Identification of a 5-gene signature panel for the prediction of prostate cancer progression.

BACKGROUND: Despite nearly 100% 5-year survival for localised prostate cancer, the survival rate for metastatic prostate cancer significantly declines to 32%. Thus, it is crucial to identify molecular indicators that reflect the progression from localised disease to metastatic prostate cancer. METHODS: To search for molecular indicators associated with prostate cancer metastasis, we performed proteomic analysis of rapid autopsy tissue samples from metastatic prostate cancer (N = 8) and localised prostate cancer (N = 2). Then, we utilised multiple independent, publicly available prostate cancer patient datasets to select candidates that also correlate with worse prostate cancer clinical prognosis. RESULTS: We identified 154 proteins with increased expressions in metastases relative to localised prostate cancer through proteomic analysis. From the subset of these candidates that correlate with prostate cancer recurrence (N = 28) and shorter disease-free survival (N = 37), we identified a 5-gene signature panel with improved performance in predicting worse clinical prognosis relative to individual candidates. CONCLUSIONS: Our study presents a new 5-gene signature panel that is associated with worse clinical prognosis and is elevated in prostate cancer metastasis on both protein and mRNA levels. Our 5-gene signature panel represents a potential modality for the prediction of prostate cancer progression towards the onset of metastasis.

Clonal Hematopoiesis Is Associated With Long-Term Adverse Outcomes Following Cardiac Surgery.

BACKGROUND: Cardiac surgery triggers sterile innate immune responses leading to postoperative complications. Clonal hematopoiesis (CH) is associated with short-term inflammation-mediated outcomes after cardiac surgery. The impact of CH on long-term postoperative outcomes remains unknown. METHODS AND RESULTS: In this cohort study, patients undergoing elective cardiac surgery were included from January 2017 to September 2019. Patients were screened for CH using a predefined gene panel of 19 genes. Recorded clinical events were all-cause death, major adverse cardiac and cerebral events including cardiovascular death, myocardial infarction or nonscheduled coronary revascularization, stroke, and hospitalization for acute heart failure. The primary study outcome was time to a composite criterion including all-cause mortality and major adverse cardiac and cerebral events. Among 314 genotyped patients (median age: 67 years; interquartile range 59-74 years), 139 (44%) presented with CH, based on a variant allelic frequency ≥1%. Carriers of CH had a higher proportion of patients with a history of atrial fibrillation (26% for CH versus 17% for non-CH carriers, P=0.022). The most frequently mutated genes were DNMT3A, TET2, and ASXL1. After a median follow-up of 1203 [813-1435] days, the primary outcome occurred in 50 patients. After multivariable adjustment, CH was independently associated with a higher risk for the primary outcome (hazard ratio, 1.88 [95% CI, 1.05-3.41], P=0.035). Most adverse events occurred in patients carrying TET2 variants. CONCLUSIONS: In patients undergoing cardiac surgery, CH is frequent and associated with a 2-fold increased long-term risk for major adverse clinical outcomes. CH is a novel risk factor for long-term postcardiac surgery complications and might be useful to personalize management decisions. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03376165.

Bacterial and fungal characterization of pancreatic adenocarcinoma from Endoscopic Ultrasound-guided biopsies.

Introduction: The tumor microbiome (TM) has been linked to pancreatic cancer prognosis. Specific microbes can confer tumor resistance to therapies. Early knowledge of the TM at time of diagnosis would be clinically relevant for precision therapy based on microbial composition. However, it is difficult to define the TM prior to surgical resection. Methods: In this pilot feasibility study, patients underwent Endoscopic Ultrasound-Fine Needle Aspiration (EUS-FNA) biopsy of pancreatic adenocarcinoma. These samples were analyzed using 16S rRNA and internal transcribed spacer (ITS) sequencing for characterization of the tumor bacteria and fungi. Result: After in silico decontamination and comparison to non-matched tumor, we were able to characterize the TM in biopsies, which was comparable to the TM from surgical specimens. Discussion: EUS-FNA biopsy may represent a feasible modality to characterize the pancreatic TM prior to surgical resection with proper decontamination strategies and improvements in matched controls.

Hematopoietic Somatic Mosaicism Is Associated With an Increased Risk of Postoperative Atrial Fibrillation.

