Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-29 (of 29 Records) |
Query Trace: Unger Elizabeth[original query] |
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Identification of Phosphoglycerate Kinase 1 (PGK1) as a reference gene for quantitative gene expression measurements in human blood RNA.
Falkenberg VR , Whistler T , Murray JR , Unger ER , Rajeevan MS . BMC Res Notes 2011 4 324 BACKGROUND: Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS). Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs), have not been described. FINDINGS: Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT)-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. Phosphoglycerate kinase 1 (PGK1) was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; Ribosomal protein large, P0 (RPLP0) for PBMC RNA and Peptidylprolyl isomerase B (PPIB) for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used. CONCLUSIONS: We have identified PGK1 as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of gene expression results from blood RNA collected and processed by different methods with the intention of biomarker discovery. Results of this study should facilitate large-scale molecular epidemiologic studies using blood RNA as the target of quantitative gene expression measurements. |
Relationships of p16 Immunohistochemistry and Other Biomarkers With Diagnoses of Cervical Abnormalities: Implications for LAST Terminology
Castle PE , Adcock R , Cuzick J , Wentzensen N , Torrez-Martinez NE , Torres SM , Stoler MH , Ronnett BM , Joste NE , Darragh TM , Gravitt PE , Schiffman M , Hunt WC , Kinney WK , Wheeler CM , New Mexico HPV Pap Registry Steering Committee and for the p16 IHC Study Panel , Bernard Vicki , Unger Elizabeth . Arch Pathol Lab Med 2020 144 (6) 725-734 CONTEXT.—: Lower Anogenital Squamous Terminology (LAST) standardization recommended p16(INK4a) immunohistochemistry (p16 IHC) for biopsies diagnosed morphologically as cervical intraepithelial neoplasia (CIN) grade 2 (CIN2) to classify them as low-grade or high-grade squamous intraepithelial lesions (HSILs). OBJECTIVE.—: To describe the relationships of p16 IHC and other biomarkers associated with cervical cancer risk with biopsy diagnoses. DESIGN.—: A statewide, stratified sample of cervical biopsies diagnosed by community pathologists (CPs), including 1512 CIN2, underwent a consensus, expert pathologist panel (EP) review (without p16 IHC results), p16 IHC interpretation by a third pathology group, and human papillomavirus (HPV) genotyping, results of which were grouped hierarchically according to cancer risk. Antecedent cytologic interpretations were also available. RESULTS.—: Biopsies were more likely to test p16 IHC positive with increasing severity of CP diagnoses, overall (P(trend) ≤ .001) and within each HPV risk group (P(trend) ≤ .001 except for low-risk HPV [P(trend) < .010]). All abnormal grades of CP-diagnosed biopsies were more likely to test p16 IHC positive with a higher HPV risk group (P(trend) < .001), and testing p16 IHC positive was associated with higher HPV risk group than testing p16 IHC negative for each grade of CP-diagnosed biopsies (P < .001). p16 IHC-positive, CP-diagnosed CIN2 biopsies were less likely than CP-diagnosed CIN3 biopsies to test HPV16 positive, have an antecedent HSIL(+) cytology, or to be diagnosed as CIN3(+) by the EP (P < .001 for all). p16 IHC-positive, CP-diagnosed CIN1 biopsies had lower HPV risk groups than p16 IHC-negative, CP-diagnosed CIN2 biopsies (P < .001). CONCLUSIONS.—: p16 IHC-positive, CP-diagnosed CIN2 appears to be lower cancer risk than CP-diagnosed CIN3. LAST classification of "HSIL" diagnosis, which includes p16 IHC-positive CIN2, should annotate the morphologic diagnosis (CIN2 or CIN3) to inform all management decisions, which is especially important for young (<30 years) women diagnosed with CIN2 for whom surveillance rather than treatment is recommended. |
NanoString Technology for Human Papillomavirus Typing.
Rajeevan MS , Patel S , Li T , Unger ER . Viruses 2021 13 (2) High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter(®) platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 10(4) copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 10(4) plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples. |
Bioinformatics Pipeline for Human Papillomavirus Short Read Genomic Sequences Classification Using Support Vector Machine.
