Evaluation of silica spin­column and magnetic bead formats for rapid DNA methylation analysis in clinical and point­of­care settings.

Late-stage cancers lack effective treatment, underscoring the need for early diagnosis to improve prognosis and decrease mortality rates. Molecular markers, such as DNA methylation, offer promise in early cancer detection. The present study compared commercial kits for analyzing DNA from cervical liquid cytology samples in cancer screening. Rapid bisulfite conversion kits using silica spin-columns and magnetic beads were assessed against standard DNA extraction and bisulfite conversion methods for profiling DNA methylation using quantitative methylation-specific PCR. ß-actin amplification indicated the suitability of small sample volumes for methylation studies using either the pellet or supernatant (cell-free DNA) parts. Comparison of Bisulfite Conversion Kit-Whole Cell (Abcam), Methylamp Bisulfite Modification (Epigentek), EpiTect Fast LyseAll Bisulfite Kit (Qiagen GmbH) and EZ DNA Methylation-Direct Kit (Zymo Research Corp.) showed no significant differences in ß-actin cycle threshold values. EZ-96 DNA Methylation-Lightning MagPrep (Zymo Research Corp.), a hybrid kit in a 96-well plate format, exhibited swift turnaround time and similar amplification efficiency. Automation with magnetic bead kits increased throughput without compromising amplification efficiency in open PCR systems. Cost analysis favored direct kits over the gold standard manual protocol. This comparison aids in selecting cost-effective DNA methylation diagnostic tests. The present study confirmed comparable kit performance in methylation-based analysis, highlighting the adequacy of cytology samples and the potential of bodily fluids as alternatives for liquid biopsy.

Evaluation of self-collected nasal, urine, and saliva samples for molecular detection of SARS-CoV-2 using an EUA approved RT-PCR assay and a laboratory developed LAMP SARS-CoV-2 test.

As the SARS-CoV-2 virus spread throughout the world, millions of positive cases of COVID-19 were registered and, even though there are millions of people already vaccinated against SARS-CoV-2, a large part of the global population remains vulnerable to contracting the virus. Massive nasopharyngeal sample collection in Puerto Rico at the beginning of the pandemic was limited by the scarcity of trained personnel and testing sites. To increase SARS-CoV-2 molecular testing availability, we evaluated the diagnostic accuracy of self-collected nasal, saliva, and urine samples using the TaqPath reverse transcription polymerase chain reaction (RT-PCR) COVID-19 kit to detect SARS-CoV-2. We also created a colorimetric loop-mediated isothermal amplification (LAMP) laboratory developed test (LDT) to detect SARS-CoV-2, as another strategy to increase the availability of molecular testing in community-based laboratories. Automated RNA extraction was performed in the KingFisher Flex instrument, followed by PCR quantification of SARS-CoV-2 on the 7500 Fast Dx RT-PCR using the TaqPath RT-PCR COVID-19 molecular test. Data was interpreted by the COVID-19 Interpretive Software from Applied Biosystems and statistically analyzed with Cohen's kappa coefficient (k). Cohen's kappa coefficient (k) for paired nasal and saliva samples showed moderate agreement (0.52). Saliva samples exhibited a higher viral load. We also observed 90% concordance between LifeGene-Biomarks' SARS-CoV-2 Rapid Colorimetric LAMP LDT and the TaqPath RT-PCR COVID-19 test. Our results suggest that self-collected saliva is superior to nasal and urine samples for COVID-19 testing. The results also suggest that the colorimetric LAMP LDT is a rapid alternative to RT-PCR tests for the detection of SARS-CoV-2. This test can be easily implemented in clinics, hospitals, the workplace, and at home; optimizing the surveillance and collection process, which helps mitigate global public health and socioeconomic upheaval caused by airborne pandemics.

Promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions are associated with tumor differentiation, nodal involvement and survival.