BACKGROUND: On-pump cardiac surgery triggers sterile inflammation and postoperative complications such as postoperative atrial fibrillation (POAF). Hematopoietic somatic mosaicism (HSM) is a recently identified risk factor for cardiovascular diseases and results in a shift toward a chronic proinflammatory monocyte transcriptome and phenotype. OBJECTIVES: The aim of this study was to assess the prevalence, characteristics, and impact of HSM on preoperative blood and myocardial myeloid cells as well as on outcomes after cardiac surgery. METHODS: Blood DNA from 104 patients referred for surgical aortic valve replacement (AVR) was genotyped using the HemePACT panel (576 genes). Four screening methods were applied to assess HSM, and postoperative outcomes were explored. In-depth blood and myocardial leukocyte phenotyping was performed in selected patients using mass cytometry and preoperative and postoperative RNA sequencing analysis of classical monocytes. RESULTS: The prevalence of HSM in the patient cohort ranged from 29%, when considering the conventional HSM panel (97 genes) with variant allelic frequencies ≥2%, to 60% when considering the full HemePACT panel and variant allelic frequencies ≥1%. Three of 4 explored HSM definitions were significantly associated with higher risk for POAF. On the basis of the most inclusive definition, HSM carriers exhibited a 3.5-fold higher risk for POAF (age-adjusted OR: 3.5; 95% CI: 1.52-8.03; P = 0.003) and an exaggerated inflammatory response following AVR. HSM carriers presented higher levels of activated CD64+CD14+CD16- circulating monocytes and inflammatory monocyte-derived macrophages in presurgery myocardium. CONCLUSIONS: HSM is frequent in candidates for AVR, is associated with an enrichment of proinflammatory cardiac monocyte-derived macrophages, and predisposes to a higher incidence of POAF. HSM assessment may be useful in the personalized management of patients in the perioperative period. (Post-Operative Myocardial Incident & Atrial Fibrillation [POMI-AF]; NCT03376165).

Clinical Importance of Clonal Hematopoiesis in Metastatic Gastrointestinal Tract Cancers.

Importance: Clonal hematopoiesis (CH) has been associated with development of atherosclerosis and leukemia and worse survival among patients with cancer; however, the association with cancer therapy efficacy, in particular immune checkpoint blockade (ICB), and toxicity has not yet been established. Given the widespread use of ICB and the critical role hematopoietic stem cell-derived lymphocytes play in mediating antitumor responses, CH may be associated with therapeutic efficacy and hematologic toxicity. Objective: To determine the association between CH and outcomes, hematologic toxicity, and therapeutic efficacy in patients with metastatic gastrointestinal tract cancers being treated with systemic therapy, both in the first-line metastatic treatment setting and in ICB. Design, Setting, and Participants: This retrospective cohort study included 633 patients with stage IV colorectal (CRC) and esophagogastric (EGC) cancer who were treated with first-line chemotherapy and/or ICB at Memorial Sloan Kettering Cancer Center. Patients underwent matched tumor and peripheral blood DNA sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets next-generation sequencing assay between January 1, 2006, and December 31, 2020. Exposures: Clonal hematopoiesis-related genetic alterations were identified by next-generation sequencing of patients' tumor and normal blood buffy coat samples, with a subset of these CH alterations annotated as likely putative drivers (CH-PD) based upon previously established criteria. Main Outcomes and Measures: Patients with CH and CH-PD in peripheral blood samples were identified, and these findings were correlated with survival outcomes (progression-free survival [PFS] and overall survival [OS]) during first-line chemotherapy and ICB, as well as baseline white blood cell levels and the need for granulocyte colony-stimulating factor (G-CSF) support. Results: Among the 633 patients included in the study (390 men [61.6%]; median age, 58 [IQR, 48-66] years), the median age was 52 (IQR, 45-63) years in the CRC group and 61 (IQR, 53-69) years in the EGC group. In the CRC group, 161 of 301 patients (53.5%) were men, compared with 229 of 332 patients (69.0%) in the EGC group. Overall, 62 patients (9.8%) were Asian, 45 (7.1%) were Black or African American, 482 (76.1%) were White, and 44 (7.0%) were of unknown race or ethnicity. Presence of CH was identified in 115 patients with EGC (34.6%) and 83 with CRC (27.6%), with approximately half of these patients harboring CH-PD (CRC group, 44 of 83 [53.0%]; EGC group, 55 of 115 [47.8%]). Patients with EGC and CH-PD exhibited a significantly worse median OS of 16.0 (95% CI, 11.6-22.3) months compared with 21.6 (95% CI, 19.6-24.3) months for those without CH-PD (P = .01). For patients with CRC and EGC, CH and CH-PD were not associated with PFS differences in patients undergoing ICB or first-line chemotherapy. Neither CH nor CH-PD were correlated with baseline leukocyte levels or increased need for G-CSF support. Conclusions and Relevance: These findings suggest CH and CH-PD are not directly associated with the treatment course of patients with metastatic gastrointestinal tract cancer receiving cancer-directed therapy.

