrophs, which may have driven the niche-filling introduction of chlorophyll d. To investigate these unique adaptations, we have sequenced the complete genome of A. marina. The DNA content of A. marina is composed of 8.3 million base pairs, which is among the largest bacterial genomes sequenced thus far. This large array of genomic data is distributed into nine single-copy plasmids that code for >25% of the putative ORFs. Heavy duplication of genes related to DNA repair and recombination (primarily recA) and transposable elements could account for genetic mobility and genome expansion. We discuss points of interest for the biosynthesis of the unusual pigments chlorophyll d and α-carotene and genes responsible for previously studied phycobilin aggregates. Our analysis also reveals that A. marina carries a unique complement of genes for these phycobiliproteins in relation to those coding for antenna proteins related to those in Prochlorococcus species. The global replacement of major photosynthetic pigments appears to have incurred only minimal specializations in reaction center proteins to accommodate these alternate pigments. These features clearly show that the genus Acaryochloris is a fitting candidate for understanding genome expansion, gene acquisition, ecological adaptation, and photosystem modification in the cyanobacteria.

The Complete Genome Sequence of Roseobacter denitrificans Reveals a Mixotrophic Rather than Photosynthetic Metabolism

Purple aerobic anoxygenic phototrophs (AAPs) are the only organisms known to capture light energy to enhance growth only in the presence of oxygen but do not produce oxygen. The highly adaptive AAPs compose more than 10% of the microbial community in some euphotic upper ocean waters and are potentially major contributors to the fixation of the greenhouse gas CO2. We present the complete genomic sequence and feature analysis of the AAP Roseobacter denitrificans, which reveal clues to its physiology. The genome lacks genes that code for known photosynthetic carbon fixation pathways, and most notably missing are genes for the Calvin cycle enzymes ribulose bisphosphate carboxylase (RuBisCO) and phosphoribulokinase. Phylogenetic evidence implies that this absence could be due to a gene loss from a RuBisCO-containing alpha-proteobacterial ancestor. We describe the potential importance of mixotrophic rather than autotrophic CO2 fixation pathways in these organisms and suggest that these pathways function to fix CO2 for the formation of cellular components but do not permit autotrophic growth. While some genes that code for the redox-dependent regulation of photosynthetic machinery are present, many light sensors and transcriptional regulatory motifs found in purple photosynthetic bacteria are absent.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus.

Despite the fact that heliobacteria are the only phototrophic representatives of the bacterial phylum Firmicutes, genomic analyses of these organisms have yet to be reported. Here we describe the complete sequence and analysis of the genome of Heliobacterium modesticaldum, a thermophilic species belonging to this unique group of phototrophs. The genome is a single 3.1-Mb circular chromosome containing 3,138 open reading frames. As suspected from physiological studies of heliobacteria that have failed to show photoautotrophic growth, genes encoding enzymes for known autotrophic pathways in other phototrophic organisms, including ribulose bisphosphate carboxylase (Calvin cycle), citrate lyase (reverse citric acid cycle), and malyl coenzyme A lyase (3-hydroxypropionate pathway), are not present in the H. modesticaldum genome. Thus, heliobacteria appear to be the only known anaerobic anoxygenic phototrophs that are not capable of autotrophy. Although for some cellular activities, such as nitrogen fixation, there is a full complement of genes in H. modesticaldum, other processes, including carbon metabolism and endosporulation, are more genetically streamlined than they are in most other low-G+C gram-positive bacteria. Moreover, several genes encoding photosynthetic functions in phototrophic purple bacteria are not present in the heliobacteria. In contrast to the nutritional flexibility of many anoxygenic phototrophs, the complete genome sequence of H. modesticaldum reveals an organism with a notable degree of metabolic specialization and genomic reduction.

A North American Yersinia pestis Draft Genome Sequence: SNPs and Phylogenetic Analysis

Background Yersinia pestis, the causative agent of plague, is responsible for some of the greatest epidemic scourges of mankind. It is widespread in the western United States, although it has only been present there for just over 100 years. As a result, there has been very little time for diversity to accumulate in this region. Much of the diversity that has been detected among North American isolates is at loci that mutate too quickly to accurately reconstruct large-scale phylogenetic patterns. Slowly-evolving but stable markers such as SNPs could be useful for this purpose, but are difficult to identify due to the monomorphic nature of North American isolates. Methodology/Principal Findings To identify SNPs that are polymorphic among North American populations of Y. pestis, a gapped genome sequence of Y. pestis strain FV-1 was generated. Sequence comparison of FV-1 with another North American strain, CO92, identified 19 new SNP loci that differ among North American isolates. Conclusions/Significance The 19 SNP loci identified in this study should facilitate additional studies of the genetic population structure of Y. pestis across North America.

