Publications by authors named "Sushma Nagaraj"

14 Publications

  • Page 1 of 1

Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

Cell Rep 2021 Nov;37(8):110047

University of Toronto Musculoskeletal Oncology Unit, Sinai Health System; Department of Surgery, University of Toronto, Toronto, ON, Canada.

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.110047DOI Listing
November 2021

Impact of the methylation classifier and ancillary methods on CNS tumor diagnostics.

Neuro Oncol 2021 Sep 23. Epub 2021 Sep 23.

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Background: Accurate CNS tumor diagnosis can be challenging, and methylation profiling can serve as an adjunct to classify diagnostically difficult cases.

Methods: An integrated diagnostic approach was employed for a consecutive series of 1,258 surgical neuropathology samples obtained primarily in a consultation practice over 2-year period. DNA methylation profiling and classification using the DKFZ/Heidelberg CNS tumor classifier was performed, as well as unsupervised analyses of methylation data. Ancillary testing, where relevant, was performed.

Results: Among the received cases in consultation, a high confidence methylation classifier score (>0.84) was reached in 66.4% of cases. The classifier impacted the diagnosis in 46.5% of these high-confidence classifier score cases, including a substantially new diagnosis in 26.9% cases. Among the 289 cases received with only a descriptive diagnosis, methylation was able to resolve approximately half (144, 49.8%) with high-confidence scores. Additional methods were able to resolve diagnostic uncertainty in 41.6% of the low-score cases. Tumor purity was significantly associated with classifier score (p = 1.15e-11). Deconvolution demonstrated that suspected GBMs matching as control/inflammatory brain tissue could be resolved into GBM methylation profiles, which provided a proof-of-concept approach to resolve tumor classification in the setting of low tumor purity.

Conclusions: This work assesses the impact of a methylation classifier and additional methods in a consultative practice by defining the proportions with concordant vs. change in diagnosis in a set of diagnostically challenging CNS tumors. We address approaches to low-confidence scores and confounding issues of low tumor purity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/neuonc/noab227DOI Listing
September 2021

Comparative Analysis of Genome of Ehrlichia sp. HF, a Model Bacterium to Study Fatal Human Ehrlichiosis.

BMC Genomics 2021 Jan 6;22(1):11. Epub 2021 Jan 6.

Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH, 43210, USA.

Background: The genus Ehrlichia consists of tick-borne obligatory intracellular bacteria that can cause deadly diseases of medical and agricultural importance. Ehrlichia sp. HF, isolated from Ixodes ovatus ticks in Japan [also referred to as I. ovatus Ehrlichia (IOE) agent], causes acute fatal infection in laboratory mice that resembles acute fatal human monocytic ehrlichiosis caused by Ehrlichia chaffeensis. As there is no small laboratory animal model to study fatal human ehrlichiosis, Ehrlichia sp. HF provides a needed disease model. However, the inability to culture Ehrlichia sp. HF and the lack of genomic information have been a barrier to advance this animal model. In addition, Ehrlichia sp. HF has several designations in the literature as it lacks a taxonomically recognized name.

Results: We stably cultured Ehrlichia sp. HF in canine histiocytic leukemia DH82 cells from the HF strain-infected mice, and determined its complete genome sequence. Ehrlichia sp. HF has a single double-stranded circular chromosome of 1,148,904 bp, which encodes 866 proteins with a similar metabolic potential as E. chaffeensis. Ehrlichia sp. HF encodes homologs of all virulence factors identified in E. chaffeensis, including 23 paralogs of P28/OMP-1 family outer membrane proteins, type IV secretion system apparatus and effector proteins, two-component systems, ankyrin-repeat proteins, and tandem repeat proteins. Ehrlichia sp. HF is a novel species in the genus Ehrlichia, as demonstrated through whole genome comparisons with six representative Ehrlichia species, subspecies, and strains, using average nucleotide identity, digital DNA-DNA hybridization, and core genome alignment sequence identity.