Lomsadze A , Li T , Rajeevan MS , Unger ER , Borodovsky M . Viruses 2020 12 (7) We recently developed a test based on the Agilent SureSelect target enrichment system capturing genomic fragments from 191 human papillomaviruses (HPV) types for Illumina sequencing. This enriched whole genome sequencing (eWGS) assay provides an approach to identify all HPV types in a sample. Here we present a machine learning algorithm that calls HPV types based on the eWGS output. The algorithm based on the support vector machine (SVM) technique was trained on eWGS data from 122 control samples with known HPV types. The new algorithm demonstrated good performance in HPV type detection for designed samples with 25 or greater HPV plasmid copies per sample. We compared the results of HPV typing made by the new algorithm for 261 residual epidemiologic samples with the results of the typing delivered by the standard HPV Linear Array (LA). The agreement between methods (97.4%) was substantial (kappa= 0.783). However, the new algorithm identified additionally 428 instances of HPV types not detectable by the LA assay by design. Overall, we have demonstrated that the bioinformatics pipeline is an accurate tool for calling HPV types by analyzing data generated by eWGS processing of DNA fragments extracted from control and epidemiological samples. |
Vaccine effectiveness on DNA prevalence of human papillomavirus infection in anal and oral specimens from men who have sex with men- United States, 2016-2018.
Meites E , Winer RL , Newcomb ME , Gorbach PM , Querec TD , Rudd J , Collins T , Lin J , Moore J , Remble T , Swanson F , Franz J , Bolan RK , Golden MR , Mustanski B , Crosby RA , Unger ER , Markowitz LE . J Infect Dis 2020 222 (12) 2052-2060 BACKGROUND: In the United States, human papillomavirus (HPV) vaccination has been recommended for young adult men who have sex with men (MSM) since 2011. METHODS: The Vaccine Impact in Men (VIM) study surveyed MSM and transgender women aged 18-26 years in 3 U.S. cities during 2016-2018. Self-collected anal swab and oral rinse specimens were assessed for 37 types of HPV DNA. We compared HPV prevalence among vaccinated and unvaccinated participants and determined adjusted prevalence ratios (aPR) and confidence intervals (CI). RESULTS: Among 1,767 participants, 704 (39.8%) self-reported receiving HPV vaccine. Median age at vaccination (18.7 years) was older than age at first sex (15.7 years). Quadrivalent vaccine-type HPV was detected in anal or oral specimens from 475 (26.9%) participants. Vaccine-type HPV prevalence was lower among vaccinated (22.9%) compared with unvaccinated (31.6%) participants; aPR for those who initiated vaccination at </=18 years was 0.41 (95% CI: 0.24-0.57) and at >18 years was 0.82 (95% CI: 0.67-0.98). Vaccine effectiveness for at least one HPV vaccine dose at age >/=18 years or >18 years was 59% and 18%, respectively. CONCLUSIONS: Findings suggest real-world effectiveness of HPV vaccination among young adult MSM. This effect was stronger with younger age at vaccination. |
Results of a Pilot Study of a Mail-Based Human Papillomavirus Self-Testing Program for Underscreened Women From Appalachian Ohio.
Reiter PL , Shoben AB , McDonough D , Ruffin MT , Steinau M , Unger ER , Paskett ED , Katz ML . Sex Transm Dis 2019 46 (3) 185-190 BACKGROUND: Human papillomavirus (HPV) self-testing is an emerging cervical cancer screening strategy, yet few mail-based HPV self-testing programs have been implemented in the United States. We report the results of a pilot study of a mail-based program, the Health Outcomes through Motivation and Education Project. METHODS: In 2015 to 2016, we recruited 103 women from Appalachian Ohio who were aged 30 to 65 years and had not received a Papanicolaou (Pap) test in at least 3 years. Women were mailed an HPV self-test and randomized to receive either (a) self-test instructions developed by the device manufacturer and a standard information brochure about cervical cancer (control group) or (b) self-test instructions developed by the Health Outcomes through Motivation and Education Project and a photo story information brochure about cervical cancer (intervention group). Logistic regression compared study arms on HPV self-test return and receipt of a Pap test. RESULTS: Overall, 80 (78%) women returned their HPV self-test. Return was similar among the intervention and control groups (78% vs. 77%; odds ratio, 1.09; 95% confidence interval, 0.43-2.76). Among returners, 26% had an oncogenic HPV type detected in their sample. Women who returned their self-test reported high levels of satisfaction and positive experiences with the self-testing process. Few women overall received a Pap test (11%), and Pap testing was similar among the intervention and control groups (14% vs. 8%; odds ratio, 1.91; 95% confidence interval, 0.52-6.97). CONCLUSIONS: Mail-based HPV self-testing programs are a potentially promising strategy for reaching underscreened women in Appalachia. Efforts are needed to better understand how to optimize the success of such programs. |
An Isothermal, Multiplex Amplification Assay for Detection and Genotyping of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues.
