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.

Previous microbiome studies at the genus level have described altered microbiota in head and neck squamous cell carcinoma (HNSCC), both in terms of taxonomic composition and metabolic capacity. We applied high-resolution microbiome profiling (Resphera Insight) to analyze 16S rRNA sequencing data in saliva and tissue samples from HNSCC patients and healthy controls. DNA extraction and amplicon library preparation was performed for saliva samples from HNSCC (n=38) and controls (n=25), as well as tissue samples from HNSCC (n=25) and controls (n=8). Raw sequences were processed for quality and length, screened for chimeras and filtered for contaminant human and chloroplast DNA. High-quality passing sequences were submitted to Resphera Insight for species-level taxonomic assignments, followed-by differential abundance analysis with the DESeq package. Samples from a subset of HNSCC patients were significantly enriched with commensal species from the vaginal flora, including Lactobacillus gasseri/johnsonii (710x higher in saliva and 1990x higher in tissue) and Lactobacillus vaginalis (52x higher in saliva). These species were not observed in normal saliva or tissue samples from Hopkins patients (n=33) nor in normal saliva samples (n=292) from the Human Microbiome Project (HMP). Interestingly, both species were only observed in saliva from Human Papilloma Virus positive (HPV+) and HPV negative (HPV-) oropharyngeal cancer patients, and we confirmed their representation in vaginal samples from the HMP (n=249). We also found that Fusobacterium nucleatum (F.nucleatum), an oral cavity flora commensal bacterium linked to colon cancer, is enriched (600x higher in saliva and 51x higher in tissue) in a subset of HNSCC patients with advanced tumors (T3 or above). F. nucleatum was detected in samples obtained before and after treatment with chemo-radiation, but not with surgery alone. Interestingly, we identified upregulation of the oncogenic Wnt/Beta catenin pathway (Wnt7B, FZD6, SFRP4) and down regulation of immune system pathways (TLR10, IRF8) with genome-wide mRNA arrays (Affymetrix) in HNSCC samples enriched for F.nucleatum. Using fluorogenic quantitative PCR we confirmed that F.nucleatum and Fusobacterium spp. are enriched in saliva samples collected prior to treatment in another cohort of HNSCC patients, and in post-treatment saliva samples obtained from HNSCC patients treated with PD-1 checkpoint blockade. We also found enrichment of HPV+ oropharyngeal tumors with F.nucleatum and Fusobacterium spp., prior to therapy, while some recurrent HPV- oropharyngeal tumors are enriched only with Fusobacterium spp. Together, these results suggest that bacteria may impact therapy in HPV+ and HPV- oropharyngeal and oral cavity cancer by altering oncogenic and immune system pathways. Citation Format: Rafael E. Guerrero-Preston, James Robert White, Filipa Godoy-Vitorino, Herminio Gonzalez, Arnold Rodríguez-Hilario, Kelvin Navarro, Gustavo A. Miranda-Carboni, Christina Michailidi, Anne Jedlicka, Stephanie Hao, Sierra Canapp, Jessica Bondy, Amanda Dziedzic, Barbara Mora Lagos, Gustavo Rivera-Alvarez, Winston Timp, William Westra, Wayne Koch, Hyunseok Kang, Luigi Marchionni, Young Kim, David Sidransky. High-resolution microbiome profiling and genome wide arrays uncover bacteria driven alterations of oncogenic and immune pathways in head and neck cancer patients treated with surgery, chemo-radiation and PD-1 checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1018. doi:10.1158/1538-7445.AM2017-1018

Spontaneous pneumomediastinum (SPM) is defined as free air in the mediastinum thought to be due to non-traumatic rupture of marginal alveoli without evidence of underlying lung disease. Secondary causes must be excluded, including perforations of the esophagus, trachea, and bronchi, due to their propensity for unfavorable outcomes. Infectious etiologies have been documented to cause pneumomediastinum, but this is most frequently observed in the setting of mechanical and noninvasive positive-pressure ventilation (NIPPV). Here we present a case of asymptomatic spontaneous pneumomediastinum in a patient with COVID-19 in absence of mechanical ventilation.