Differentially methylated regions (DMRs) can be used as head and neck squamous cell carcinoma (HNSCC) diagnostic, prognostic and therapeutic targets in precision medicine workflows. DNA from 21 HNSCC and 10 healthy oral tissue samples was hybridized to a genome-wide tiling array to identify DMRs in a discovery cohort. Downstream analyses identified differences in promoter DNA methylation patterns in oral, laryngeal and oropharyngeal anatomical regions associated with tumor differentiation, nodal involvement and survival. Genome-wide DMR analysis showed 2,565 DMRs common to the three subsites. A total of 738 DMRs were unique to laryngeal cancer (n=7), 889 DMRs were unique to oral cavity cancer (n=10) and 363 DMRs were unique to pharyngeal cancer (n=6). Based on the genome-wide analysis and a Gene Ontology analysis, 10 candidate genes were selected to test for prognostic value and association with clinicopathological features. TIMP3 was associated with tumor differentiation in oral cavity cancer (P=0.039), DAPK1 was associated with nodal involvement in pharyngeal cancer (P=0.017) and PAX1 was associated with tumor differentiation in laryngeal cancer (P=0.040). A total of five candidate genes were selected, DAPK1, CDH1, PAX1, CALCA and TIMP3, for a prevalence study in a larger validation cohort: Oral cavity cancer samples (n=42), pharyngeal cancer tissues (n=25) and laryngeal cancer samples (n=52). PAX1 hypermethylation differed across HNSCC anatomic subsites (P=0.029), and was predominantly detected in laryngeal cancer. Kaplan-Meier survival analysis (P=0.043) and Cox regression analysis of overall survival (P=0.001) showed that DAPK1 methylation is associated with better prognosis in HNSCC. The findings of the present study showed that the HNSCC subsites oral cavity, pharynx and larynx display substantial differences in aberrant DNA methylation patterns, which may serve as prognostic biomarkers and therapeutic targets.

Precision health diagnostic and surveillance network uses S gene target failure (SGTF) combined with sequencing technologies to track emerging SARS-CoV-2 variants.

INTRODUCTION: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic revealed a worldwide lack of effective molecular surveillance networks at local, state, and national levels, which are essential to identify, monitor, and limit viral community spread. SARS-CoV-2 variants of concern (VOCs) such as Alpha and Omicron, which show increased transmissibility and immune evasion, rapidly became dominant VOCs worldwide. Our objective was to develop an evidenced-based genomic surveillance algorithm, combining reverse transcription polymerase chain reaction (RT-PCR) and sequencing technologies to quickly identify highly contagious VOCs, before cases accumulate exponentially. METHODS: Deidentified data were obtained from 508,969 patients tested for coronavirus disease 2019 (COVID-19) with the TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) in four CLIA-certified clinical laboratories in Puerto Rico (n = 86,639) and in three CLIA-certified clinical laboratories in the United States (n = 422,330). RESULTS: TaqPath data revealed a frequency of S Gene Target Failure (SGTF) > 47% for the last week of March 2021 in both, Puerto Rico and US laboratories. The monthly frequency of SGTF in Puerto Rico steadily increased exponentially from 4% in November 2020 to 47% in March 2021. The weekly SGTF rate in US samples was high (>8%) from late December to early January and then also increased exponentially through April (48%). The exponential increase in SGFT prevalence in Puerto Rico was concurrent with a sharp increase in VOCs among all SARS-CoV-2 sequences from Puerto Rico uploaded to Global Influenza Surveillance and Response System (GISAID) (n = 461). Alpha variant frequency increased from <1% in the last week of January 2021 to 51.5% of viral sequences from Puerto Rico collected in the last week of March 2021. CONCLUSIONS: According to the proposed evidence-based algorithm, approximately 50% of all SGTF patients should be managed with VOCs self-quarantine and contact tracing protocols, while WGS confirms their lineage in genomic surveillance laboratories. Our results suggest this workflow is useful for tracking VOCs with SGTF.

Integration of sociocultural and behavioral factors into the clinical framework of cardiovascular studies in Hispanic/Latino populations: Relevance during the SARS-COV-2 pandemic.

Recent reports on the burden of cardiovascular disease (CVD) in the USA indicate that despite significant declines in CVD mortality in the late 20th century, this decline is now decelerating and may be worsened by inequalities in health care. Social factors contribute to most of the cardiovascular health disparities documented to date. Hispanics/Latinos and African-Americans share a higher prevalence of cardiovascular risk factors and experience higher rates of poverty and social stressors than non-Hispanic Whites. We propose that the use of social and behavioral data beyond basic and sometimes loose identifiers of race/ethnicity, educational attainment, and occupation would inform clinical practice and greatly facilitate the provision of adequate guidance and support to patients regarding continuity of care, adherence to medications and treatment plans, and engagement of participants in future research. This perspective briefly highlights factors deemed to be critical for the advancement of Hispanic/Latino health and delineates pathways toward future applications.