Tang YW , Lozano L , Chen X , Querec TD , Katabi N , Moreno-Docon A , Wang H , Fix D , De Brot L , McMillen TA , Yoon JY , Torroba A , Wang Y , Unger ER , Park KJ . J Mol Diagn 2020 22 (3) 419-428 Rapid and accurate identification of human papillomavirus (HPV) is important for both clinical management and population screening. We performed analytic validation of Atila AmpFire Multiplex HPV assays on formalin-fixed, paraffin-embedded (FFPE) cervix/vulva and oropharynx diagnostic tissue samples. The AmpFire assay incorporates a novel isothermal multiplex amplification coupled with real-time fluorescent detection to detect and genotype 15 high-risk (HR) HPV genotypes. Limits of detection determined by plasmids cloned with HPV genotype-specific sequences were 2 copies/reaction for HPV16, HPV18, and some HR HPV genotypes, and 20 copies/reaction for the remaining HR HPV genotypes. The performance of the AmpFire assays in clinical samples was evaluated using 214 FFPE specimens. The AmpFire assay failed in one clinical specimen for an invalid rate of 0.5%. The AmpFire assay detected HPV in clinical samples with positive percent agreements of 100.0% for HPV16, 100.0% for HPV18, and 94.7% for non-16/18 HR-HPV, and 100% negative percent agreements for HPV16, HPV18, and non-16/18 HR-HPV. Qualitative detection agreement was obtained in the reproducibility study. In summary, the Atila AmpFire HPV assay demonstrated excellent analytic sensitivity and specificity for detection and genotyping of 15 HR HPV genotypes. Assay parameters of simple specimen processing, small sample size requirement, rapid turnaround time, and being near instrument-free render it well suited for HPV detection and genotyping in FFPE specimens. |
Universal human papillomavirus typing by whole genome sequencing following target enrichment: evaluation of assay reproducibility and limit of detection.
Li T , Unger ER , Rajeevan MS . BMC Genomics 2019 20 (1) 231 BACKGROUND: We recently described a method for unbiased detection of all known human papillomaviruses (HPV) types with the potential for the determination of their variant and integration from the resulting whole genome sequence data. Considering the complex workflow for target-enriched next generation sequencing (NGS), we focused on the reproducibility and limit of detection (LOD) of this new universal HPV typing assay in this study. RESULTS: We evaluated the reproducibility and LOD for HPV genotyping based on our recently published method that used RNA-baits targeting whole genomes of 191 HPV types, Agilent SureSelect protocol for target enrichment and Illumina HiSeq 2500 for sequencing (eWGS, enriched whole genome sequencing). Two libraries, prepared from pooled plasmids representing 9 vaccine HPV types at varying input (1-625 copies/reaction), were sequenced twice giving four replicates for evaluating reproducibility and LOD. eWGS showed high correlation in the number of reads mapped to HPV reference genomes between the two flow-cell lanes within (R(2) = 1) and between experiments (R(2) = 0.99). The number of mapped reads was positively correlated to copy number (beta = 13.9, p < 0.0001). The limit of blank (LOB) could be calculated based on mapped reads to HPV types not included in each sample. HPV genotyping was reproducible for all 9 types at 625 copies using multiple cut-off criteria but LOD was 25 copies based on number of reads above LOB even when multiple types were present. eWGS showed no bias for HPV genotyping under single or multiple infection (p = 0.16-0.99). CONCLUSIONS: The universal eWGS method for HPV genotyping has sensitivity, competitive with widely used consensus PCR methods with reduced type competition, and with the potential for determination of variant and integration status. The protocol used in this study, using defined samples varying in complexity and copy number, analyzed in replicate and duplicate assays, is applicable to most WGS methods. |
Simultaneous extraction of mRNA and microRNA from whole blood stabilized in tempus tubes.