Metabolic flexibility revealed in the genome of the cyst-forming α-1 proteobacterium Rhodospirillum centenum

BackgroundRhodospirillum centenum is a photosynthetic non-sulfur purple bacterium that favors growth in an anoxygenic, photosynthetic N2-fixing environment. It is emerging as a genetically amenable model organism for molecular genetic analysis of cyst formation, photosynthesis, phototaxis, and cellular development. Here, we present an analysis of the genome of this bacterium.ResultsR. centenum contains a singular circular chromosome of 4,355,548 base pairs in size harboring 4,105 genes. It has an intact Calvin cycle with two forms of Rubisco, as well as a gene encoding phosphoenolpyruvate carboxylase (PEPC) for mixotrophic CO2 fixation. This dual carbon-fixation system may be required for regulating internal carbon flux to facilitate bacterial nitrogen assimilation. Enzymatic reactions associated with arsenate and mercuric detoxification are rare or unique compared to other purple bacteria. Among numerous newly identified signal transduction proteins, of particular interest is a putative bacteriophytochrome that is phylogenetically distinct from a previously characterized R. centenum phytochrome, Ppr. Genes encoding proteins involved in chemotaxis as well as a sophisticated dual flagellar system have also been mapped.ConclusionsRemarkable metabolic versatility and a superior capability for photoautotrophic carbon assimilation is evident in R. centenum.

Clinical Implementation of Integrated Genomic Profiling in Patients with Advanced Cancers

DNA focused panel sequencing has been rapidly adopted to assess therapeutic targets in advanced/refractory cancer. Integrated Genomic Profiling (IGP) utilising DNA/RNA with tumour/normal comparisons in a Clinical Laboratory Improvement Amendments (CLIA) compliant setting enables a single assay to provide: therapeutic target prioritisation, novel target discovery/application and comprehensive germline assessment. A prospective study in 35 advanced/refractory cancer patients was conducted using CLIA-compliant IGP. Feasibility was assessed by estimating time to results (TTR), prioritising/assigning putative therapeutic targets, assessing drug access, ascertaining germline alterations, and assessing patient preferences/perspectives on data use/reporting. Therapeutic targets were identified using biointelligence/pathway analyses and interpreted by a Genomic Tumour Board. Seventy-five percent of cases harboured 1–3 therapeutically targetable mutations/case (median 79 mutations of potential functional significance/case). Median time to CLIA-validated results was 116 days with CLIA-validation of targets achieved in 21/22 patients. IGP directed treatment was instituted in 13 patients utilising on/off label FDA approved drugs (n = 9), clinical trials (n = 3) and single patient IND (n = 1). Preliminary clinical efficacy was noted in five patients (two partial response, three stable disease). Although barriers to broader application exist, including the need for wider availability of therapies, IGP in a CLIA-framework is feasible and valuable in selection/prioritisation of anti-cancer therapeutic targets.

Comprehensive Genomic Analysis of Metastatic Mucinous Urethral Adenocarcinoma Guides Precision Oncology Treatment: Targetable EGFR Amplification Leading to Successful Treatment with Erlotinib

Alan H. Bryce1,3,4, Mitesh J. Borad1,3,4, Jan B. Egan4, Rachel M. Condjella3, Winnie S. Liang6, Rafael Fonseca1,3,4, Ann E. McCullough7, Katherine S. Hunt1, Nicole R. Ritacca3, Michael T. Barrett3,6, Maitray D. Patel8, Scott W. Young8, Alvin C. Silva8, Thai H. Ho1,3,4, Thorvardur R. Halfdanarson1,3,4, Melissa L. Stanton7, John Cheville5, Scott Swanson2, Daniel E. Schneider2, Robert R. McWilliams4,9, Angela Baker6, Jessica Aldrich6, Ahmet Kurdoglu6, Tyler Izatt6, Alexis Christoforides6, Irene Cherni6, Sara Nasser6, Rebecca Reiman6, Lori Cuyugan6, Jacquelyn McDonald6, Jonathan Adkins6, Stephen D. Mastrian6, Daniel D. Von Hoff6, David W. Craig6, A. Keith Stewart1,3,4, John D. Carpten6 1Division of Hematology/Oncology

