Search

GenoScape - example of HPV genotype profiling report and visualization. Number, characteristics and percentage of reads that map to thirteen (13) HPV types, human, bacteria and unknown genomes after profiling reads of the clinical sample TrDNA-456 by tiered read mapping.

Scatter plots of HPV 16-L1 methylation in cervical brush (S7a) and urine ccfDNA (S7b). HPV16-L1 qMSP methylation can discriminate bisulfite treated cervical epithelium DNA and urine ccfDNA from patients with normal cytology, from women with dysplastic cytology, and premalignant cervical lesions with high Sensitivity and Specificity.

Data Integration modules and systems interface.s

GenoScape - HPV genotype profiling from high-throughput sequencing samples consists of four analytic steps: (1) High-throughput sequencing (454, MiSeq) of HPV TrDNA; (2) Profiling by comparing sequences against several databases such as Human genome (bowtie), HPV databases -GenBank, PaVE, Enterix-HPV Hi-lo risk (sim4db) and RefSeq bacterial genomes (blast); (3) Assembly into contigs, for AlignScape visualization (newbler,minimus, trinity); and SNP discovery (samtools + filter).

Circulating cell free DNA isolation results.

Primer and probe list of genes used for quantitative Methylation Specific PCR in urine ccfDNA.

Mapping efficiency of cervical cancer cell lines, head and neck squamous cell carcinoma cell lines and clinical samples to HPV16-L1.

Somatic mutations and single nucleotide variants (SNVs) in paired tissue from cervical swabs, serum/plasma and urine from patient TG01

Mutational profile in Black HNSCC patients by anatomic site

Supplementary Figure 1

Supplementary Figure 4

Dual Sequence capture of high risk HPV in TrDNA aligned to all high risk HPV types and low risk HPV types from PaVE database.

AlignScape - Pairwise alignment of the HPV16 genome with reads obtained from urine ccfDNA from seven clinical samples: TrDNA-445, TrDNA-455, TrDNA-456, TrDNA-481, TrDNA-504, TrDNA-513, and TrDNA-571. The large-scale HPV DNA server can produce a graphical 'large-scale' view of the pairwise alignments of up to twenty HPV genomes against a reference genome (HPV16 in this demo), together with annotations of genome rearrangement events.

Supplementary Figure 2

Patient characteristics.

Differences in the frequency of somatic mutations observed in Non-Latino Whites and Black HNSCC patients.

Reads of the HPV positive cervical cancer, head and neck cancer squamous cell carcinoma (HNSCC) cell lines and clinical samples that mapped uniquely to the reference genomes in the PAVE database.

Supplementary Methods.

The top differentially altered pathways revealed by Gene Ontology (GO) clustering when comparing Black and NLW HNSCC patients.

Phase 1 Biomarker Development Trial to identify genes or clusters of genes that appear to be differentially methylated in cervical cancer biopsy tissues relative to control cervical brush epithelium tissue taken from women seen in the same hospital. The search for cervical cancer screening methylation biomarkers began with preclinical exploratory studies to identify characteristics unique to tumor tissue that might lead to methylated biomarkers for clinical tests that can discriminate cancer from pre-malignant and normal lesions. We used Methylated DNA Immunoprecipitation (MeDIP) and oligonucleotide sequencing arrays (Nimblegen 385K CpG Island Plus Promoter Arrays) to unveil the cervical cancer promoter region methylome (MeDIP-chip). We then used bisulfite sequencing and quantitative Methylation Specific Polymerase Chain Reaction (qMSP) to identify potential biomarkers of progression from normal cervical mucosa epithelium in cervical brush samples from women with abnormal cytology. MeDIP-chip Discovery identified 2,044 differentially methylated probes associated to total of 444 gene loci differentially methylated when comparing tumor and normal samples. Gene loci were subdivided according to two cutoffs defined for the maximal distance between a methylation peak and the TSS: -1000 to +1000, called the standard cut-off; -500 to +500, called the narrow cut-off. We found 255 unique gene loci that are cancer specific methylated according to the standard cutoff and 189 according to the narrow cutoff. More than half of the methylated gene promoters identified with MeDIP-chip (60%) were hypermethylated in all cancer samples and not in normal samples. One hundred and sixty two (162) genes were jointly identified by the intersection between both lists. These stringent criteria ensured that all 10 genes selected for biomarker validation had methylated peaks within a CPG island located in the promoter region, 500 base pairs upstream from the TSS in all the hybridized tumor samples and none in the normal samples hybridized to the arrays. The top three genes in the list that contained CpG islands in their promoter regions FKBP6, ZNF516 and INTS1) were selected for further analysis with MSP and qMSP in Validation and Prevalence cohorts. Samples were tsted for HPV presence using the reverse line blot assay.