Association of Antineoplastic Therapy With Decreased SARS-CoV-2 Infection Rates in Patients With Cancer.

Importance: Novel therapies for SARS-CoV-2 infection are urgently needed. Antineoplastic compounds that target cellular machinery used by SARS-CoV-2 for entry and replication, including angiotensin-converting enzyme 2 (ACE2), may disrupt SARS-CoV-2 activity. Objectives: To determine whether patients with cancer treated with potential ACE2-lowering antineoplastic compounds exhibit lower SARS-CoV-2 infection rates. Design, Setting, and Participants: We used the Library of Integrated Network-Based Cellular Signatures database to identify antineoplastic compounds associated with decreased ACE2 gene expression across cell lines. We then evaluated a retrospective cohort of 1701 patients who were undergoing antineoplastic therapy at Memorial Sloan Kettering Cancer Center in New York, New York, during the COVID-19 pandemic to determine if treatment with an ACE2-lowering antineoplastic was associated with a decreased odds ratio (OR) of SARS-CoV-2 infection. Patients included in the analysis underwent active treatment for cancer and received a SARS-CoV-2 test between March 10 and May 28, 2020. Main Outcome and Measure: The association between potential ACE2-lowering antineoplastic treatment and a positive SARS-CoV-2 test. Results: In the cohort of 1701 patients, SARS-CoV-2 infection rates were determined for 949 (55.8%) female and 752 (44.2%) male patients (mean [SD] age, 63.1 [13.1] years) with diverse cancers receiving antineoplastic therapy. In silico analysis of gene expression signatures after drug treatment identified 91 compounds associated with downregulation of ACE2 across cell lines. Of the total cohort, 215 (12.6%) patients were treated with 8 of these compounds, including 3 mTOR/PI3K inhibitors and 2 antimetabolites. In a multivariable analysis of patients who received an ACE2-lowering antineoplastic adjusting for confounders, 15 of 215 (7.0%) patients had a positive SARS-CoV-2 test compared with 191 of 1486 (12.9%) patients who received other antineoplastic therapies (OR, 0.53; 95% CI, 0.29-0.88). Findings were confirmed in additional sensitivity analyses including cancer type, steroid use, and a propensity-matched subcohort. Gemcitabine treatment was associated with reduced SARS-CoV-2 infection (OR, 0.42; 95% CI, 0.17-0.87). Conclusions and Relevance: In this cohort study, in silico analysis of drug-associated gene expression signatures identified potential ACE2-lowering antineoplastic compounds, including mTOR/PI3K inhibitors and antimetabolites. Patients who received these compounds exhibited statistically significantly lower rates of SARS-CoV-2 infection compared with patients given other antineoplastics. Further evaluation of the biological and clinical anti-SARS-CoV-2 properties of identified antineoplastic compounds is warranted.

Comparative Analysis of Colon Cancer-Derived Fusobacterium nucleatum Subspecies: Inflammation and Colon Tumorigenesis in Murine Models.

