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).

The distributions of relative QMSP values of PAX5 methylation are shown.

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.

Supplementary Table S1: List of primers and probes used for QMSP and ddQMSP Supplementary Table S2: Analysis of clinicopathological and molecular factors in association with clinical outcomes of imprint cases Supplementary Table S3: Analysis of clinicopathological and molecular factors in association with clinical outcomes of tissue cases Supplementary Table S4: QMSP assay of 6 HNSCC surgical margin tissues which were histologically cancer-positive

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.

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.

Supplementary Table S1.The PCR condition and sequences of the primer and the fluorescent probe used in this study.

Cervical cancer is one of the most common cancers in women. Despite progress in prevention through Human Papilloma Virus [HPV] vaccination and success in early detection of cervical cancer through cytologic screening and HPV detection, there remains an unequal burden in low-resource settings in developed and developing countries. Applying novel methylation-based detection methods, we validated a panel of three human methylated genes (ZNF516, FKBP6 and INTS1) on discarded cervical liquid cytology samples from clinical laboratories in the United States during the development of the CervicalMethDx test. We hypothesized that the CervicalMethDx test can identify HPV positive women most likely to be diagnosed with Cervical Intraepithelial Neoplasia (CIN) grades 2 and 3 by anatomic pathologists, before they are referred to colposcopy-driven biopsies. We assessed DNA methylation by Quantitative Real Time Methylation Specific PCR (QMSP) analysis of sodium bisulfite-modified genomic DNA. Primers and probes were previously designed to specifically amplify the promoters of the 3 genes of interest and the promoter of a reference gene, β-actin, to assess DNA input. We performed blinded retrospective studies on well-characterized, discarded, HPV-positive clinical samples in PreservCyt sample transport media (Thin Prep, Hologic), comparing DNA methylation levels in samples from CIN2 and CIN3 cases (346), and 120 controls without evidence of Intraepithelial Lesions or Malignancy (NILM) as an endpoint. Our results showed that the CervicalMethDx test can correctly classify 96% of CIN2 (n=197) samples with 93% Sensitivity, 100% Specificity, and an AUC of 0.96 as well as 95% of CIN3 (n=149) samples with 91% Sensitivity, 100% Specificity, and an AUC of 0.96. Moreover, the assay correctly classified 94% of CIN2-CIN3 samples combined (n=346) with 92% Sensitivity, 100% Specificity, an AUC of 0.96, and a 100% positive predictive value (PPV), when compared to samples with NILM (n=120). The CervicalMethDx test high PPV supports the addition of this low-cost molecular test to cervical cancer screening algorithms worldwide. Our results suggest that the CervicalMethDx test is a new and valuable tool to stratify HPV positive women prior to colposcopy-driven biopsies in developed countries and ablative treatment in developing countries, most of which are unnecessary. These results warrant further evaluation of the CervicalMethDX test in prospective, population-based studies, assessing the use of precision DNA methylation algorithms to triage women before colposcopy-driven biopsies or ablative treatments worldwide. Citation Format: Laura Palmieri, Fernando T. Zamuner, Dieila Giomo De Lima, Keerthana Gosala, Eli Winkler, Yash Prashar, Ana Purcell-Wiltz, Amanda García-Negrón, Ashley Ramos-Lopez, David Sidransky, Mariana Brait, Rafael Guerrero-Preston. CervicalMethDx: A precision DNA methylation test to identify advanced disease risk in cervical cancer screening algorithms. [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 4192.

Several genomic epidemiology tools have been developed to track the public and population health impact of SARS-CoV-2 community spread worldwide. A SARS-CoV-2 Variant of Concern (VOC) B.1.1.7, known as 501Y.V1, which shows increased transmissibility, has rapidly become the dominant VOC in the United States (US). Our objective was to develop an evidenced-based genomic surveillance algorithm that combines RT-PCR and sequencing technologies to identify VOCs. Deidentified data were obtained from 508,969 patients tested for 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 US (n=422,330). 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 is concurrent with a sharp increase in VOCs among all SARS-CoV-2 sequences from Puerto Rico uploaded to GISAID (n=461). B.1.1.7 frequency increased from

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 (

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12079-018-00503-5) contains supplementary material, which is available to authorized users.

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.

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.