Legends for Supplementary Figures 1-4

Goals and Primary Aims of the five phases of the National Cancer Institue Early Detection Research Network biomarker development trials.

Amplification curves for the HPV TrDNA-qPCR assay on TrDNA from women with Cervical Intraepithelial Neoplasia 1 (CIN1), Cervical Intraepithelial Neoplasia 2-3 (CIN2-3) and women with no Intraepithelial Lesions or Neoplasia (NILM). TrDNA samples were analyzed for high risk HPV DNA using our HPV TrDNA SYBR green qPCR assay. HeLa genomic DNA was included as a positive control. Ct ranges are variable, but are in a similar range as the genomic control. NILM samples did not amplify.

Patient cohorts analyzed in this project.

Survival alleles at SNV level

Supplementary Figure 3

Aircraft Tracking Analogy that integrates Contextual, Bio-psychosocial and Molecular Determinants of Population Health in a Patient Centered Trajectory that can be used in Personalized Medicine workflows.

Methylation and mutation associations

Supplemental Methods, Figures and Tables

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.

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.

Cox regression analysis of TCGA Head and Neck Squamous Cell Carcinoma patients (n=279).

Somatic mutations and single nucleotide variants (SNVs) in paired tissue from cervical swabs, serum/plasma and urine from patient TG05

Primer and probe list of genes used for quantitative Methylation Specific PCR.

Hispanics/Latinos, Black Americans, and poor Non-Latino Whites in the United States are at higher risk of developing head and neck squamous cell carcinoma (HNSCC). The incidence of HNSCC in Puerto Rico is 2.5 higher than for Hispanics/Latinos living in the United States. These health disparities are a serious public health concern due to HNSCC high mortality and morbidity rates, higher treatment costs, and significant quality of life burden. We hypothesized that the discovery of actionable targets for HNSCC early detection, diagnosis, prognostication. Differentially Methylated Regions (DMRs) can be used as HNSCC diagnostic, prognostic, and therapeutic targets in precision medicine workflows. DNA from 23 HNSCC samples and 10 healthy oral tissue samples from patients in Puerto Rico were 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. We identified 738 DMRs unique to laryngeal cancer (n=7), 889 DMRs unique to oral cavity cancer (n=10), and 363 DMRs unique to pharyngeal cancer (n=6). Based on the genome-wide analysis, and a gene ontology analysis, we selected 10 candidate genes 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). We selected 5 candidate genes, DAPK1, CDH1, PAX1, CALCA, and TIMP3, for a prevalence study in a larger validation cohort: 42 oral cavity; 25 pharyngeal; and 52 laryngeal cancer samples. PAX1 hypermethylation differed across HNSCC anatomic sub-sites (p= 0.029), and 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. Our findings show that HNSCC sub-sites (oral cavity, pharynx, and larynx) display substantial differences in aberrant DNA methylation patterns among Hispanic/Latinos HNSCC patients in the US. These data suggest further research may lead to the development of diagnostic medical devices, prognostic biomarkers and novel therapeutic targets that may lead to a reduction in HNSCC disparities among Hispanic/Latinos in the US. Citation Format: BIanca Rivera-Peña, Oluwasina Folawiyo, Nitesh Turaga, Rosa J. Rodríguez-Benítez, Marco E. Felici, Jaime A. Aponte-Ortiz, Francesca Pirini, Sebastián Rodríguez-Torres, Roger Vázquez, Ricardo López, David Sidransky, Rafael E. Guerrero-Preston, Adriana Báez. Promoter DNA methylation patterns in oral, laryngeal, and oropharyngeal anatomical regions are associated with tumor differentiation, nodal involvement, and survival in Latinos. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5765.