HIST1H2BB and MAGI2 Methylation and Somatic Mutations as Precision Medicine Biomarkers for Diagnosis and Prognosis of High-grade Serous Ovarian Cancer.

Molecular alterations that contribute to long-term (LT) and short-term (ST) survival in ovarian high-grade serous carcinoma (HGSC) may be used as precision medicine biomarkers. DNA promoter methylation is an early event in tumorigenesis, which can be detected in blood and urine, making it a feasible companion biomarker to somatic mutations for early detection and targeted treatment workflows. We compared the methylation profile in 12 HGSC tissue samples to 30 fallopian tube epithelium samples, using the Infinium Human Methylation 450K Array. We also used 450K methylation arrays to compare methylation among HGSCs long-term survivors (more than 5 years) and short-term survivors (less than 3 years). We verified the array results using bisulfite sequencing and methylation-specific PCR (qMSP). in another cohort of HGSC patient samples (n = 35). Immunoblot and clonogenic assays after pharmacologic unmasking show that HIST1H2BB and MAGI2 promoter methylation downregulates mRNA expression levels in ovarian cancer cells. We then used qMSP in paired tissue, ascites, plasma/serum, vaginal swabs, and urine from a third cohort of patients with HGSC cancer (n = 85) to test the clinical potential of HIST1H2BB and MAGI2 in precision medicine workflows. We also performed next-generation exome sequencing of 50 frequently mutated in human cancer genes, using the Ion AmpliSeqCancer Hotspot Panel, to show that the somatic mutation profile found in tissue and plasma can be quantified in paired urine samples from patients with HGSC. Our results suggest that HIST1H2BB and MAGI2 have growth-suppressing roles and can be used as HGSC precision medicine biomarkers.

JAK3 Variant, Immune Signatures, DNA Methylation, and Social Determinants Linked to Survival Racial Disparities in Head and Neck Cancer Patients.

To inform novel personalized medicine approaches for race and socioeconomic disparities in head and neck cancer, we examined germline and somatic mutations, immune signatures, and epigenetic alterations linked to neighborhood determinants of health in Black and non-Latino White (NLW) patients with head and neck cancer. Cox proportional hazards revealed that Black patients with squamous cell carcinoma of head and neck (HNSCC) with PAX5 (P = 0.06) and PAX1 (P = 0.017) promoter methylation had worse survival than NLW patients, after controlling for education, zipcode, and tumor-node-metastasis stage (n = 118). We also found that promoter methylation of PAX1 and PAX5 (n = 78), was correlated with neighborhood characteristics at the zip-code level (P < 0.05). Analyses also showed differences in the frequency of TP53 mutations (n = 32) and tumor-infiltrating lymphocyte (TIL) counts (n = 24), and the presence of a specific C → A germline mutation in JAK3, chr19:17954215 (protein P132T), in Black patients with HNSCC (n = 73; P < 0.05), when compared with NLW (n = 37) patients. TIL counts are associated (P = 0.035) with long-term (>5 years), when compared with short-term survival (<2 years). We show bio-social determinants of health associated with survival in Black patients with HNSCC, which together with racial differences shown in germline mutations, somatic mutations, and TIL counts, suggests that contextual factors may significantly inform precision oncology services for diverse populations.

PIM1 kinase promotes gallbladder cancer cell proliferation via inhibition of proline-rich Akt substrate of 40 kDa (PRAS40).

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC.

INSIG2 rs7566605 single nucleotide variant and global DNA methylation index levels are associated with weight loss in a personalized weight reduction program.

