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Freeman, Sam

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Freeman

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Sam

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Freeman, Sam
Author's Publications: search.filters.isAuthorOfPublication.d38ac43a-05a4-4682-b0a2-cb12bc5a78db

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    Metagenomic Characterization of Microbial Communities In Situ Within the Deeper Layers of the Ileum in Crohn’s Disease
    (Elsevier, 2016) Pedamallu, Chandra Sekhar; Bhatt, Ami S.; Bullman, Susan; Fowler, Sharyle; Freeman, Sam; Durand, Jacqueline; Jung, Joonil; Duke, Fujiko; Manzo, Veronica; Cai, Diana; Ananthakrishnan, Ashwin; Ojesina, Akinyemi I.; Ramachandran, Aruna; Gevers, Dirk; Xavier, Ramnik; Bhan, Atul; Meyerson, Matthew; Yajnik, Vijay
    Background & Aims Microbial dysbiosis and aberrant host–microbe interactions in the gut are believed to contribute to the development and progression of Crohn’s disease (CD). Microbiome studies in CD typically have focused on microbiota in feces or superficial mucosal layers of the colon because accessing DNA from deeper layers of the bowel is challenging. In this study, we analyzed the deep tissue microbiome in patients who underwent surgical resection of the small intestine. Methods: Paraffin blocks were obtained from 12 CD patients undergoing ileocecal resection, and healthy ileum samples (inflammatory bowel disease–free controls) were obtained from 12 patients undergoing surgery for right-sided colon cancer. Diseased and healthy-appearing ileum was identified using microscopy, and paraffin blocks were macrodissected using a core needle to specifically isolate DNA. Illumina Whole Genome Sequencing was used for microbial sequence identification and subsequent taxonomic classification using the PathSeq tool. Results: We observed significant differences between the microbiome of CD samples vs inflammatory bowel disease–free controls, including depletion of Bacteroidetes and Clostridia. Notably, microbial composition at the phyla level did not differ markedly between healthy and diseased areas of CD patients. However, we observed enrichment of potentially pathogenic organisms at the species level. Conclusions: Our study showed dysbiosis within deeper layers of the ileum of CD patients, specifically enrichment of enterotoxigenic Staphylococcus aureus and an environmental Mycobacterium species not described previously. Future studies with larger cohort sizes are warranted to confirm these findings. Studies would benefit from effective microbial DNA extraction methods from paraffin sections and host nucleic acid depletion approaches to increase microbial read coverage.
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    Sequence-Based Discovery of Bradyrhizobium enterica in Cord Colitis Syndrome
    (New England Journal of Medicine (NEJM/MMS), 2013) Bhatt, Ami; Freeman, Sam; Herrera, Alex Francisco; Pedamallu, Chandra Sekhar; Gevers, Dirk; Duke, Fujiko; Jung, Joonil; Michaud, Monia; Walker, Bruce; Young, Sarah; Earl, Ashlee M.; Kostic, Aleksander D.; Ojesina, Akinyemi Ifedapo; Hasserjian, Robert; Ballen, Karen Kuhn; Chen, Yi-Bin; Hobbs, Gabriela; Antin, Joseph; Soiffer, Robert; Baden, Lindsey; Garrett, Wendy; Hornick, Jason; Marty, Francisco; Meyerson, Matthew
    BACKGROUND—Immunosuppression is associated with a variety of idiopathic clinical syndromes that may have infectious causes. It has been hypothesized that the cord colitis syndrome, a complication of umbilical-cord hematopoietic stem-cell transplantation, is infectious in origin. METHODS—We performed shotgun DNA sequencing on four archived, paraffin-embedded endoscopic colon-biopsy specimens obtained from two patients with cord colitis. Computational subtraction of human and known microbial sequences and assembly of residual sequences into a bacterial draft genome were performed. We used polymerase-chain-reaction (PCR) assays and fluorescence in situ hybridization to determine whether the corresponding bacterium was present in additional patients and controls. RESULTS—DNA sequencing of the biopsy specimens revealed more than 2.5 million sequencing reads that did not match known organisms. These sequences were computationally assembled into a 7.65-Mb draft genome showing a high degree of homology with genomes of bacteria in the bradyrhizobium genus. The corresponding newly discovered bacterium was provisionally named Bradyrhizobium enterica. PCR identified B. enterica nucleotide sequences in biopsy specimens from all three additional patients with cord colitis whose samples were tested, whereas B. enterica sequences were absent in samples obtained from healthy controls and patients with colon cancer or graft-versus-host disease. CONCLUSIONS—We assembled a novel bacterial draft genome from the direct sequencing of tissue specimens from patients with cord colitis. Association of these sequences with cord colitis suggests that B. enterica may be an opportunistic human pathogen.
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    The Mutational Landscape of Circulating Tumor Cells in Multiple Myeloma
    (2017) Mishima, Yuji; Paiva, Bruno; Shi, Jiantao; Park, Jihye; Manier, Salomon; Takagi, Satoshi; Massoud, Mira; Perilla-Glen, Adriana; Aljawai, Yosra; Huynh, Daisy; Roccaro, Aldo M.; Sacco, Antonio; Capelletti, Marzia; Detappe, Alexandre; Alignani, Diego; Anderson, Kenneth; Munshi, Nikhil; Prosper, Felipe; Lohr, Jens; Ha, Gavin; Freeman, Sam; Van Allen, Eliezer; Adalsteinsson, Viktor A.; Michor, Franziska; San Miguel, Jesus F.; Ghobrial, Irene
    Summary The development of sensitive and non-invasive “liquid biopsies” presents new opportunities for longitudinal monitoring of tumor dissemination and clonal evolution. The number of circulating tumor cells (CTCs) is prognostic in multiple myeloma (MM), but there is little information on their genetic features. Here, we have analyzed the genomic landscape of CTCs from 29 MM patients, including eight cases with matched/paired bone marrow (BM) tumor cells. Our results show that 100% of clonal mutations in patient BM were detected in CTCs and that 99% of clonal mutations in CTCs were present in BM MM. These include typical driver mutations in MM such as in KRAS, NRAS, or BRAF. These data suggest that BM and CTC samples have similar clonal structures, as discordances between the two were restricted to subclonal mutations. Accordingly, our results pave the way for potentially less invasive mutation screening of MM patients through characterization of CTCs.
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    Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors
    (Nature Publishing Group UK, 2017) Adalsteinsson, Viktor A.; Ha, Gavin; Freeman, Sam; Choudhury, Atish D.; Stover, Daniel G.; Parsons, Heather; Gydush, Gregory; Reed, Sarah C.; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M.; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S.; Santiago, Rebecca A.; O’Connor, Edward P.; Jeong, Seong H.; Leeson, Rachel; Barry, Rachel M.; Kramkowski, Joseph F.; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M.; Marini, Lori; Waks, Adrienne; Harshman, Lauren C.; Tolaney, Sara M.; Van Allen, Eliezer; Winer, Eric P.; Lin, Nancy U.; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M.; Garraway, Levi; Golub, Todd; Boehm, Jesse S.; Wagle, Nikhil; Getz, Gad; Love, J. Christopher; Meyerson, Matthew
    Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.