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 (

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.

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.

Human Papilloma Virus (HPV) testing is increasingly used for cervical cancer screening in conjunction with cervical cytology. Although privacy, cultural, and infrastructure issues challenge the effective implementation of HPV testing for cervical cancer screening worldwide, several countries have already implemented HPV testing in their screening protocols. There are currently no tests that can reliably identify the patients with abnormal cytology and positive oncogenic HPV results (HPV+) that need to be referred for colposcopy. A useful triage test from cytology to colposcopy must discriminate the patients with a Cervical Intraepithelial Neoplasia (CIN) lesion that are more likely to progress to cervical cancer (CIN2+), from those that are less likely to progress. We set out to identify a panel of methylated HPV and human genes that can discriminate between CIN2+ and normal/CIN1 patients, first in cytology samples and then in urine samples. The Reverse Line Blot Assay identified high-risk HPV types in study participants from a Discovery cohort (n=96): 31 women with normal cervical cytology and 65 women with dysplasia (LSIL, n=43) and (HSIL, n=22). We developed custom sequence capture pools of baits to pull down genomic (Roche SeqCap EZ) and bisulfite converted high-risk HPV DNA (Agilent SureSelect) from urine, before library prep for NGS in a Roche 454 and MiSeq instruments, respectively. We also optimzed a qPCR assay to identify high risk HPV types, and quantitative Methylation Specific PCR (qMSP) assays to examine methylated HPV and human genes, in Pap smear DNA and TrDNA samples from women with normal Pap and women with CIN1, CIN2 and CIN3 lesions (n=80). The human genes selected for the qMSP assays, ZNF516, FKBP6, and INTS1, were identified in a previously published genome-wide analysis of the cevical cancer methylome using Nimblegen 385K CpG plus Promoters oligonucleotide tiling arrays. Presence of high-risk HPV in cervical epithelium correctly classified 37% of HSIL when compared with LSIL patients, with 61% sensitivity and 22% specificity. Promoter methylation of INTS1 correctly classified 67% (31% sensitivity, 92% specificity), promoter methylation of ZNF516 correctly classified 54% (11% sensitivity, 84% specificity), promoter methylation of FKBP6 correctly classified 59% (19% sensitivity, 88% specificity), and methylation of the HPVL1 gene correctly classified 61% (22% sensitivity, 90% specificity) of HSIL patients when compared with LSIL patients. A molecular panel comprised of HPV16-L1 methylation and promoter methylation of INTS1, ZNF516 and FKBP6 correctly classified 70% of HSIL patients with an Area Under the Curve of 0.66, 44% sensitivity, 88% specificity and 72% Positive Predictive value. While HPV16-L1 methylation discriminated patients with normal cytology from women with cervical dysplasia with close to 100% sensitivity and specificity, the high-risk HPV TrDNA qPCR assay had a pre-capture and post-capture sensitivity of 79% with 50% specificity. The detection of circulating HPV DNA in urine is a cost-effective and non-invasive alternative for cervical cancer screening. We have shown that a panel of genomic and epigenomic alterations in human and high risk HPV DNA can discriminate normal from cervical dysplasia in cervical epithelium DNA and TrDNA isolated from patients seen in a high risk cervical cancer screening setting. Citation Format: Rafael Guerrero-Preston, Anne Jedlicka, Oluwasina Folawiyo, Francesca Pirini, Blanca L. Valle, Fahcina Lawson, Angelo Vergura, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Díaz, Liliana Florea, Teresa Díaz-Montes, Josefina Romaguera, David Sidransky. Genomic and epigenomic alterations in human and high-risk HPV DNA can discriminate normal from cervical dysplasia patients in urine. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-50.