Single nucleotide polymorphisms associated with lipid metabolism and energy balance are implicated in the weight loss response caused by nutritional interventions. Diet­induced weight loss is also associated with differential global DNA methylation. DNA methylation has been proposed as a predictive biomarker for weight loss response. Personalized biomarkers for successful weight loss may inform clinical decisions when deciding between behavioral and surgical weight loss interventions. The aim of the present study was to investigate the association between global DNA methylation, genetic variants associated with energy balance and lipid metabolism, and weight loss following a non­surgical weight loss regimen. The present study included 105 obese participants that were enrolled in a personalized weight loss program based on their allelic composition of the following five energy balance and lipid metabolism­associated loci: Near insulin­induced gene 2 (INSIG2); melanocortin 4 receptor; adrenoceptor ß2; apolipoprotein A5; and G­protein subunit ß3. The present study investigated the association between a global DNA methylation index (GDMI), the allelic composition of the five energy balance and lipid metabolism­associated loci, and weight loss during a 12 month program, after controlling for age, sex and body mass index (BMI). The results demonstrated a significant association between the GDMI and near INSIG2 locus, after adjusting for BMI and weight loss, and significant trends were observed when stratifying by gender. In conclusion, a combination of genetic and epigenetic biomarkers may be used to design personalized weight loss interventions, enabling adherence and ensuring improved outcomes for obesity treatment programs. Precision weight loss programs designed based on molecular information may enable the creation of personalized interventions for patients, that use genomic biomarkers for treatment design and for treatment adherence monitoring, thus improving response to treatment.

PAX5 gene as a novel methylation marker that predicts both clinical outcome and cisplatin sensitivity in esophageal squamous cell carcinoma.

Therapeutic strategies for esophageal cancer largely depend on histopathological assessment. To select appropriate treatments of individual patients, we examined the background molecular characteristics of tumor malignancy and sensitivity to multidisciplinary therapy. Seventy-eight surgically-resected esophageal squamous cell carcinoma (ESCC) cases during 2001-2013 were examined. PAX5, a novel gene methylation marker in ESCC, was evaluated in the specimens, as methylation of this gene was identified as an extremely tumor-specific event in squamous cell carcinogenesis of head and neck. PAX5 methylation status was evaluated by quantitative MSP (QMSP) assays. Mean QMSP value was 15.7 (0-136.3) in ESCCs and 0.3 (0-8.6) in adjacent normal tissues (P < 0.001). The 78 cases were divided into high QMSP value (high QMSP, n = 26) and low QMSP value (low QMSP, n = 52). High QMSP cases were significantly associated with downregulated PAX5 expression (P = 0.040), and showed significantly poor recurrence-free survival [Hazard Ratio (HR) = 2.84; P = 0.005; 95% Confidence Interval (CI): 1.39-5.81] and overall survival (HR = 3.23; P = 0.002; 95%CI: 1.52-7.01) in multivariable analyses with histopathological factors. PAX5-knockdown cells exhibited significantly increased cell proliferation and cisplatin resistance. PAX5 gene methylation can predict poor survival outcomes and cisplatin sensitivity in ESCCs and could be a useful diagnostic tool for cancer therapy selection.

Personalized medicine and Hispanic health: improving health outcomes and reducing health disparities - a National Heart, Lung, and Blood Institute workshop report.

Persons of Hispanic/Latino descent may represent different ancestries, ethnic and cultural groups and countries of birth. In the U.S., the Hispanic/Latino population is projected to constitute 29% of the population by 2060. A personalized approach focusing on individual variability in genetics, environment, lifestyle and socioeconomic determinants of health may advance the understanding of some of the major factors contributing to the health disparities experienced by Hispanics/Latinos and other groups in the U.S., thus leading to new strategies that improve health care outcomes. However, there are major gaps in our current knowledge about how personalized medicine can shape health outcomes among Hispanics/Latinos and address the potential factors that may explain the observed differences within this heterogeneous group, and between this group and other U.S. demographic groups. For that purpose, the National Heart, Lung, and Blood Institute (NHLBI), in collaboration with the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and the Food and Drug Administration (FDA), held a workshop in which experts discussed (1) potential approaches to study medical treatments and health outcomes among Hispanics/Latinos and garner the necessary evidence to fill gaps of efficacy, effectiveness and safety of therapies for heart, lung, blood and sleep (HLBS) disorders and conditions--and their risk factors; (2) research opportunities related to personalized medicine to improve knowledge and develop effective interventions to reduce health disparities among Hispanics/Latinos in the U.S.; and (3) the incorporation of expanded sociocultural and socioeconomic data collection and genetic/genomic/epigenetic information of Hispanic/Latino patients into their clinical assessments, to account for individual variability in ancestry; physiology or disease risk; culture; environment; lifestyle; and socioeconomic determinants of health. The experts also provided recommendations on: sources of Hispanic/Latino health data and strategies to enhance its collection; policy; genetics, genomics and epigenetics research; and integrating Hispanic/Latino health research within clinical settings.