Abstract 4694: Indices of actionability and clinical utility in a CLIA-enabled study of whole genome/exome/RNA sequencing in 33 cancer patients: Actionable vs. utility

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Whole genome/exome/RNA sequencing has revolutionized the ability to assess the genomic landscape of cancer and is increasingly being utilized for clinical decision-making. Initial clinical applications have been constrained by specimen quantity, analyte quality and the time from sample acquisition to results report. Methods: Patients with advanced cancers underwent surgical resection, excisional/core biopsies, or bone marrow biopsy. Samples were analyzed by whole genome or exome sequencing in addition to RNA sequencing, bioinformatics analysis, and therapeutic target prioritization by a multi-disciplinary Clinical Genomics Board. All prioritized targets were CLIA validated using Sanger sequencing, RT-qPCR, FISH, or IHC as appropriate. Treatment was delivered using off-label FDA approved drugs, clinical trials, or single patient INDs. Results: We have enrolled 40 patients for whom sequencing data is available on 33. The initial 6 patients were evaluated in a non-CLIA pilot phase and 27 in a CLIA-enabled phase. Tumor types in the CLIA-enabled phase with the highest enrollment were pancreatic cancer (n=8) and cholangiocarcinoma (n=8). We sought to quantify the targets identified along with clinical benefit, defining these as the “Actionable Index” (AI) (proportion of patients with ≥ 1 putative drug target) and “Utility Index” (UI) (proportion of patients who derive clinical benefit). Putative therapeutic targets were identified in 7/8 (AI=0.88) cholangiocarcinoma (CC) patients and in 5/8 (AI=0.63) pancreatic cancer (PC) patients. All 3 CC patients who received target directed treatment achieved a partial response (UI=0.38). In contrast, none of the 4 PC patients who received target directed therapy had treatment response (UI=0.0). Interestingly no actionable targets were identified in 1 CC and in 2 PCs. One CC with an identified target was unable to access the drug and subsequently died. A CC patient and a PC patient, each with identified targets, expired prior to the initiation of therapy. Conclusions: While whole genome/exome/RNA sequencing is providing unparalleled detail of tumor genomes, the application to the clinic must be carefully considered. Actionability of targets will eventually need to be defined in close relation to eventual clinical utility and appropriate refinements to disease-gene-drug databases implemented. Preliminary observations in pancreatic cancer and cholangiocarcinoma demonstrate disparity in correlation between utility indices and actionable indices. Application of these tools in larger cohorts and types of tumors will need to be conducted to ascertain more precise estimates. Additional measures that are organ-site agnostic but pertain to specific targets (e.g. BRAF) will also need to be developed in order to facilitate more judicious application of sequencing in the clinical setting. Citation Format: Jan B. Egan, Alan H. Bryce, Mia D. Champion, Winnie S. Liang, Rafael Fonseca, Ann E. McCullough, Michael T. Barrett, Katherine Hunt, Rachel M. Condjella, Robert R. McWilliams, Stephen D. Mastrian, Janine LoBello, Daniel Von Hoff, David W. Craig, A. Keith Stewart, John D. Carpten, Mitesh J. Borad. Indices of actionability and clinical utility in a CLIA-enabled study of whole genome/exome/RNA sequencing in 33 cancer patients: Actionable vs. utility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4694. doi:10.1158/1538-7445.AM2014-4694

DNA Sequencing: An Outsourcing Guide

DNA sequencing, the process of determining the precise order of nucleotides in a DNA strand, is a fundamental and ubiquitous method in molecular biology. Examples of its many uses include characterization of unknown DNA, mutation detection, allele identification, and clone construct confirmation. Advances in sequencing technology, motivated by the successful International Human Genome Project, have resulted in unprecedented access to high‐quality and cost‐effective automated analysis. It is increasingly common for researchers to delegate routine DNA sequencing needs to dedicated laboratories or companies specializing in this technology. In this unit, we discuss key protocols for maximizing the success of DNA sequencing when working with such service providers and describe detailed troubleshooting advice for analyzing anomalous results.