// Rafael Guerrero-Preston 1, 2 , Filipa Godoy-Vitorino 3 , Anne Jedlicka 4 , Arnold Rodriguez-Hilario 3 , Herminio Gonzalez 3 , Jessica Bondy 1 , Fahcina Lawson 1 , Oluwasina Folawiyo 1 , Christina Michailidi 1 , Amanda Dziedzic 4 , Rajagowthamee Thangavel 5 , Tal Hadar 1 , Maartje G. Noordhuis 1, 6 , William Westra 7 , Wayne Koch 1 , David Sidransky 1 1 Department of Otolaryngology and Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 2 Department of Obstetrics and Gynecology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico 3 Natural Sciences Department, Microbial Ecology and Genomics Laboratory, Inter American University of Puerto Rico, Metropolitan Campus, San Juan, Puerto Rico 4 Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Public Health, Baltimore, Maryland, USA 5 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA 6 Department of Otorhinolaryngology-Head and Neck Surgery, University of Groningen, University Medical Center, Groningen, The Netherlands 7 Department of Pathology-Surgical Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Correspondence to: Rafael Guerrero-Preston, email: rguerre3@jhmi.edu David Sidransky, email: dsidrans@jhmi.edu Keywords: microbiome, 16s rRNA, oral cancer, oropharyngeal cancer, human papilloma virus (HPV) Received: February 23, 2016 Accepted: May 16, 2016 Published: May 30, 2016 ABSTRACT 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

Somatic mutations play an important role in cancer prognosis and can pave the way for precision medicine to overcome cancer disparities. Current models hold that solid tumors evolve from microscopic clonal cellular proliferations driven through progressive histologic stages by the acquisition of somatic alterations. The accumulation of somatic mutations is associated to varying rates of stem cell divisions, leading to replicative errors that arise in response to hereditary burden, environmental stressors, or random error. Environmental, lifestyle, and neighborhood factors track closely with the acquisition of DNA methylation alterations and may partly explain the differential accumulation of genetic alterations in cancer patients. However, the combined roles of germline and somatic mutation burden, promoter methylation, and neighborhood characteristics in cancer disparities are unknown, in large part because most of the genomic studies have been performed on genomes of European descendants. Here we show that neighborhood characteristics linked to genetic, epigenetic, and tumor-associated immune signatures differ in Black and non-Latino White (NLW) head and neck squamous cell carcinoma (HNSCC) patients. Promoter methylation in PAX genes is associated to neighborhood characteristics at the zip code level (p A germline mutation in JAK3, chr19:17954215 (protein P132T), differ in Black when compared to NLW HNSCC patients (p Citation Format: Rafael Guerrero-Preston, Fahcina Lawson, Laura Manuel, Blanca Valle, Tal Hadar, Bianca Rivera, Oluwasina Folawiyo, Adriana Baez Bermejo, Luigi Marchionni, Wayne Koch, William Westra, David Sidransky. Germline and somatic mutations, immune signatures, and epigenetic alterations linked to neighborhood determinants of health differ in Black and non-Latino White head and neck cancer patients [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B43.