Richards J , Unger ER , Rajeevan MS . BMC Res Notes 2019 12 (1) 39 OBJECTIVE: Studies of mRNA and miRNA expression profiling increasingly use stabilized whole blood. Commercial RNA extraction kits do not provide information about the simultaneous recovery of both mRNA and miRNA. This study evaluated yield, quality, integrity and representation of mRNA and miRNA from whole blood stabilized in Tempus tubes using three RNA extraction kits; two filter-based (Tempus and Norgen) and one bead-based (MagMax; manual vs. semi-automated, and with and without DNase treatment). RESULTS: All RNA extraction kits and methods resulted in similar yields of mRNA (total RNA yield, quality, integrity and representation) whereas there were differences in yields of miRNA. MagMax, either manual or semi-automated, with or without DNase treatment, yielded 1.6-2.2-fold more miRNA than Tempus and Norgen kits. In addition, MagMax and Norgen methods gave greater than 12-fold more and 3.3-fold less enrichment of specific miRNA targets, respectively, in comparison to Tempus extraction reagents. This study identified MagMax kit for simultaneous recovery of both mRNA and miRNA from whole blood collected in Tempus tubes. |
Population-Based Assessment of HPV Genotype-Specific Cervical Cancer Survival: CDC Cancer Registry Sentinel Surveillance System.
Hallowell BD , Saraiya M , Thompson TD , Unger ER , Lynch CF , Tucker T , Copeland G , Hernandez BY , Peters ES , Wilkinson E , Goodman MT . JNCI Cancer Spectr 2018 2 (3) Background: Human papillomavirus (HPV) genotype influences the development of invasive cervical cancer (ICC); however, there is uncertainty regarding the association of HPV genotype with survival among ICC patients. Method(s): Follow-up data were collected from 693 previously selected and HPV-typed ICC cases that were part of the Centers for Disease Control and Prevention Cancer Registry Surveillance System. Cases were diagnosed between 1994 and 2005. The Kaplan-Meier method was used to estimate five-year all-cause survival. A multivariable Cox proportional hazards model was used to estimate the effect of HPV genotype on survival after adjusting for demographic, tumor, and treatment characteristics. Result(s): Five-year all-cause survival rates varied by HPV status (HPV 16: 66.9%, HPV 18: 65.7%, HPV 31/33/45/52/58: 70.8%, other oncogenic HPV genotypes: 79.0%, nononcogenic HPV: 69.3%, HPV-negative: 54.0%). Following multivariable adjustment, no statistically significant survival differences were found for ICC patients with HPV 16-positive tumors compared with women with tumors positive for HPV 18, other oncogenic HPV types, or HPV-negative tumors. Women with detectable HPV 31/33/33/ 45/52/58 had a statistically significant 40% reduced hazard of death at five years (95% confidence interval [CI] = 0.38 to 0.95), and women who tested positive for nononcogenic HPV genotypes had a statistically significant 57% reduced hazard of death at five years (95% CI = 0.19 to 0.96) compared with women with HPV 16 tumors. Few statistically significant differences in HPV positivity, tumor characteristics, treatment, or survival were found by race/ethnicity. Conclusion(s): HPV genotype statistically significantly influenced five-year survival rates among women with ICC; however, screening and HPV vaccination remain the most important factors to improve patient prognosis and prevent future cases. |
Association of chronic fatigue syndrome with premature telomere attrition.
Rajeevan MS , Murray J , Oakley L , Lin JS , Unger ER . J Transl Med 2018 16 (1) 44 BACKGROUND: Chronic fatigue syndrome (CFS), also known as myalgic encephalomyelitis (ME), is a severely debilitating condition of unknown etiology. The symptoms and risk factors of ME/CFS share features of accelerated aging implicated in several diseases. Using telomere length as a marker, this study was performed to test the hypothesis that ME/CFS is associated with accelerated aging. METHODS: Participant (n = 639) data came from the follow-up time point of the Georgia CFS surveillance study. Using the 1994 CFS Research Case Definition with questionnaire-based subscale thresholds for fatigue, function, and symptoms, participants were classified into four illness groups: CFS if all criteria were met (n = 64), CFS-X if CFS with exclusionary conditions (n = 77), ISF (insufficient symptoms/fatigue) if only some criteria were met regardless of exclusionary conditions (n = 302), and NF (non-fatigued) if no criteria and no exclusionary conditions (n = 196). Relative telomere length (T/S ratio) was measured using DNA from whole blood and real-time PCR. General linear models were used to estimate the association of illness groups or T/S ratio with demographics, biological measures and covariates with significance set at p < 0.05. RESULTS: The mean T/S ratio differed significantly by illness group (p = 0.0017); the T/S ratios in CFS (0.90 +/- 0.03) and ISF (0.94 +/- 0.02) were each significantly lower than in NF (1.06 +/- 0.04). Differences in T/S ratio by illness groups remained significant after adjustment for covariates of age, sex, body mass index, waist-hip ratio, post-exertional malaise and education attainment. Telomere length was shorter by 635, 254 and 424 base pairs in CFS, CFS-X and ISF, respectively, compared to NF. This shorter telomere length translates to roughly 10.1-20.5, 4.0-8.2 and 6.6-13.7 years of additional aging in CFS, CFS-X and ISF compared to NF respectively. Further, stratified analyses based on age and sex demonstrated that the association of ME/CFS with short telomeres is largely moderated by female subjects < 45 years old. CONCLUSIONS: This study found a significant association of ME/CFS with premature telomere attrition that is largely moderated by female subjects < 45 years old. Our results indicate that ME/CFS could be included in the list of conditions associated with accelerated aging. Further work is needed to evaluate the functional significance of accelerated aging in ME/CFS. |
Precision Medicine Requires Precision Laboratories.