Fusobacteria are commonly associated with human colorectal cancer (CRC), but investigations are hampered by the absence of a stably colonized murine model. Further, Fusobacterium nucleatum subspecies isolated from human CRC have not been investigated. While F. nucleatum subspecies are commonly associated with CRC, their ability to induce tumorigenesis and contributions to human CRC pathogenesis are uncertain. We sought to establish a stably colonized murine model and to understand the inflammatory potential and virulence genes of human CRC F. nucleatum, representing the 4 subspecies, animalis, nucleatum, polymorphum, and vincentii. Five human CRC-derived and two non-CRC derived F. nucleatum strains were tested for colonization, tumorigenesis, and cytokine induction in specific-pathogen-free (SPF) and/or germfree (GF) wild-type and ApcMin/+ mice, as well as in vitro assays and whole-genome sequencing (WGS). SPF wild-type and ApcMin/+ mice did not achieve stable colonization with F. nucleatum, whereas certain subspecies stably colonized some GF mice but without inducing colon tumorigenesis. F. nucleatum subspecies did not form in vivo biofilms or associate with the mucosa in mice. In vivo inflammation was inconsistent across subspecies, whereas F. nucleatum induced greater cytokine responses in a human colorectal cell line, HCT116. While F. nucleatum subspecies displayed genomic variability, no distinct virulence genes associated with human CRC strains were identified that could reliably distinguish these strains from non-CRC clinical isolates. We hypothesize that the lack of F. nucleatum-induced tumorigenesis in our model reflects differences in human and murine biology and/or a synergistic role for F. nucleatum in concert with other bacteria to promote carcinogenesis. IMPORTANCE Colon cancer is a leading cause of cancer morbidity and mortality, and it is hypothesized that dysbiosis in the gut microbiota contributes to colon tumorigenesis. Fusobacterium nucleatum, a member of the oropharyngeal microbiome, is enriched in a subset of human colon tumors. However, it is unclear whether this genetically varied species directly promotes tumor formation, modulates mucosal immune responses, or merely colonizes the tumor microenvironment. Mechanistic studies to address these questions have been stymied by the lack of an animal model that does not rely on daily orogastric gavage. Using multiple murine models, in vitro assays with a human colon cancer cell line, and whole-genome sequencing analysis, we investigated the proinflammatory and tumorigenic potential of several F. nucleatum clinical isolates. The significance of this research is development of a stable colonization model of F. nucleatum that does not require daily oral gavages in which we demonstrate that a diverse library of clinical isolates do not promote tumorigenesis.

Therapeutic administration of Tregitope-Human Albumin Fusion with Insulin Peptides to promote Antigen-Specific Adaptive Tolerance Induction.

Type 1 Diabetes (T1D) is an autoimmune disease that is associated with effector T cell (Teff) destruction of insulin-producing pancreatic beta-islet cells. Among the therapies being evaluated for T1D is the restoration of regulatory T cell (Treg) activity, specifically directed toward down-modulation of beta-islet antigen-specific T effector cells. This is also known as antigen-specific adaptive tolerance induction for T1D (T1D ASATI). Tregitopes (T regulatory cell epitopes) are natural T cell epitopes derived from immunoglobulin G (IgG) that were identified in 2008 and have been evaluated in several autoimmune disease models. In the T1D ASATI studies presented here, Tregitope peptides were administered to non-obese diabetic (NOD) mice at the onset of diabetes within two clinically-relevant delivery systems (liposomes and in human serum albumin [HSA]-fusion products) in combination with preproinsulin (PPI) target antigen peptides. The combination of Tregitope-albumin fusions and PPI peptides reduced the incidence of severe diabetes and reversed mild diabetes, over 49 days of treatment and observation. Combining HSA-Tregitope fusions with PPI peptides is a promising ASATI approach for therapy of T1D.

Tumor Microbiome Diversity and Composition Influence Pancreatic Cancer Outcomes.

Most patients diagnosed with resected pancreatic adenocarcinoma (PDAC) survive less than 5 years, but a minor subset survives longer. Here, we dissect the role of the tumor microbiota and the immune system in influencing long-term survival. Using 16S rRNA gene sequencing, we analyzed the tumor microbiome composition in PDAC patients with short-term survival (STS) and long-term survival (LTS). We found higher alpha-diversity in the tumor microbiome of LTS patients and identified an intra-tumoral microbiome signature (Pseudoxanthomonas-Streptomyces-Saccharopolyspora-Bacillus clausii) highly predictive of long-term survivorship in both discovery and validation cohorts. Through human-into-mice fecal microbiota transplantation (FMT) experiments from STS, LTS, or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration. Our study demonstrates that PDAC microbiome composition, which cross-talks to the gut microbiome, influences the host immune response and natural history of the disease.

Gut microbiome meta-analysis reveals dysbiosis is independent of body mass index in predicting risk of obesity-associated CRC.