A Panel of Novel Detection and Prognostic Methylated DNA Markers in Primary Non-Small Cell Lung Cancer and Serum DNA.

Purpose: To establish a novel panel of cancer-specific methylated genes for cancer detection and prognostic stratification of early-stage non-small cell lung cancer (NSCLC).Experimental Design: Identification of differentially methylated regions (DMR) was performed with bumphunter on "The Cancer Genome Atlas (TCGA)" dataset, and clinical utility was assessed using quantitative methylation-specific PCR assay in multiple sets of primary NSCLC and body fluids that included serum, pleural effusion, and ascites samples.Results: A methylation panel of 6 genes (CDO1, HOXA9, AJAP1, PTGDR, UNCX, and MARCH11) was selected from TCGA dataset. Promoter methylation of the gene panel was detected in 92.2% (83/90) of the training cohort with a specificity of 72.0% (18/25) and in 93.0% (40/43) of an independent cohort of stage IA primary NSCLC. In serum samples from the later 43 stage IA subjects and population-matched 42 control subjects, the gene panel yielded a sensitivity of 72.1% (31/41) and specificity of 71.4% (30/42). Similar diagnostic accuracy was observed in pleural effusion and ascites samples. A prognostic risk category based on the methylation status of CDO1, HOXA9, PTGDR, and AJAP1 refined the risk stratification for outcomes as an independent prognostic factor for an early-stage disease. Moreover, the paralog group for HOXA9, predominantly overexpressed in subjects with HOXA9 methylation, showed poor outcomes.Conclusions: Promoter methylation of a panel of 6 genes has potential for use as a biomarker for early cancer detection and to predict prognosis at the time of diagnosis. Clin Cancer Res; 23(22); 7141-52. ©2017 AACR.

Early detection of gastric cancer using global, genome-wide and IRF4, ELMO1, CLIP4 and MSC DNA methylation in endoscopic biopsies.

Clinically useful molecular tools to triage gastric cancer patients are not currently available. We aimed to develop a molecular tool to predict gastric cancer risk in endoscopy-driven biopsies obtained from high-risk gastric cancer clinics in low resource settings.We discovered and validated a DNA methylation biomarker panel in endoscopic samples obtained from 362 patients seen between 2004 and 2009 in three high-risk gastric cancer clinics in Lima, Perú, and validated it in 306 samples from the Cancer Genome Atlas project ("TCGA"). Global, epigenome wide and gene-specific DNA methylation analyses were used in a Phase I Biomarker Development Trial to identify a continuous biomarker panel that combines a Global DNA Methylation Index (GDMI) and promoter DNA methylation levels of IRF4, ELMO1, CLIP4 and MSC.We observed an inverse association between the GDMI and histological progression to gastric cancer, when comparing gastritis patients without metaplasia (mean = 5.74, 95% CI, 4.97-6.50), gastritis patients with metaplasia (mean = 4.81, 95% CI, 3.77-5.84), and gastric cancer cases (mean = 3.38, 95% CI, 2.82-3.94), respectively (p < 0.0001). Promoter methylation of IRF4 (p < 0.0001), ELMO1 (p < 0.0001), CLIP4 (p < 0.0001), and MSC (p < 0.0001), is also associated with increasing severity from gastritis with no metaplasia to gastritis with metaplasia and gastric cancer.Our findings suggest that IRF4, ELMO1, CLIP4 and MSC promoter methylation coupled with a GDMI>4 are useful molecular tools for gastric cancer risk stratification in endoscopic biopsies.

Whole Blood Cytokine Response to Local Traffic-Related Particulate Matter in Peruvian Children With and Without Asthma.

