Amanda Dziedzic

16S rRNA amplicon sequencing identifies microbiota associated with oral cancer, human papilloma virus infection and surgical treatment

Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropharyngeal (OPSCC), Oral Cavity Squamous Cell Carcinoma (OCSCC) patients and normal epithelium controls, to characterize the HNSCC saliva microbiota and examine their abundance before and after surgical resection. The analyses identified a predominance of Firmicutes, Proteobacteria and Bacteroidetes, with less frequent presence of Actinobacteria and Fusobacteria before surgery. At lower taxonomic levels, the most abundant genera were Streptococcus, Prevotella, Haemophilus, Lactobacillus and Veillonella, with lower numbers of Citrobacter and Neisseraceae genus Kingella. HNSCC patients had a significant loss in richness and diversity of microbiota species (p<0.05) compared to the controls. Overall, the Operational Taxonomic Units network shows that the relative abundance of OTUs within genus Streptococcus, Dialister, and Veillonella can be used to discriminate tumor from control samples (p<0.05). Tumor samples lost Neisseria, Aggregatibacter (Proteobacteria), Haemophillus (Firmicutes) and Leptotrichia (Fusobacteria). Paired taxa within family Enterobacteriaceae, together with genus Oribacterium, distinguish OCSCC samples from OPSCC and normal samples (p<0.05). Similarly, only HPV positive samples have an abundance of genus Gemellaceae and Leuconostoc (p<0.05). Longitudinal analyses of samples taken before and after surgery, revealed a reduction in the alpha diversity measure after surgery, together with an increase of this measure in patients that recurred (p<0.05). These results suggest that microbiota may be used as HNSCC diagnostic and prognostic biomonitors.

Performance assessment of copy number microarray platforms using a spike-in experiment

MOTIVATION Changes in the copy number of chromosomal DNA segments [copy number variants (CNVs)] have been implicated in human variation, heritable diseases and cancers. Microarray-based platforms are the current established technology of choice for studies reporting these discoveries and constitute the benchmark against which emergent sequence-based approaches will be evaluated. Research that depends on CNV analysis is rapidly increasing, and systematic platform assessments that distinguish strengths and weaknesses are needed to guide informed choice. RESULTS We evaluated the sensitivity and specificity of six platforms, provided by four leading vendors, using a spike-in experiment. NimbleGen and Agilent platforms outperformed Illumina and Affymetrix in accuracy and precision of copy number dosage estimates. However, Illumina and Affymetrix algorithms that leverage single nucleotide polymorphism (SNP) information make up for this disadvantage and perform well at variant detection. Overall, the NimbleGen 2.1M platform outperformed others, but only with the use of an alternative data analysis pipeline to the one offered by the manufacturer. AVAILABILITY The data is available from http://rafalab.jhsph.edu/cnvcomp/. CONTACT pevsner@jhmi.edu; fspencer@jhmi.edu; rafa@jhu.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Genetic predisposition to natural rubber latex allergy differs between health care workers and high-risk patients.

BACKGROUND: In health care workers, the natural rubber latex (NRL) allergy phenotype has been shown to be associated with promoter polymorphisms in interleukins 13 and 18 (IL13 and IL18) when compared with nonatopic controls. However, it is not known whether high-risk patient populations, such as those born with neural tube defects or genitourinary abnormalities, demonstrate a heightened propensity toward the same genetic/immunologic risk factors that have been reported for health care workers. In this study, we tested the hypothesis that single-nucleotide polymorphisms in genes encoding IL13 and IL18 occur at an increased frequency in NRL allergic patients with spina bifida (SB) or bladder exstrophy (BE). METHODS: One hundred twenty subjects (40 SB, 40 BE, and 40 control) were screened using a clinical history questionnaire and NRL-specific immunoglobulin E (IgE) antibody measurements in the blood. Genomic DNA was extracted from peripheral blood lymphocytes and analyzed for single-nucleotide polymorphisms in candidate genes of interest. Univariate and multivariate analyses were performed to identify significant variables with significance defined as P < 0.05. RESULTS: Sensitization (IgE antibody positivity) to NRL allergens was associated with atopic history and number of prior operations and was prevented by the avoidance of NRL beginning at birth. However, unlike health care workers, the NRL allergy phenotype was not significantly associated with promoter polymorphisms in IL13 or IL18 when comparing NRL allergic SB and BE patients with nonsensitized patients and with atopic and nonatopic controls. CONCLUSIONS: In patients born with SB or BE, environmental factors seem to play a greater role in the development of NRL sensitization and overt allergic symptoms than the IL polymorphisms in IL13 and IL18 previously shown to be associated with NRL allergy in health care workers.