Conclusions: The genome of Ehrlichia sp. HF encodes all known virulence factors found in E. chaffeensis, substantiating it as a model Ehrlichia species to study fatal human ehrlichiosis. Comparisons between Ehrlichia sp. HF and E. chaffeensis will enable identification of in vivo virulence factors that are related to host specificity, disease severity, and host inflammatory responses. We propose to name Ehrlichia sp. HF as Ehrlichia japonica sp. nov. (type strain HF), to denote the geographic region where this bacterium was initially isolated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-020-07309-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789307PMC
January 2021

High level MYCN amplification and distinct methylation signature define an aggressive subtype of spinal cord ependymoma.

Acta Neuropathol Commun 2020 07 8;8(1):101. Epub 2020 Jul 8.

Neuro-Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

We report a novel group of clinically aggressive spinal cord ependymomas characterized by Grade III histology, MYCN amplification, an absence of NF2 alterations or other recurrent pathogenic mutations, and a unique methylation classifier profile. Seven cases were found to have MYCN amplification in the course of routine mutational profiling of 552 patients with central nervous system tumors between December 2016 and July of 2019 and an eighth patient was identified from an unrelated set of cases. Methylation array analysis revealed that none of the 8 cases clustered with any of the nine previously described ependymoma methylation subgroups, and 7 of 8 formed their own tight unique cluster. Histologically all cases showed grade III features, and all demonstrated aggressive clinical behavior. These findings are presented in the context of data from three other studies describing similar cases. Therefore, a combined total of 27 MYCN amplified spinal cord ependymoma cases have now been reported in the literature, warranting their consideration as a distinctive subtype of spinal cord ependymoma (SP-EPN-MYCN) with their unique molecular characteristics and aggressive clinical behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40478-020-00973-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346356PMC
July 2020

Genome and Functional Characterization of Colonization Factor Antigen I- and CS6-Encoding Heat-Stable Enterotoxin-Only Enterotoxigenic Reveals Lineage and Geographic Variation.

mSystems 2019 Jan-Feb;4(1). Epub 2019 Jan 15.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of childhood diarrhea and is a leading cause of traveler's diarrhea. ETEC strains encoding the heat-stable enterotoxin (ST) are more often associated with childhood diarrhea than ETEC strains that encode only the heat-labile enterotoxin (LT). Colonization factors (CFs) also have a demonstrated role in ETEC virulence, and two of the most prevalent CFs among ETEC that have caused diarrhea are colonization factor antigen I (CFA/I) and CS6. In the current report, we describe the genomes of 269 CS6- or CFA/I-encoding ST-only ETEC isolates that were associated with human diarrhea. While the CS6 and CFA/I ETEC were identified in at least 13 different ETEC genomic lineages, a majority (85%; 229/269) were identified in only six lineages. Complete genome sequencing of selected isolates demonstrated that a conserved plasmid contributed to the dissemination of CFA/I whereas at least five distinct plasmids were involved in the dissemination of ST and/or CS6. Additionally, there were differences in gene content between CFA/I and CS6 ETEC at the phylogroup and lineage levels and in association with their geographic location of isolation as well as lineage-related differences in ST production. Thus, we demonstrate that genomically diverse E. coli strains have acquired ST, as well as CFA/I or CS6, via one or more plasmids and that, in some cases, isolates of a particular lineage or geographic location have undergone additional modifications to their genome content. These findings will aid investigations of virulence and the development of improved diagnostics and vaccines against this important human diarrheal pathogen. Comparative genomics and functional characterization were used to analyze a global collection of CFA/I and CS6 ST-only ETEC isolates associated with human diarrhea, demonstrating differences in the genomic content of CFA/I and CS6 isolates related to CF type, lineage, and geographic location of isolation and also lineage-related differences in ST production. Complete genome sequencing of selected CFA/I and CS6 isolates enabled descriptions of a highly conserved ST-positive (ST) CFA/I plasmid and of at least five diverse ST and/or CS6 plasmids among the CS6 ETEC isolates. There is currently no approved vaccine for ST-only ETEC, or for any ETEC for that matter, and as such, the current report provides functional verification of ST and CF production and antimicrobial susceptibility testing and an in-depth genomic characterization of a collection of isolates that could serve as representatives of CFA/I- or CS6-encoding ST-only ETEC strains for future studies of ETEC pathogenesis, vaccine studies, and/or clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mSystems.00329-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446980PMC
January 2019