This study sought to investigate if acute phase immune responses of whole blood from Peruvian children with controlled and uncontrolled asthma differed from children without asthma, following exposure to traffic-related particulate matter (TRPM). TRPM, including particulate matter from diesel combustion, has been shown to stimulate acute airway inflammation in individuals with and without asthma. For this study, a whole blood assay (WBA) was used to test peripheral whole blood samples from 27 children with asthma, and 12 without asthma. Participant blood samples were stimulated, ex vivo, for 24-h with an aqueous extract of TRPM that was collected near study area highways in Lima, Peru. All participant blood samples were tested against the same TRPM extract, in addition to purified bacterial endotoxin and pyrogen-free water, which served as positive and negative WBA controls, respectively. The innate and adaptive cytokine responses were evaluated in cell-free supernatants of the whole blood incubations. Comparatively similar levels were recorded for nine out of the 10 cytokines measured [e.g., - Interleukin (IL)-1ß, IL-6, IL-10], regardless of study participant asthma status. However, IL-8 levels in TRPM-stimulated blood from children with uncontrolled asthma were diminished, compared to subjects without asthma (633 pg/ml vs. 1,023 pg/ml, respectively; p < 0.01); IL-8 responses for subjects with controlled asthma were also reduced, but to a lesser degree (799 pg/ml vs. 1,023 pg/ml, respectively; p = 0.10). These relationships were present before, and after, adjusting for age, sex, obesity/overweight status, C-reactive protein levels, and residential proximity to the study area's major roadway. For tests conducted with endotoxin, there were no discernible differences in cytokine response between groups, for all cytokines measured. The WBA testing conducted for this study highlighted the capacity of the TRPM extract to potently elicit the release of IL-8 from the human whole blood system. Although the small sample size of the study limits the capacity to draw definitive conclusions, the IL-8 responses suggest that that asthma control may be associated with the regulation of a key mediator in neutrophil chemotaxis, at a systemic level, following exposure to PM derived from traffic-related sources.

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.

Prenatal exposure to tobacco smoke leads to increased mitochondrial DNA content in umbilical cord serum associated to reduced gestational age.

We investigated if prenatal exposures to tobacco smoke lead to changes in mitochondrial DNA content (mtDNA) in cord serum and adversely affect newborns' health. Umbilical cord serum cotinine levels were used to determine in utero exposure to smoking. Cord serum mtDNA was measured by quantitative polymerase chain reaction analysis of the genes coding for cytochrome c oxidase1 (MT-CO1) and cytochrome c oxidase2 (MT-CO2). Log transformed levels of mtDNA coding for MT-CO1 and MT-CO2 were significantly higher among infants of active smokers with higher serum level of cotinine (p < 0.05) and inversely associated with gestational age (p = 0.08; p = 0.02). Structural equation modeling results confirmed a positive association between cotinine and MT-CO1 and2 (p < 0.01) and inverse associations with gestational age (p = 0.02) and IGF-1 (p < 0.01). We identified a dose-dependent increase in the level of MT-CO1 and MT-CO2 associated to increased cord serum cotinine and decreased gestational age.

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.

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 OTU's 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.

Macrophage migration inhibitory factor - a therapeutic target in gallbladder cancer.

BACKGROUND: Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. METHODS: Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. RESULTS: The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. CONCLUSIONS: Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.

Chronic exposure to chewing tobacco selects for overexpression of stearoyl-CoA desaturase in normal oral keratinocytes.

Chewing tobacco is a common practice in certain socio-economic sections of southern Asia, particularly in the Indian subcontinent and has been well associated with head and neck squamous cell carcinoma. The molecular mechanisms of chewing tobacco which leads to malignancy remains unclear. In large majority of studies, short-term exposure to tobacco has been evaluated. From a biological perspective, however, long-term (chronic) exposure to tobacco mimics the pathogenesis of oral cancer more closely. We developed a cell line model to investigate the chronic effects of chewing tobacco. Chronic exposure to tobacco resulted in higher cellular proliferation and invasive ability of the normal oral keratinocytes (OKF6/TERT1). We carried out quantitative proteomic analysis of OKF6/TERT1 cells chronically treated with chewing tobacco compared to the untreated cells. We identified a total of 3,636 proteins among which expression of 408 proteins were found to be significantly altered. Among the overexpressed proteins, stearoyl-CoA desaturase (SCD) was found to be 2.6-fold overexpressed in the tobacco treated cells. Silencing/inhibition of SCD using its specific siRNA or inhibitor led to a decrease in cellular proliferation, invasion and colony forming ability of not only the tobacco treated cells but also in a panel of head and neck cancer cell lines. These findings suggest that chronic exposure to chewing tobacco induced carcinogenesis in non-malignant oral epithelial cells and SCD plays an essential role in this process. The current study provides evidence that SCD can act as a potential therapeutic target in head and neck squamous cell carcinoma, especially in patients who are users of tobacco.