Rajeevan MS , Li T , Unger ER . J Mol Diagn 2017 19 (2) 226-229 This commentary highlights the validation study by Lih et al that supports the use of precision medicine for improved clinical trials. |
Universal Human Papillomavirus Typing Assay: Whole Genome Sequencing Following Target Enrichment.
Li T , Unger ER , Batra D , Sheth M , Steinau M , Jasinski J , Jones J , Rajeeven MS . J Clin Microbiol 2016 55 (3) 811-823 We designed a universal HPV typing assay based on target enrichment and whole genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 targeting beta-globin as control. We used Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing and CLC genomics workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall-type specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology. |
Population-Level Effects of Human Papillomavirus Vaccination Programs on Infections with Nonvaccine Genotypes.
Mesher D , Soldan K , Lehtinen M , Beddows S , Brisson M , Brotherton JM , Chow EP , Cummings T , Drolet M , Fairley CK , Garland SM , Kahn JA , Kavanagh K , Markowitz L , Pollock KG , Soderlund-Strand A , Sonnenberg P , Tabrizi SN , Tanton C , Unger E , Thomas SL . Emerg Infect Dis 2016 22 (10) 1732-40 We analyzed human papillomavirus (HPV) prevalences during prevaccination and postvaccination periods to consider possible changes in nonvaccine HPV genotypes after introduction of vaccines that confer protection against 2 high-risk types, HPV16 and HPV18. Our meta-analysis included 9 studies with data for 13,886 girls and women ≤19 years of age and 23,340 women 20-24 years of age. We found evidence of cross-protection for HPV31 among the younger age group after vaccine introduction but little evidence for reductions of HPV33 and HPV45. For the group this same age group, we also found slight increases in 2 nonvaccine high-risk HPV types (HPV39 and HPV52) and in 2 possible high-risk types (HPV53 and HPV73). However, results between age groups and vaccines used were inconsistent, and the increases had possible alternative explanations; consequently, these data provided no clear evidence for type replacement. Continued monitoring of these HPV genotypes is important. |
Validation of a standardized extraction method for formalin-fixed paraffin-embedded tissue samples.
Lagheden C , Eklund C , Kleppe SN , Unger ER , Dillner J , Sundstrom K . J Clin Virol 2016 80 36-39 BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) samples can be DNA-extracted and used for human papillomavirus (HPV) genotyping. The xylene-based gold standard for extracting FFPE samples is laborious, suboptimal and involves health hazards for the personnel involved. OBJECTIVES: To compare extraction with the standard xylene method to a xylene-free method used in an HPV LabNet Global Reference Laboratory at the Centers for Disease Control (CDC); based on a commercial method with an extra heating step. STUDY DESIGN: Fifty FFPE samples were randomly selected from a national audit of all cervical cancer cases diagnosed in Sweden during 10 years. For each case-block, a blank-block was sectioned, as a control for contamination. For xylene extraction, the standard WHO Laboratory Manual protocol was used. For the CDC method, the manufacturers' protocol was followed except for an extra heating step, 120 degrees C for 20min. Samples were extracted and tested in parallel with beta-globin real-time PCR, HPV16 real-time PCR and HPV typing using modified general primers (MGP)-PCR and Luminex assays. RESULTS: For a valid result the blank-block had to be betaglobin-negative in all tests and the case-block positive for beta-globin. Overall, detection was improved with the heating method and the amount of HPV-positive samples increased from 70% to 86% (p=0.039). For all samples where HPV type concordance could be evaluated, there was 100% type concordance. CONCLUSIONS: A xylene-free and robust extraction method for HPV-DNA typing in FFPE material is currently in great demand. Our proposed standardized protocol appears to be generally useful. |
Human papillomavirus genotype and oropharynx cancer survival in the United States of America.