We set out to develp novel workflows to identify panels of methylated human papilloma virus (HPV) and human genes that can discriminate between CIN2+ and normal/CIN1 patients in liquid prep samples and in Transrenal DNA (TrDNA) isolated from urine. Using Human DNA methylation arrays and massively parallel Next Generation Sequencing (NGS) for Discovery and quantitative Methylation Specific PCR (qMSP) for validation, we found that promoter methylation of ZNF516, FKBP6, and INST1 discriminates samples with CIN2+ lesions from no intraepithelial lesions or malignancy (NILM) in cervical brush samples: 88.3% sensitivity, 88.9% specificity, 93.2 Area Under the Curve (AUC), 86.9% positive predictive value (PPV) and 90.2% negative predictive value (NPV). Using custom sequence capture pools of baits, we pulled down genomic and bisulfite converted high-risk HPV DNA before library prep for massively parallel Next Generation Sequencing (NGS) in 454 and MiSeq instruments, respectively to identify high risk HPV and the HPV methylome. Using our NGS results, we designed Syber Greeen qPCR assays to detect high risk HPV DNA in tissue and in Trans Renal DNA (TrDNA). We also designed qMSP primers-probe sets to detect methylated DNA in the HPV16-L1 gene region and ZNF516, FKBP6, and INST1 in TrDNA. TrDNA is circulating DNA that gets filtered by the kidneys and we isolate from urine as fragmented DNA ranging is size from 50 to 250 bp. We replicated the cervical brush epithelium results in liquid prep samples from another cohort, by adding HPV16-L1 methylation to the panel of methylated host genes (ZNF516, FKBP6, and INST1): 90.9% sensitivity, 60.9% specificity, AUC = 90.1, 52.6% positive predictive value (PPV) and 93.3% NPV. These results were verified in plasma DNA (AUC = 80.7) and TrDNA (AUC = 86.1) isolated from a subset of patients who provided liquid prep samples. Our results suggest that a biomarker panel that quantifies DNA methylation in the HPV-LI gene, together with promoter methylation of three host genes, ZNF516, FKBP6, and INST1, may be used as a reflex test in liquid prep to triage high-risk HPV positive women into colposcopy in countries with established cervical cancer screening programs. Our results also suggest that a panel of human and viral DNA methylation markers can be combined with a high-risk HPV DNA assay, to develop urine screening and triage tests in countries where cervical cancer screening programs are not effective due to cultural or infrastructure factors. Country specific DNA methylomes of normal and premalignant cervical epithelium for different age groups can be created as reference methylomes, to better understand the cultural-, socio-economic- and age-specific variations in the cervical epithelium and their correlation with HPV DNA methylomes. Citation Format: Rafael E. Guerrero-Preston, Blanca L. Valle, Anne Jedlicka, Nitesh Turaga, Liliana Florea, Oluwasina Folawiyo, Francesca Pirini, Angelo Vergura, Maartje Noordhuis, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Díaz, Edgardo De Jesus-Rodríguez, Keimari Méndez-Martínez, José Rodríguez-Orengo, Teresa Díaz-Montes, David Sidransky, Josefina Romaguera. Triage of high risk HPV positive women before colposcopy with reflex tests in pap smears and screening of high risk HPV in Transrenal DNA isolated from urine, using novel workflows to identify panels of methylated viral and human DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-133.