Intratumor genetic heterogeneity in squamous cell carcinoma of the oral cavity.

Head Neck 2019 08 14;41(8):2514-2524. Epub 2019 Mar 14.

Department of Medicine, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland.

Background: We sought to evaluate intratumor heterogeneity in squamous cell carcinoma of the oral cavity (OCC) and specifically determine the effect of physical separation and histologic differentiation within the same tumor.

Methods: We performed whole exome sequencing on five biopsy sites-two from well-differentiated, two from poorly differentiated regions, and one from normal parenchyma-from five primary OCC specimens.

Results: We found high levels of intratumor heterogeneity and, in four primary tumors, identified only 0 to 2 identical mutations in all subsites. We found that the heterogeneity inversely correlated with physical separation and that pairs of well-differentiated samples were more similar to each other than analogous poorly differentiated specimens. Only TP53 mutations, but not other purported "driver mutations" in head and neck squamous cell carcinoma, were found in multiple biopsy sites.

Conclusion: These data highlight the challenges to characterization of the mutational landscape of OCC with single site biopsy and have implications for personalized medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hed.25719DOI Listing
August 2019

Diversity among bla-containing plasmids in Escherichia coli and other bacterial species isolated from the same patients.

Sci Rep 2018 07 6;8(1):10291. Epub 2018 Jul 6.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Carbapenem resistant Enterobacteriaceae are a significant public health concern, and genes encoding the Klebsiella pneumoniae carbapenemase (KPC) have contributed to the global spread of carbapenem resistance. In the current study, we used whole-genome sequencing to investigate the diversity of bla-containing plasmids and antimicrobial resistance mechanisms among 26 bla-containing Escherichia coli, and 13 bla-containing Enterobacter asburiae, Enterobacter hormaechei, K. pneumoniae, Klebsiella variicola, Klebsiella michiganensis, and Serratia marcescens strains, which were isolated from the same patients as the bla-containing E. coli. A bla-containing IncN and/or IncFII plasmid was identified in 77% (30/39) of the E. coli and other bacterial species analyzed. Complete genome sequencing and comparative analysis of a bla-containing IncN plasmid from one of the E. coli strains demonstrated that this plasmid is present in the K. pneumoniae and S. marcescens strains from this patient, and is conserved among 13 of the E. coli and other bacterial species analyzed. Interestingly, while both IncFII and IncN plasmids were prevalent among the strains analyzed, the IncN plasmids were more often identified in multiple bacterial species from the same patients, demonstrating a contribution of this IncN plasmid to the inter-genera dissemination of the bla genes between the E. coli and other bacterial species analyzed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-28085-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035167PMC
July 2018

Aligner optimization increases accuracy and decreases compute times in multi-species sequence data.

Microb Genom 2017 09 8;3(9):e000122. Epub 2017 Jul 8.

2Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA.