Goodman MT , Saraiya M , Thompson TD , Steinau M , Hernandez BY , Lynch CF , Lyu CW , Wilkinson EJ , Tucker T , Copeland G , Peters ES , Altekruse S , Unger ER . Eur J Cancer 2015 51 (18) 2759-67 BACKGROUND: The presence of human papillomavirus (HPV) DNA in oropharyngeal squamous cell cancer (OPSCC) tissue appears to be a strong predictor of improved prognosis, but this observation has not been explored in a population-based sample with generalisable findings. METHODS: Follow-up data from a large sample of OPSCC patients identified through six population-based cancer registries in the United States of America (USA) were used to characterise the association of tumour HPV status with survival. RESULTS: HPV DNA was detected in tumour tissue from 71% (378 in 529) of the OPSCC patients. A total of 65% of patients with HPV16-associated tumours survived 5 years compared to 46% of patients with other HPV types and 28% of patients with HPV-negative tumours (p log-rank test <0.0001). The OPSCC patients with detectable HPV16 DNA had a 62% reduced hazard of death at 5 years, and patients with other HPV types had a 42% reduced hazard of death at 5 years compared to HPV-negative patients. Compared to non-Hispanic Whites, Blacks with OPSCC had a 2.6-fold greater risk of death at 5 years after adjustment for HPV status and other prognostic variables. Both surgery and radiation therapy were associated with a reduced 5-year risk of death, but no evidence was found for an interaction between HPV status and radiotherapy or surgery on survival time. CONCLUSIONS: Data from this US study suggest that HPV16-positive OPSCC patients survive longer than HPV-negative patients regardless of treatment, highlighting the prognostic importance of HPV status for this malignancy. Optimal treatment regimens for OPSCC could be tailored to each patient's HPV status and prognostic profile. |
Pathway-Focused Genetic Evaluation of Immune and Inflammation Related Genes with Chronic Fatigue Syndrome.
Rajeevan MS , Dimulescu I , Murray J , Falkenberg VR , Unger ER . Hum Immunol 2015 76 (8) 553-60 Recent evidence suggests immune and inflammatory alterations are important in chronic fatigue syndrome (CFS). This study was done to explore the association of functionally important genetic variants in inflammation and immune pathways with CFS. Peripheral blood DNA was isolated from 50 CFS and 121 non-fatigued (NF) control participants in a population-based study. Genotyping was performed with the Affymetrix Immune and Inflammation Chip that covers 11K single nucleotide polymorphisms (SNP) following the manufacturer's protocol. Genotyping accuracy for specific genes was validated by pyrosequencing. Golden Helix SVS software was used for genetic analysis. SNP functional annotation was done using SPOT and GenomePipe programs. CFS was associated with 32 functionally important SNPs: 11 missense variants, 4 synonymous variants, 11 untranslated regulatory region (UTR) variants and 6 intronic variants. Some of these SNPs were in genes within pathways related to complement cascade (SERPINA5, CFB, CFH, MASP1 and C6), chemokines (CXCL16, CCR4, CCL27), cytokine signaling (IL18, IL17B, IL2RB), and toll-like receptor signaling (TIRAP, IRAK4). Of particular interest is association of CFS with two missense variants in genes of complement activation, rs4151667 (L9H) in CFB and rs1061170 (Y402H) in CFH. A 5'UTR polymorphism (rs11214105) in IL18 also associated with physical fatigue, body pain and score for CFS case defining symptoms. This study identified new associations of CFS with genetic variants in pathways including complement activation providing additional support for altered innate immune response in CFS. Additional studies are needed to validate the findings of this exploratory study. |
Human papillomavirus genotype prevalence in invasive penile cancers from a registry-based United States population.