Introduction: Head and neck squamous cell carcinoma (HNSCC) survival has steadily increased over the last 50 years, but the survival gap between African Americans (AAs) and Non-Latino Whites (NLWs) has remained practically unchanged. Most of the survival differences in HNSCC survival are due to HPV infection among oropharyngeal patients, which is mostly seen in younger white males and has better prognosis than HPV-negative disease. HPV-negative HNSCC survival among AAs is associated with stage at presentation, treatment compliance, and other health services related factors. Little is known about how molecular and neighborhood-level effects are linked to HNSCC survival. Our objective is to determine the extent to which molecular alterations; clinical factors and neighborhood-level interactions are linked with HNSCC survival. We hypothesize that HNSCC survival is associated with promoter methylation and neighborhood zip code level measures of education, income, health insurance and housing variables. Methods: We conducted a retrospective analysis in specimens collected between 1985 and 2010 from the Johns Hopkins Head and Neck Cancer tumor bank. Neighborhood-level characteristics at the zip code level were collected from the US Census Bureau. We estimated HNSCC survival linked to molecular, clinical, and neighborhood variables using the Kaplan-Meier method. The log-rank test was used to test the significance of the association between survival and the following categorical variables: promoter methylation of EDNRB, NID2, PAX5, PAX1 and tumor stage; and the following determinants of health at the zip code level: high school diploma, median family income, health insurance, home ownership rate and home vacancy rate. Cox proportional hazard regression was used to test the significance of the association between survival and the following continuous variables: promoter methylation of EDNRB, NID2, PAX5, PAX1 and the following determinants of health at the zip code level: high school diploma, median family income, health insurance, home ownership rate and home vacancy rate. We then used Cox proportional hazard models for univariate and multivariate analyses. Multiple regression models were developed to describe the relationship between HNSCC survival and differential promoter methylation, clinical factors and neighborhood-level interactions within the entire study population and among racial strata. Analyses were performed using STATA version 13. Results: Of 117 patients, 63 AAs and 56 NLWs, AAs had significantly lower 5-year HNSCC cancer survival rates compared to NLWs (57% versus 67%). Within racial strata, multivariate analysis revealed that at the molecular level promoter methylation of EDNRB, NID2 and PAX5 are significant. At the individual level, tumor stage is a significantly strong predictor of HNSCC survival and has an increased HR for tumors that are at stage I (HR 4.3; 95% CI .94, 20) and II (HR 6.0; 95% CI 1.4, 26). At the neighborhood-level, health insurance, home ownership rate and home vacancy rate displayed an association with survival (P Conclusion: We conclude that HNSCC survival is driven by differences in promoter methylation of EDNRB, NID2 and PAX5 at the molecular-level, tumor stage at the individual-level and neighborhood-level effects such as health insurance, home ownership rate and home vacancy rate. Iterations of this work will improve our understanding of how molecular, individual and neighborhood-level factors influence cancer risk and survival, potentially leading to improved prevention and treatment strategies and the elimination of cancer health disparities. Citation Format: Fahcina P. Lawson, Oluwasina Folawiyo, Blanca L. Valle, Francesca Pirini, Nitesh Turaga, David Sidransky, Rafael E. Guerrero-Preston. Promoter methylation, stage at presentation, and neighborhood-level effects are linked to survival disparities in HNSCC patients. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B52.