Molecular Triage of Premalignant Lesions in Liquid-Based Cervical Cytology and Circulating Cell-Free DNA from Urine, Using a Panel of Methylated Human Papilloma Virus and Host Genes

Clinically useful molecular tools to triage women for a biopsy upon referral to colposcopy are not available. We aimed to develop a molecular panel to detect cervical intraepithelial neoplasia (CIN) grade 2 or higher lesions (CIN2+) in women with abnormal cervical cytology and high-risk HPV (HPV+). We tested a biomarker panel in cervical epithelium DNA obtained from 211 women evaluated in a cervical cancer clinic in Chile from 2006 to 2008. Results were verified in a prospective cohort of 107 women evaluated in a high-risk clinic in Puerto Rico from 2013 to 2015. Promoter methylation of ZNF516, FKBP6, and INTS1 discriminated cervical brush samples with CIN2+ lesions from samples with no intraepithelial lesions or malignancy (NILM) with 90% sensitivity, 88.9% specificity, 0.94 area under the curve (AUC), 93.1% positive predictive value (PPV), and 84.2% negative predictive value (NPV). The panel results were verified in liquid-based cervical cytology samples from an independent cohort with 90.9% sensitivity, 60.9% specificity, 0.90 AUC, 52.6% PPV, and 93.3% NPV, after adding HPV16-L1 methylation to the panel. Next-generation sequencing results in HPV+ cultured cells, and urine circulating cell-free DNA (ccfDNA) were used to design assays that show clinical feasibility in a subset (n = 40) of paired plasma (AUC = 0.81) and urine (AUC = 0.86) ccfDNA samples obtained from the prospective cohort. Viral and host DNA methylation panels can be tested in liquid cytology and urine ccfDNA from women referred to colposcopy, to triage CIN2+ lesions for biopsy and inform personalized screening algorithms. Cancer Prev Res; 9(12); 915–24. ©2016 AACR.

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.

Mycobacteria induce TPL-2 mediated IL-10 in IL-4-generated alternatively activated macrophages

IL-4 drives expansion of Th2 cells that cause generation of alternatively activated macrophages (AAMs). Filarial infections are established early in life, induce increased IL-4 production are co-endemic with tuberculosis (TB). We sought to understand, therefore, how mycobacteria are handled in the context of IL-4-induced AAM. Comparing IL-4 generated in vitro monocyte derived human AAMs to LPS and IFN-γ generated classically macrophages (CAMs), both infected with mycobacteria (BCG), we demonstrated increased early BCG uptake and increased IL-10 production in AAMs compared to CAMs. We further demonstrated that increased IL-10 production is mediated by upregulation of tumor progression locus 2 (TPL-2), an upstream activator of extracellular signal related kinases (ERKs) in AAMs but not in CAMs, both at the transcript as well as the protein level. Pharmacologic inhibition of TPL-2 significantly diminished IL-10 production only in BCG-infected AAMs. Finally, we validated our findings in an in vivo C57Bl/6 model of filarial infection, where an exaggerated Th2 induced lung-specific alternative activation led to TPL-2 and IL-10 upregulation on subsequent TB infection. These data show that in response to mycobacterial infection, IL-4 generated AAMs in chronic filarial infections have impaired immune responses to TB infection by increasing IL-10 production in a TPL-2 mediated manner.

Copy Number Variants Associated with 14 Cases of Self-Injurious Behavior.

Copy number variants (CNVs) were detected and analyzed in 14 probands with autism and intellectual disability with self-injurious behavior (SIB) resulting in tissue damage. For each proband we obtained a clinical history and detailed behavioral descriptions. Genetic anomalies were observed in all probands, and likely clinical significance could be established in four cases. This included two cases having novel, de novo copy number variants and two cases having variants likely to have functional significance. These cases included segmental trisomy 14, segmental monosomy 21, and variants predicted to disrupt the function of ZEB2 (encoding a transcription factor) and HTR2C (encoding a serotonin receptor). Our results identify variants in regions previously implicated in intellectual disability and suggest candidate genes that could contribute to the etiology of SIB.

Abstract 1533: 16S rRNA saliva analysis unveils differences in the head and neck squamous cell carcinoma microbiome before and after surgical resection

Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We compared the saliva microbiome in DNA isolated from 38 patients and 25 normal oral cavity epithelium controls to characterize the HNSCC microbiota before and after surgical resection. PCR amplification of the 16S rRNA V3-V5 gene region was performed using the 357F/926R primer set prior to multiplexing on the Roche/454 GS Junior sequencing platform. Data were screened for chimeric sequences and contaminant chloroplast DNA after pre-processing. Passing sequences were characterized for diversity and taxonomic composition using QIIME and R before cross-tabulation analyses were performed. After preprocessing 142,887 reads were obtained with an average length of 491 bp. The number of sequences per sample was rarefied at 3,487 to guarantee equal depth. Bacteroidetes, Firmicutes, and Proteobacteria dominated the microbiome in our sample set with less frequent presence of Actinobacteria and Fusobacteria members. At lower taxonomic levels, the most abundant genera observed were Streptococcus, Prevotella, Haemophilus and Veillonella with lower numbers of Citrobacter and Neisseraceae genus Kingella. We found that 46 OTUs changed significantly in HNSCC patients (p Longitudinal analyses (3 time periods) of samples taken before and after surgery revealed a reduction in the alpha diversity measure after surgery, together with an increase of this measure in patients that recurred. We also observed statistically significant differences (p We are the first to observe that OTUs and several microbial communities at different taxonomic levels discriminate HNSCC from control samples; HPV positive and HPV negative samples; and pre- vs postsurgical treatment samples. Future work will determine the correlation of microbial communities in paired tissue and saliva HNSCC samples, as well as their link to treatment response and survival. Citation Format: Rafael E. Guerrero-Preston, Filipa Godoy-Vitorino, Herminio Gonzalez, Christina Michailidi, Anne Jedlicka, Amanda Dziedzic, Rajagowthamee Thangavel, Tal Hadar, Maartje G. Noordhuis, William Westra, Wayne Koch, David Sidransky. 16S rRNA saliva analysis unveils differences in the head and neck squamous cell carcinoma microbiome before and after surgical resection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1533. doi:10.1158/1538-7445.AM2015-1533