As sequencing technologies have evolved, the tools to analyze these sequences have made similar advances. However, for multi-species samples, we observed important and adverse differences in alignment specificity and computation time for bwa- mem (Burrows-Wheeler aligner-maximum exact matches) relative to bwa-aln. Therefore, we sought to optimize bwa-mem for alignment of data from multi-species samples in order to reduce alignment time and increase the specificity of alignments. In the multi-species cases examined, there was one majority member (i.e. or ) and one minority member (i.e. human or the endosymbiont Bm) of the sequence data. Increasing bwa-mem seed length from the default value reduced the number of read pairs from the majority sequence member that incorrectly aligned to the reference genome of the minority sequence member. Combining both source genomes into a single reference genome increased the specificity of mapping, while also reducing the central processing unit (CPU) time. In , at a seed length of 18 nt, 24.1 % of reads mapped to the human genome using 1.7±0.1 CPU hours, while 83.6 % of reads mapped to the genome using 0.2±0.0 CPU hours (total: 107.7 % reads mapping; in 1.9±0.1 CPU hours). In contrast, 97.1 % of the reads mapped to a combined human reference in only 0.7±0.0 CPU hours. Overall, the results suggest that combining all references into a single reference database and using a 23 nt seed length reduces the computational time, while maximizing specificity. Similar results were found for simulated sequence reads from a mock metagenomic data set. We found similar improvements to computation time in a publicly available human-only data set.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mgen.0.000122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643015PMC
September 2017

Characterization of a Large Antibiotic Resistance Plasmid Found in Enteropathogenic Escherichia coli Strain B171 and Its Relatedness to Plasmids of Diverse E. coli and Shigella Strains.

Antimicrob Agents Chemother 2017 09 24;61(9). Epub 2017 Aug 24.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA

Enteropathogenic (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline (), sulfonamides (), and mercury, as well as several virulence-associated genes, including the transcriptional regulator and the putative calcium sequestration inhibitor (). detection of the pB171_90 genes among 4,798 publicly available genome assemblies indicates that the unique genes of pB171_90 ( and ) are primarily restricted to genomes identified as EPEC or enterotoxigenic However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00995-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571317PMC
September 2017

Analysis of complete genome sequence and major surface antigens of Neorickettsia helminthoeca, causative agent of salmon poisoning disease.

Microb Biotechnol 2017 07 6;10(4):933-957. Epub 2017 Jun 6.

Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road, Columbus, OH, 43210, USA.

Neorickettsia helminthoeca, a type species of the genus Neorickettsia, is an endosymbiont of digenetic trematodes of veterinary importance. Upon ingestion of salmonid fish parasitized with infected trematodes, canids develop salmon poisoning disease (SPD), an acute febrile illness that is particularly severe and often fatal in dogs without adequate treatment. We determined and analysed the complete genome sequence of N. helminthoeca: a single small circular chromosome of 884 232 bp encoding 774 potential proteins. N. helminthoeca is unable to synthesize lipopolysaccharides and most amino acids, but is capable of synthesizing vitamins, cofactors, nucleotides and bacterioferritin. N. helminthoeca is, however, distinct from majority of the family Anaplasmataceae to which it belongs, as it encodes nearly all enzymes required for peptidoglycan biosynthesis, suggesting its structural hardiness and inflammatory potential. Using sera from dogs that were experimentally infected by feeding with parasitized fish or naturally infected in southern California, Western blot analysis revealed that among five predicted N. helminthoeca outer membrane proteins, P51 and strain-variable surface antigen were uniformly recognized. Our finding will help understanding pathogenesis, prevalence of N. helminthoeca infection among trematodes, canids and potentially other animals in nature to develop effective SPD diagnostic and preventive measures. Recent progresses in large-scale genome sequencing have been uncovering broad distribution of Neorickettsia spp., the comparative genomics will facilitate understanding of biology and the natural history of these elusive environmental bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1751-7915.12731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481527PMC
July 2017

Complete Genome Sequences of Strains Isolated from Rhesus Macaques.

Genome Announc 2017 Apr 20;5(16). Epub 2017 Apr 20.

Department of Pathology, and Department of Epidemiology and Public Health, Program of Comparative Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

is a nontuberculous mycobacterium. It causes opportunistic infections with pulmonary and extrapulmonary manifestations. We report here the complete genome sequences of two strains isolated from rhesus macaques. We performed genome comparisons with human and environmental isolates of to assess the genomic diversity of this species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/genomeA.00187-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399255PMC
April 2017

Use of Comparative Genomics To Characterize the Diversity of Acinetobacter baumannii Surveillance Isolates in a Health Care Institution.