OBJECTIVE: Obesity is a risk factor for colorectal cancer (CRC), accounting for more than 14% of CRC incidence. Microbial dysbiosis and chronic inflammation are common characteristics in both obesity and CRC. Human and murine studies, together, demonstrate the significant impact of the microbiome in governing energy metabolism and CRC development; yet, little is understood about the contribution of the microbiome to development of obesity-associated CRC as compared to individuals who are not obese. DESIGN: In this study, we conducted a meta-analysis using five publicly available stool and tissue-based 16S rRNA and whole genome sequencing (WGS) data sets of CRC microbiome studies. High-resolution analysis was employed for 16S rRNA data, which allowed us to achieve species-level information to compare with WGS. RESULTS: Characterisation of the confounders between studies, 16S rRNA variable region and sequencing method did not reveal any significant effect on alpha diversity in CRC prediction. Both 16S rRNA and WGS were equally variable in their ability to predict CRC. Results from diversity analysis confirmed lower diversity in obese individuals without CRC; however, no universal differences were found in diversity between obese and non-obese individuals with CRC. When examining taxonomic differences, the probability of being classified as CRC did not change significantly in obese individuals for all taxa tested. However, random forest classification was able to distinguish CRC and non-CRC stool when body mass index was added to the model. CONCLUSION: Overall, microbial dysbiosis was not a significant factor in explaining the higher risk of colon cancer among individuals with obesity.

Cervicovaginal Fungi and Bacteria Associated With Cervical Intraepithelial Neoplasia and High-Risk Human Papillomavirus Infections in a Hispanic Population.

The human cervicovaginal microbiota resides at an interface between the host and the environment and may affect susceptibility to disease. Puerto Rican women have high human papillomavirus (HPV) infection and cervical cancer rates. We hypothesized that the population structure of the cervicovaginal bacterial and fungal biota changed with cervical squamous intraepithelial lesions and HPV infections. DNA was extracted from cervix, introitus, and anal sites of 62 patients attending high-risk San Juan clinics. The 16S rRNA V4 region and ITS-2 fungal regions were amplified and sequenced using Illumina technology. HPV genotyping was determined by reverse hybridization with the HPV SPF10-LiPA25 kit. HPV prevalence was 84% of which ∼44% subjects were infected with high-risk HPV, ∼35% were co-infected with as many as 9 HPV types and ∼5% were infected with exclusively low-risk HPV types. HPV diversity did not change with cervical dysplasia. Cervical bacteria were more diverse in patients with CIN3 pre-cancerous lesions. We found enrichment of Atopobium vaginae and Gardnerella vaginalis in patients with CIN3 lesions. We found no significant bacterial biomarkers associated with HPV infections. Fungal diversity was significantly higher in cervical samples with high-risk HPV and introitus samples of patients with Atypical Squamous Cells of Undetermined Significance (ASCUS). Fungal biomarker signatures for vagina and cervix include Sporidiobolaceae and Sacharomyces for ASCUS, and Malassezia for high-risk HPV infections. Our combined data suggests that specific cervicovaginal bacterial and fungal populations are related to the host epithelial microenvironment, and could play roles in cervical dysplasia.

Metabolic Biosynthesis Pathways Identified from Fecal Microbiome Associated with Prostate Cancer.

BACKGROUND: The fecal microbiome is associated with prostate cancer risk factors (obesity, inflammation) and can metabolize and produce various products that may influence cancer but have yet to be defined in prostate cancer. OBJECTIVE: To investigate gut bacterial diversity, identify specific metabolic pathways associated with disease, and develop a microbiome risk profile for prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: After prospective collection of 133 rectal swab samples 2 wk before the transrectal prostate biopsy, we perform 16S rRNA amplicon sequencing on 105 samples (64 with cancer, 41 without cancer). Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was applied to infer functional categories associated with taxonomic composition. The p values were adjusted using the false discovery rate. The α- and ß-diversity analyses were performed using QIIME. The Mann-Whitney U test was employed to evaluate the statistical significance of ß-diversity distances within and between groups of interest, and least absolute shrinkage and selection operator (LASSO) regression analysis was used to determine pathway significance. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The detection of prostate cancer on transrectal prostate needle biopsy and 16s microbiome profile. RESULTS AND LIMITATIONS: We identified significant associations between total community composition and cancer/non-cancer status (Bray-Curtis distance metric, p<0.01). We identified significant differences in enrichments of Bacteroides and Streptococcus species in cancer (all p<0.04). Folate (LDA 3.8) and arginine (LDA 4.1) were the most significantly altered pathways. We formed a novel microbiome-derived risk factor for prostate cancer based on 10 aberrant metabolic pathways (area under curve=0.64, p=0.02). CONCLUSIONS: Microbiome analyses on men undergoing prostate biopsy noted mostly similar bacterial species diversity among men diagnosed with and without prostate cancer. The microbiome may have subtle influences on prostate cancer but are likely patient-specific and would require paired analysis and precise manipulation, such as improvement of natural bacterial folate production. PATIENT SUMMARY: Microbiome evaluation may provide patients with personalized data regarding the presence or absence of particular bacteria that have metabolic functions and implications regarding prostate cancer risk. The study provides a basis to investigate the manipulation of aberrant microbiomes to reduce prostate cancer risk.