Hernandez BY , Goodman MT , Unger ER , Steinau M , Powers A , Lynch CF , Cozen W , Saber MS , Peters ES , Wilkinson EJ , Copeland G , Hopenhayn C , Huang Y , Watson M , Altekruse SF , Lyu C , Saraiya M . Front Oncol 2014 4 9 BACKGROUND: Human papillomavirus (HPV) is estimated to play an etiologic role in 40-50% of penile cancers worldwide. Estimates of HPV prevalence in U.S. penile cancer cases are limited. METHODS: HPV DNA was evaluated in tumor tissue from 79 invasive penile cancer patients diagnosed in 1998-2005 within the catchment areas of seven U.S. cancer registries. HPV was genotyped using PCR-based Linear Array and INNO-LiPA assays and compared by demographic, clinical, and pathologic characteristics and survival. Histological classification was also obtained by independent pathology review. RESULTS: HPV DNA was present in 50 of 79 (63%) of invasive penile cancer cases. Sixteen viral genotypes were detected. HPV 16, found in 46% (36/79) of all cases (72% of HPV-positive cases) was the most prevalent genotype followed equally by HPV 18, 33, and 45, each of which comprised 5% of all cases. Multiple genotypes were detected in 18% of viral positive cases. HPV prevalence did not significantly vary by age, race/ethnicity, population size of geographic region, cancer stage, histology, grade, penile subsite, or prior cancer history. Penile cases diagnosed in more recent years were more likely to be HPV-positive. Overall survival did not significantly vary by HPV status. CONCLUSION: The relatively high prevalence of HPV in our study population provides limited evidence of a more prominent and, possibly, increasing role of infection in penile carcinogenesis in the U.S. compared to other parts of the world. |
Urine-based human papillomavirus DNA testing as a screening tool for cervical cancer in high-risk women.
Mendez K , Romaguera J , Ortiz AP , Lopez M , Steinau M , Unger ER . Int J Gynaecol Obstet 2014 124 (2) 151-5 OBJECTIVE: To test the hypothesis that self-collected urine could be used to detect high-risk human papillomavirus (HPV) DNA with sensitivity and specificity comparable to those of standard cervical testing. METHODS: Women attending a gynecology clinic for evaluation of abnormal cytology were recruited. Fifty-two participants (21-60years of age) collected urine samples, and clinicians collected cervical brush samples. When appropriate, cervical biopsies were obtained during colposcopy. HPV detection and typing were performed on DNA extracts from each sample, using commercial reagents for L1 consensus polymerase chain reaction (PCR) and type-specific hybridization. HPV 16 viral load was determined by quantitative PCR in HPV 16-positive samples. A diagnostic test analysis was conducted for urine samples. RESULTS: Fifty paired samples were analyzed, with 76% agreement between samples. The 12 discrepant pairs were all urine negative/cervix positive. The most common HPV types detected were 16, 51, 53, and 62. The urine test correctly identified 100% of the uninfected and 65% of the infected patients. CONCLUSION: The results indicate that HPV DNA detection using urine is less sensitive than cervical sampling in a population with abnormal cytology. Further exploration is warranted to determine clinical utility when other options are unavailable. |
Human papillomavirus genotypes in high-grade cervical lesions in the United States.
Hariri S , Unger ER , Powell SE , Bauer HM , Bennett NM , Bloch KC , Niccolai LM , Schafer S , Steinau M , Markowitz LE . J Infect Dis 2012 206 (12) 1878-86 BACKGROUND: Two vaccines protect against human papillomaviruses (HPV) 16 and 18 that cause 70% of cervical cancer and 50% of cervical intraepithelial neoplasia 2/3 and adenocarcinoma in situ (CIN2+). Monitoring HPV types in CIN2+ may be used to assess HPV vaccine impact. METHODS: As part of a multi-site vaccine impact monitoring project (HPV-IMPACT), biopsy specimens used to diagnose CIN2+ were obtained for HPV DNA typing for women aged 18-39 years. RESULTS: Among 4,121 CIN2+ cases reported from 2008-2009 in 18-39 year-old women 3,058 (74.2%) were tested; 96% were HPV DNA positive. HPV16 was most common (49.1%), followed by HPV31 (10.4%) and HPV52 (9.7%). HPV18 prevalence was 5.5% overall. Proportion of CIN2+ cases associated with HPV16/18 was highest (56.3%) in 25-29 year-old women. HPV16/18-associated lesions were less common in non-Hispanic blacks (41.9%) and Hispanics (46.3%) compared to non-Hispanic whites (59.1%) (p<.0001); the difference remained significant when adjusted for covariates. Compared to non-Hispanic white, HPV35 and 58 were significantly more common in non-Hispanic black (14.5% vs. 4.2%; 12.3% vs. 3.4%) and HPV45 was higher in Hispanic cases (3.7% vs. 1.5%). CONCLUSION: Age and racial/ethnic differences in HPV type distribution may have implications for vaccine impact, and must be considered in monitoring trends. |
HPV genotypes in high grade cervical lesions and invasive cervical carcinoma as detected by two commercial DNA assays, North Carolina, 2001-2006.