Antimicrob Agents Chemother 2016 10 23;60(10):5933-41. Epub 2016 Sep 23.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA

Despite the increasing prevalence of the nosocomial pathogen Acinetobacter baumannii, little is known about which genomic components contribute to clinical presentation of this important pathogen. Most whole-genome comparisons of A. baumannii have focused on specific genomic regions associated with phenotypes in a limited number of genomes. In this work, we describe the results of a whole-genome comparative analysis of 254 surveillance isolates of Acinetobacter species, 203 of which were A. baumannii, isolated from perianal swabs and sputum samples collected as part of an infection control active surveillance program at the University of Maryland Medical Center. The collection of surveillance isolates includes both carbapenem-susceptible and -resistant isolates. Based on the whole-genome phylogeny, the A. baumannii isolates collected belong to two major phylogenomic lineages. Results from multilocus sequence typing indicated that one of the major phylogenetic groups of A. baumannii was comprised solely of strains from the international clonal lineage 2. The genomic content of the A. baumannii isolates was examined using large-scale BLAST score ratio analysis to identify genes that are associated with carbapenem-susceptible and -resistant isolates, as well as genes potentially associated with the source of isolation. This analysis revealed a number of genes that were exclusive or at greater frequency in each of these classifications. This study is the most comprehensive genomic comparison of Acinetobacter isolates from a surveillance study to date and provides important information that will contribute to our understanding of the success of A. baumannii as a human pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.00477-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038335PMC
October 2016

Concurrent Mutations in ATM and Genes Associated with Common γ Chain Signaling in Peripheral T Cell Lymphoma.

PLoS One 2015 4;10(11):e0141906. Epub 2015 Nov 4.

Program in Oncology, Greenebaum Cancer Center, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, United States of America.

Peripheral T cell lymphoma (PTCL) is a heterogeneous malignancy with poor response to current therapeutic strategies and incompletely characterized genetics. We conducted whole exome sequencing of matched PTCL and non-malignant samples from 12 patients, spanning 8 subtypes, to identify potential oncogenic mutations in PTCL. Analysis of the mutations identified using computational algorithms, CHASM, PolyPhen2, PROVEAN, and MutationAssessor to predict the impact of these mutations on protein function and PTCL tumorigenesis, revealed 104 somatic mutations that were selected as high impact by all four algorithms. Our analysis identified recurrent somatic missense or nonsense mutations in 70 genes, 9 of which contained mutations predicted significant by all 4 algorithms: ATM, RUNX1T1, WDR17, NTRK3, TP53, TRMT12, CACNA2D1, INTS8, and KCNH8. We observed somatic mutations in ATM (ataxia telangiectasia-mutated) in 5 out of the 12 samples and mutations in the common gamma chain (γc) signaling pathway (JAK3, IL2RG, STAT5B) in 3 samples, all of which also harbored mutations in ATM. Our findings contribute insights into the genetics of PTCL and suggest a relationship between γc signaling and ATM in T cell malignancy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141906PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633051PMC
June 2016

Draft Genome Sequences of Human Pathogenic Fungus Geomyces pannorum Sensu Lato and Bat White Nose Syndrome Pathogen Geomyces (Pseudogymnoascus) destructans.

Genome Announc 2013 Dec 19;1(6). Epub 2013 Dec 19.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA.

We report the draft genome sequences of Geomyces pannorum sensu lato and Geomyces (Pseudogymnoascus) destructans. G. pannorum has a larger proteome than G. destructans, containing more proteins with ascribed enzymatic functions. This dichotomy in the genomes of related psychrophilic fungi is a valuable target for defining their distinct saprobic and pathogenic attributes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/genomeA.01045-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868853PMC
December 2013
-->