High-resolution profiling of the gut microbiome reveals the extent of Clostridium difficile burden.

Microbiome profiling through 16S rRNA gene sequence analysis has proven to be a useful research tool in the study of C. difficile infection (CDI); however, CDI microbiome studies typically report results at the genus level or higher, thus precluding identification of this pathogen relative to other members of the gut microbiota. Accurate identification of C. difficile relative to the overall gut microbiome may be useful in assessments of colonization in research studies or as a prognostic indicator for patients with CDI. To investigate the burden of C. difficile at the species level relative to the overall gut microbiome, we applied a high-resolution method for 16S rRNA sequence assignment to previously published gut microbiome studies of CDI and other patient populations. We identified C. difficile in 131 of 156 index cases of CDI (average abundance 1.78%), and 18 of 211 healthy controls (average abundance 0.008%). We further detected substantial levels of C. difficile in a subset of infants that persisted over the first two to 12 months of life. Correlation analysis of C. difficile burden compared to other detected species demonstrated consistent negative associations with C. scindens and multiple Blautia species. These analyses contribute insight into the relative burden of C. difficile in the gut microbiome for multiple patient populations, and indicate that high-resolution 16S rRNA gene sequence analysis may prove useful in the development and evaluation of new therapies for CDI.

High-resolution microbiome profiling uncovers Fusobacterium nucleatum, Lactobacillus gasseri/johnsonii, and Lactobacillus vaginalis associated to oral and oropharyngeal cancer in saliva from HPV positive and HPV negative patients treated with surgery and chemo-radiation.

Microbiome studies show altered microbiota in head and neck squamous cell carcinoma (HNSCC), both in terms of taxonomic composition and metabolic capacity. These studies utilized a traditional bioinformatics methodology, which allows for accurate taxonomic assignment down to the genus level, but cannot accurately resolve species level membership. We applied Resphera Insight, a high-resolution methodology for 16S rRNA taxonomic assignment that is able to provide species-level context in its assignments of 16S rRNA next generation sequencing (NGS) data. Resphera Insight applied to saliva samples from HNSCC patients and healthy controls led to the discovery that a subset of HNSCC saliva samples is significantly enriched with commensal species from the vaginal flora, including Lactobacillus gasseri/johnsonii (710x higher in saliva) and Lactobacillus vaginalis (52x higher in saliva). These species were not observed in normal saliva from Johns Hopkins patients, nor in 16S rRNA NGS saliva samples from the Human Microbiome Project (HMP). Interestingly, both species were only observed in saliva from Human Papilloma Virus (HPV) positive and HPV negative oropharyngeal cancer patients. We confirmed the representation of both species in HMP data obtained from mid-vagina (n=128) and vaginal introitus (n=121) samples. Resphera Insight also led to the discovery that Fusobacterium nucleatum, an oral cavity flora commensal bacterium linked to colon cancer, is enriched (600x higher) in saliva from a subset of HNSCC patients with advanced tumors stages. Together, these high-resolution analyses on 583 samples suggest a possible role for bacterial species in the therapeutic outcome of HPV positive and HPV negative HNSCC patients.

Using a Control to Better Understand Phyllosphere Microbiota.

An important data gap in our understanding of the phyllosphere surrounds the origin of the many microbes described as phyllosphere communities. Most sampling in phyllosphere research has focused on the collection of microbiota without the use of a control, so the opportunity to determine which taxa are actually driven by the biology and physiology of plants as opposed to introduced by environmental forces has yet to be fully realized. To address this data gap, we used plastic plants as inanimate controls adjacent to live tomato plants (phyllosphere) in the field with the hope of distinguishing between bacterial microbiota that may be endemic to plants as opposed to introduced by environmental forces. Using 16S rRNA gene amplicons to study bacterial membership at four time points, we found that the vast majority of all species-level operational taxonomic units were shared at all time-points. Very few taxa were unique to phyllosphere samples. A higher taxonomic diversity was consistently observed in the control samples. The high level of shared taxonomy suggests that environmental forces likely play a very important role in the introduction of microbes to plant surfaces. The observation that very few taxa were unique to the plants compared to the number that were unique to controls was surprising and further suggests that a subset of environmentally introduced taxa thrive on plants. This finding has important implications for improving our approach to the description of core phytobiomes as well as potentially helping us better understand how foodborne pathogens may become associated with plant surfaces.