Latino women in the United States have the highest cervical cancer incidence rate, yet the highest death rate from cervical cancer is among Black women. The disproportionate burden of cervical cancer in the United States is primarily due to lack of screening, among the medically underserved, regardless of race or ethnicity. Rural and inner city women living in poverty have lower rates of screening and higher rates of cervical cancer in the US. The advent of molecular diagnostics provides an opportunity to develop novel cervical cancer screening and triage tools that can be packaged as point of care and self-testing solutions. We set out to identify a panel of methylated HPV DNA and human genes that can discriminate between CIN2+ and normal/CIN1 patients in liquid prep samples and in Transrenal DNA (TrDNA) isolated from urine. We used three independent cohorts, from Chile, Baltimore and Puerto Rico, to develop a method that can be used to triage into colposcopy, HPV+ women with abnormal cytology. Participants were women with no cervical intraepithelial lesions or malignancy and women with abnormal cervical biopsies– Cervical Intraepithelial Neoplasia (CIN), carcinoma in-situ and cervical cancer. Using DNA methylation arrays for Discovery and quantitative Methylation Specific PCR (qMSP) for Validation, we found that promoter methylation of ZNF516, FKBP6, and INST1 discriminates samples with CIN2+ lesions from samples with no intraepithelial lesions or malignancy (NILM): 88.3% sensitivity, 88.9% specificity, 93.2 Area Under the Curve (AUC), 86.9% positive predictive value (PPV) and 90.2% negative predictive value (NPV). Using custom sequence capture pools of baits, we pulled down genomic and bisulfite converted high-risk HPV DNA before library prep for NGS in 454 and MiSeq instruments, respectively. Using our NGS results, we optimized a Syber Greeen qPCR assay to detect high risk HPV DNA and a qMSP primer-probe set to detect methylated HPV. We replicated the results in liquid prep samples (n=67), adding HPV16 methylation to the panel: 90.9% sensitivity, 60.9% specificity, 90.1 (AUC), 52.6% positive predictive value (PPV) and 93.3% NPV. These results were verified in plasma DNA (AUC=80.7) and TrDNA (AUC=86.1) isolated from a subset of patients who provided the liquid prep samples. Our results suggest that our panel of viral and host gene methylation markers may be used as a reflex test in liquid prep to triage high risk HPV positive women into colposcopy, or as a screening and triage test in TrDNA, in combination with our high risk HPV test. There are several commercial co-testing options for identification of oncogenic high-risk HPV types (HPV+) in patients with abnormal cytology. However, there are currently no tests that can reliably identify the HPV+ patients with abnormal cytology that need to be referred for colposcopy. Consequently, more than half of the women that are referred to colposcopy either have a negative biopsy, or a grade 1 Cervical Intraepithelial Neoplasia (CIN1) diagnosis, which usually reverts to normal in 12-24 months. A triage test from cytology to colposcopy that can identify in the liquid prep sample those patients with a CIN grade more likely to progress to cervical cancer (CIN2+), would decrease the number of unnecessary cervical mucosa biopsies performed today, decreasing health care cost and improving the quality of health care delivery. These molecular tools can also be packaged as point of care and self-testing solutions to eliminate cervical cancer disparities in medically underserved women. Note: This abstract was not presented at the conference. Citation Format: Rafael Guerrero-Preston, Anne Jedlicka, Blanca L. Valle, Nitesh Turaga, Liliana Florea, Oluwasina Folawiyo, Francesca Pirini, Fahcina Lawson, Angelo Vergura, Maartje Noordhuis, Gabriela Perez, Marisa Renehan, Carolina Guerrero-Díaz, Edgardo De Jesus, Teresa Diaz-Montes, Bruce Trock, Keimari Mendez, Josefina Romaguera, David Sidransky. Viral and host gene methylation in liquid prep: novel molecular screening and triage tools to reduce cervical cancer disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B11.

Background: Biobanks serve as essential resources for genetic and public health research. Despite their potential utility in elucidation of cancer susceptibility and development of novel cancer surveillance, diagnostic, and therapeutic approaches, most genomic research studies rely on samples with limited ethnic diversity. There is also a suggestion that disparities in participation in cancer biospecimen research have already translated into fewer efficacious treatments for under-represented groups. Inclusion of more diverse populations in biospecimen banks could ultimately contribute to a reduction in disparities in cancer morbidity and mortality that adversely impact U.S. minority groups. Research Objectives: We hypothesized that a deliberative group consent process related to cancer biobank donation would increase African American community engagement, foster an open dialogue between community members and researchers present at the discussion, allow participants to have their concerns addressed, and improve community members' knowledge and attitudes, leading to donation of biospecimens. Methods: We employed a two-stage study design. During the first stage, the Johns Hopkins Center to Reduce Cancer Disparities (JHCRCD) used a Community Based Participatory Research (CBPR) framework that explored the knowledge and attitudes of African American adults in Maryland regarding participation in a cancer biospecimen repository. We completed eight focus groups with primarily African American communities in Baltimore City and Prince George's County. Results: Stage-one results indicate that African American residents of Baltimore and Prince Georges County face limited understanding and lack of awareness in genetics and genomics. Our baseline analyses revealed limited community knowledge about biobanking and initial reluctance to donate biospecimen samples. Some participants expressed distrust in the research enterprise. After some discussion, however, participants were able to identify personal, familial, and societal benefits to participation. Moreover, focus group participants identified a number of strategies to overcome concerns and to enhance participation, including mechanisms for enhanced communication with researchers and suggestions related to building the biobank's community presence. In spite of initial concerns expressed during the focus groups, most participants stated their willingness to donate a specimen in the future if their concerns were properly addressed. Conclusions and Next Steps: Future reductions in cancer morbidity and mortality among minority groups will depend upon increasing the diversity of populations donating samples. The proposed project will provide data to support early communication and transparency to build community trust from the earliest stages of the cancer research process. Stage two of the project will commence in September of 2014. During the second stage, we propose: (a) To examine the impact of a deliberative group consent process on community members' attitudes and knowledge related to donating specimens to a pilot cancer biobank; (b) To identify participant characteristics (including genetic literacy, educational status, gender, personal cancer history, family cancer history, attitudes, and knowledge) that predict actual donation to a pilot cancer biobank; and (c) To create a pilot (small scale) cancer biobank with samples from the African American community for use in future genomic and epigenomic studies Citation Format: Fahcina P. Lawson, Oluwasina Folawiyo, Francesca Pirini, Blanca L. Valle, David Sidransky, Jean Ford, Lori Erby, Rafael Guerrero-Preston. Enrolling African Americans into a cancer-related biobank. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A28.

