Validation and Field Testing of Library-Independent Microbial Source Tracking Methods in the Gulf of Mexico

Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10−5–10−6 dilution). LODs for HPyVs and M. smithii assays were similar to each other (10−3–10−4), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces among \u3e300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples. An experts’ workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2

Segregation of HPV-positive vulvar neoplastic lesions by an anal cancer progression methylation signature

Background: Human papillomavirus (HPV) has been implicated in the development of different cancers and has been associated with genome-wide epigenetic alterations. We have demonstrated an 86-gene methylomic signature to be associated with the progression of anal cancer (AC) from normal mucosa. The signature is also able to classify AIN3 as either cancer-like or normal-like. We sought to evaluate whether this AC signature is applicable to HPV-associated vulvar cancer (VC) progression. Methods: Our 16 formalin-fixed paraffin-embedded study specimens consisted of 8 VC, 6 VIN3, and 2 normal vulvar tissues (NV). Extracted DNA was subjected to HPV genotyping (SPF10-LiPA25 kit) as well as genome-wide methylation profiling (Illumina Methylation EPIC array). Differential methylation was defined by false discovery rate of 0.3. The AC methylation signature was applied to vulvar tissues. Results: All 16 tissues except for one VC were found to be HPV+. By principal components analysis, distinct separations between NV, VIN3 and VC were observed. Application of the AC signature to the vulvar tissues demonstrated strong separation of NV and VC cases with segregation of VIN3 cases into normal-like (n=2) and cancer-like (n=4) types. By both analyses, the HPV-negative VC case presented as a distinct outlier. Conclusions: Methylomic alterations can distinguish between HPV+ NV, VIN3 and VC. Successful segregation of vulvar lesions by an AC signature suggests that HPV-driven oncogenesis may be associated with similar methylation events across different tissue types. Our findings may have implications for development of shared early detection and prevention biomarkers

Primers for Bisulfite Sequencing,

<p>Primers for Bisulfite Sequencing,</p

Chromosomal Mapping of Genes with Differentially Methylated CpG Loci.

<p>Chromosomal Mapping of Genes with Differentially Methylated CpG Loci.</p

Genome-wide host methylation profiling of anal and cervical carcinoma.

HPV infection results in changes in host gene methylation which, in turn, are thought to contribute to the neoplastic progression of HPV-associated cancers. The objective of this study was to identify joint and disease-specific genome-wide methylation changes in anal and cervical cancer as well as changes in high-grade pre-neoplastic lesions. Formalin-fixed paraffin-embedded (FFPE) anal tissues (n = 143; 99% HPV+) and fresh frozen cervical tissues (n = 28; 100% HPV+) underwent microdissection, DNA extraction, HPV genotyping, bisulfite modification, DNA restoration (FFPE) and analysis by the Illumina HumanMethylation450 Array. Differentially methylated regions (DMR; t test q0.3) were compared between normal and cancer specimens in partial least squares (PLS) models and then used to classify anal or cervical intraepithelial neoplasia-3 (AIN3/CIN3). In AC, an 84-gene PLS signature (355 significant probes) differentiated normal anal mucosa (NM; n = 9) from AC (n = 121) while a 36-gene PLS signature (173 significant probes) differentiated normal cervical epithelium (n = 10) from CC (n = 9). The CC progression signature was validated using three independent publicly available datasets (n = 424 cases). The AC and CC progression PLS signatures were interchangeable in segregating normal, AIN3/CIN3 and AC and CC and were found to include 17 common overlapping hypermethylated genes. Moreover, these signatures segregated AIN3/CIN3 lesions similarly into cancer-like and normal-like categories. Distinct methylation changes occur across the genome during the progression of AC and CC with overall similar profiles and add to the evidence suggesting that HPV-driven oncogenesis may result in similar non-random methylomic events. Our findings may lead to identification of potential epigenetic drivers of HPV-associated cancers and also, of potential markers to identify higher risk pre-cancerous lesions

Gender Heatmap of X-linked CpG Loci.

<p>DNA methylation is involved in the transcriptional activation of genes on one of the two X chromosomes in female somatic cells. In general, male and female cases clustered together with methylation levels of the majority of X-linked genes correlating well with the gender of the tissue source (i.e. little-to-no methylation in male samples and hemi-methylation in female samples). Sample gender is represented above the heatmap (pink = female, blue = male). Methylation is represented by the beta value, or percent of total signal with green representing low methylation and red representing hemi-methylation. 51 of 84 loci within X-linked genes were differentially methylated (p<0.05) between males and females.</p

HPV Genotyping of Anal Tissues: Overall Prevalence and Across the Spectrum of Anal Neoplasia.

*<p>A total of 38 HPV infections were detected in 29 tissues, due to multiple infections<b>.</b></p