The potential role of lung microbiota in lung cancer attributed to household coal burning exposures.

Bacteria influence site-specific disease etiology and the host's ability to metabolize xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs). Lung cancer in Xuanwei, China has been attributed to PAH-rich household air pollution from burning coal. This study seeks to explore the role of lung microbiota in lung cancer among never smoking Xuanwei women and how coal burning may influence these associations. DNA from sputum and buccal samples of never smoking lung cancer cases (n = 8, in duplicate) and controls (n = 8, in duplicate) in two Xuanwei villages was extracted using a multi-step enzymatic and physical lysis, followed by a standardized clean-up. V1-V2 regions of 16S rRNA genes were PCR-amplified. Purified amplicons were sequenced by 454 FLX Titanium pyrosequencing and high-quality sequences were evaluated for diversity and taxonomic membership. Bacterial diversity among cases and controls was similar in buccal samples (P = 0.46), but significantly different in sputum samples (P = 0.038). In sputum, Granulicatella (6.1 vs. 2.0%; P = 0.0016), Abiotrophia (1.5 vs. 0.085%; P = 0.0036), and Streptococcus (40.1 vs. 19.8%; P = 0.0142) were enriched in cases compared with controls. Sputum samples had on average 488.25 species-level OTUs in the flora of cases who used smoky coal (PAH-rich) compared with 352.5 OTUs among cases who used smokeless coal (PAH-poor; P = 0.047). These differences were explained by the Bacilli species (Streptococcus infantis and Streptococcus anginosus). Our small study suggests that never smoking lung cancer cases have differing sputum microbiota than controls. Further, bacteria found in sputum may be influenced by environmental exposures associated with the type of coal burned in the home.

Bacteria-human somatic cell lateral gene transfer is enriched in cancer samples.

There are 10× more bacterial cells in our bodies from the microbiome than human cells. Viral DNA is known to integrate in the human genome, but the integration of bacterial DNA has not been described. Using publicly available sequence data from the human genome project, the 1000 Genomes Project, and The Cancer Genome Atlas (TCGA), we examined bacterial DNA integration into the human somatic genome. Here we present evidence that bacterial DNA integrates into the human somatic genome through an RNA intermediate, and that such integrations are detected more frequently in (a) tumors than normal samples, (b) RNA than DNA samples, and (c) the mitochondrial genome than the nuclear genome. Hundreds of thousands of paired reads support random integration of Acinetobacter-like DNA in the human mitochondrial genome in acute myeloid leukemia samples. Numerous read pairs across multiple stomach adenocarcinoma samples support specific integration of Pseudomonas-like DNA in the 5'-UTR and 3'-UTR of four proto-oncogenes that are up-regulated in their transcription, consistent with conversion to an oncogene. These data support our hypothesis that bacterial integrations occur in the human somatic genome and may play a role in carcinogenesis. We anticipate that the application of our approach to additional cancer genome projects will lead to the more frequent detection of bacterial DNA integrations in tumors that are in close proximity to the human microbiome.

CloVR-ITS: Automated internal transcribed spacer amplicon sequence analysis pipeline for the characterization of fungal microbiota.