Introduction Several groups have identified hundreds of genes differentially methylated in head and neck squamous cell carcinoma (HNSCC) using genome-wide technologies. However, there is a paucity of studies examining differential methylation in HNSCC anatomic subsites. Objective Our principal objective is to determine DNA methylation differences between Head and Neck Squamous Cell Carcinoma (HNSCC) anatomic subsite: larynx, oral cavity and pharynx. Methods DNA was extracted from 24 HNSCC samples and 10 normal oral epithelium tissue samples of Puerto Rican Hispanic patients. To unveil the methylome of HNSCC anatomic subsites genomic DNA was enriched with Methylated DNA immunoprecipitation prior to labeling and hybridization to genome-wide oligonucleotide tilting arrays (3 × 720K Human CpG Island-Plus-Promoter Array, Roche/NimbleGen). Differentially methylated regions were identified with CHARM using the bumphunter algorithm. Downstream analyses were performed to identify differences in the methylation landscape of the three anatomic subsites: larynx, oral cavity and pharynx. Results When datasets were analyzed by anatomic site using DAVID v6.7 we identified 2565 differentially methylated genes common to the three subsites. We also identified 738 differentially methylated CpG loci in laryngeal cancer, 889 differentially methylated CpG loci in oral cavity and 363 differentially methylated CpG loci in pharyngeal cancer. We identified 11 key genes which were uniquely methylated in the larynx (CDH1, PAX1, BID, SMAD4, FGF19, BMP2, ITGA2, RXRA, IGFBP1, CDKN2D and RBL2). BCL2, AKT2, TRAF3, STAT5B, PTCH1, WNT9B, AXIN2, DVBL2, and APC were differentially methylated in oral cavity. In the pharynx, a comparatively lower number of differentially methylated genes were detected including B-catenin, RAF, BRAF, ATR, CTNNA1, FOXO1 and MED15. Conclusion We found evidence that HNSCC subsites (larynx, oral cavity and pharynx) manifest different methylation patterns that may be consistent with the clinical expression of the disease. A panel of differentially methylated genes identified will be further tested in a large cohort of HNSCC samples to determine their utility as prognostic biomarkers of HNSCC according to subsite. Citation Format: Bianca L. Rivera, Oluwasina Folawiyo, Nitesh Turaga, Francesca Pirini, Ricardo Lopez, Roger Vazquez, Rafael Guerrero, Adriana Baez. Genome-wide analysis identified differentially methylated regions in head and neck squamous cell carcinoma anatomic subsites. [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 LB-154. doi:10.1158/1538-7445.AM2015-LB-154