BACKGROUND: Besides the development of comprehensive tools for high-throughput 16S ribosomal RNA amplicon sequence analysis, there exists a growing need for protocols emphasizing alternative phylogenetic markers such as those representing eukaryotic organisms. RESULTS: Here we introduce CloVR-ITS, an automated pipeline for comparative analysis of internal transcribed spacer (ITS) pyrosequences amplified from metagenomic DNA isolates and representing fungal species. This pipeline performs a variety of steps similar to those commonly used for 16S rRNA amplicon sequence analysis, including preprocessing for quality, chimera detection, clustering of sequences into operational taxonomic units (OTUs), taxonomic assignment (at class, order, family, genus, and species levels) and statistical analysis of sample groups of interest based on user-provided information. Using ITS amplicon pyrosequencing data from a previous human gastric fluid study, we demonstrate the utility of CloVR-ITS for fungal microbiota analysis and provide runtime and cost examples, including analysis of extremely large datasets on the cloud. We show that the largest fractions of reads from the stomach fluid samples were assigned to Dothideomycetes, Saccharomycetes, Agaricomycetes and Sordariomycetes but that all samples were dominated by sequences that could not be taxonomically classified. Representatives of the Candida genus were identified in all samples, most notably C. quercitrusa, while sequence reads assigned to the Aspergillus genus were only identified in a subset of samples. CloVR-ITS is made available as a pre-installed, automated, and portable software pipeline for cloud-friendly execution as part of the CloVR virtual machine package (http://clovr.org). CONCLUSION: The CloVR-ITS pipeline provides fungal microbiota analysis that can be complementary to bacterial 16S rRNA and total metagenome sequence analysis allowing for more comprehensive studies of environmental and host-associated microbial communities.

Metagenomics of the subsurface Brazos-Trinity Basin (IODP site 1320): comparison with other sediment and pyrosequenced metagenomes.

The Brazos-Trinity Basin on the slope of the Gulf of Mexico passive margin was drilled during Integrated Ocean Drilling Progam Expedition 308. The buried anaerobic sediments of this basin are largely organic-poor and have few microbial inhabitants compared with the organic-rich sediments with high cell counts from the Peru Margin that were drilled during Ocean Drilling Program Leg 201. Nucleic acids were extracted from Brazos-Trinity Basin sediments and were subjected to whole-genome amplification and pyrosequencing. A comparison of the Brazos-Trinity Basin metagenome, consisting of 105 Mbp, and the existing Peru Margin metagenome revealed trends linking gene content, phylogenetic content, geological location and geochemical regime. The major microbial groups (Proteobacteria, Firmicutes, Euryarchaeota and Chloroflexi) occur consistently throughout all samples, yet their shifting abundances allow for discrimination between samples. The cluster of orthologous groups category abundances for some classes of genes are correlated with geochemical factors, such as the level of ammonia. Here we describe the sediment metagenome from the oligotrophic Brazos-Trinity Basin (Site 1320) and show similarities and differences with the dataset from the Pacific Peru Margin (Site 1229) and other pyrosequenced datasets. The microbial community found at Integrated Ocean Drilling Program Site 1320 likely represents the subsurface microbial inhabitants of turbiditic slopes that lack substantial upwelling.

Gut microbial gene expression in mother-fed and formula-fed piglets.

BACKGROUND: Effects of diet on the structure and function of gut microbial communities in newborn infants are poorly understood. High-resolution molecular studies are needed to definitively ascertain whether gut microbial communities are distinct in milk-fed and formula-fed infants. METHODOLOGY/PRINCIPAL FINDINGS: Pyrosequencing-based whole transcriptome shotgun sequencing (RNA-seq) was used to evaluate community wide gut microbial gene expression in 21 day old neonatal piglets fed either with sow's milk (mother fed, MF; n = 4) or with artificial formula (formula fed, FF; n = 4). Microbial DNA and RNA were harvested from cecal contents for each animal. cDNA libraries and 16S rDNA amplicons were sequenced on the Roche 454 GS-FLX Titanium system. Communities were similar at the level of phylum but were dissimilar at the level of genus; Prevotella was the dominant genus within MF samples and Bacteroides was most abundant within FF samples. Screened cDNA sequences were assigned functional annotations by the MG-RAST annotation pipeline and based upon best-BLASTX-hits to the NCBI COG database. Patterns of gene expression were very similar in MF and FF animals. All samples were enriched with transcripts encoding enzymes for carbohydrate and protein metabolism, as well as proteins involved in stress response, binding to host epithelium, and lipopolysaccharide metabolism. Carbohydrate utilization transcripts were generally similar in both groups. The abundance of enzymes involved in several pathways related to amino acid metabolism (e.g., arginine metabolism) and oxidative stress response differed in MF and FF animals. CONCLUSIONS/SIGNIFICANCE: Abundant transcripts identified in this study likely contribute to a core microbial metatranscriptome in the distal intestine. Although microbial community gene expression was generally similar in the cecal contents of MF and FF neonatal piglets, several differentially abundant gene clusters were identified. Further investigations of gut microbial gene expression will contribute to a better understanding of normal and abnormal enteric microbiology in animals and humans.