Hariri S , Steinau M , Rinas A , Gargano JW , Ludema C , Unger ER , Carter AL , Grant KL , Bamberg M , McDermott JE , Markowitz LE , Brewer NT , Smith JS . PLoS One 2012 7 (3) e34044 BACKGROUND: HPV typing using formalin fixed paraffin embedded (FFPE) cervical tissue is used to evaluate HPV vaccine impact, but DNA yield and quality in FFPE specimens can negatively affect test results. This study aimed to evaluate 2 commercial assays for HPV detection and typing using FFPE cervical specimens. METHODS: Four large North Carolina pathology laboratories provided FFPE specimens from 299 women ages18 and older diagnosed with cervical disease from 2001 to 2006. For each woman, one diagnostic block was selected and unstained serial sections were prepared for DNA typing. Extracts from samples with residual lesion were used to detect and type HPV using parallel and serial testing algorithms with the Linear Array and LiPA HPV genotyping assays. FINDINGS: LA and LiPA concordance was 0.61 for detecting any high-risk (HR) and 0.20 for detecting any low-risk (LR) types, with significant differences in marginal proportions for HPV16, 51, 52, and any HR types. Discordant results were most often LiPA-positive, LA-negative. The parallel algorithm yielded the highest prevalence of any HPV type (95.7%). HR type prevalence was similar using parallel (93.1%) and serial (92.1%) approaches. HPV16, 33, and 52 prevalence was slightly lower using the serial algorithm, but the median number of HR types per woman (1) did not differ by algorithm. Using the serial algorithm, HPV DNA was detected in >85% of invasive and >95% of pre-invasive lesions. The most common type was HPV16, followed by 52, 18, 31, 33, and 35; HPV16/18 was detected in 56.5% of specimens. Multiple HPV types were more common in lower grade lesions. CONCLUSIONS: We developed an efficient algorithm for testing and reporting results of two commercial assays for HPV detection and typing in FFPE specimens, and describe HPV type distribution in pre-invasive and invasive cervical lesions in a state-based sample prior to HPV vaccine introduction. |
A real-time PCR assay for HPV52 detection and viral load quantification.
Onyekwuluje JM , Steinau M , Swan DC , Unger ER . Clin Lab 2012 58 61-6 BACKGROUND: Human papillomavirus (HPV) 52 is one of the most frequent high risk HPV types found in cervical and anal infections. Reliable and well characterized methods for HPV 52 detection are therefore of great importance. Unfortunately one of the most widely used commercially available HPV typing assays, the Roche Linear Array (LA), detects type 52 only through its XR probe which cross-reacts with types 33, 35 and 58 and fails to give unambiguous detection of HPV 52. METHODS: To address this problem a real-time TaqMan PCR assay for HPV 52 targeting the E6/E7 region was developed and validated, which can be applied as robust duplex assay simultaneously detecting beta-globin as genomic control and reference or as highly sensitive single target detection assay. RESULTS: Optimized primer and probe concentrations produced linear PCR amplifications over seven logs of targets (10(1) - 10(7)). The detection limit for HPV 52 was reproducibly at 10 copies per reaction for the duplex assay format and 5 copies for the single-plex format. The assay was very type-specific and no amplification signal was observed with 10(7) copies of the related HPV33, 35, and 58 DNA. Of 89 samples that tested unambiguously positive for HPV 52 in the LA, 75 were confirmed in the duplex format and 88 in the single-plex format. An additional 100 samples negative for HPV 52 in LA were all negative in both HPV 52 real-time PCR assay formats. CONCLUSIONS: These results indicate 92.6% and 99.5% accuracy relative to LA for the duplex and single-plex formats, respectively. In ongoing testing of 18,484 from various studies, 10.8% required the HPV52 TaqMan assay to unequivocally determine the status. Including the HPV 52 duplex assay provides the ability to monitor variations in the cell yield in various methods of sample collection and processing. This additional information is useful in QC monitoring of epidemiologic studies. |
Performance of commercial reverse line blot assays for human papillomavirus genotyping.
Steinau M , Onyekwuluje JM , Scarbrough MZ , Unger ER , Dillner J , Zhou T . J Clin Microbiol 2012 50 (5) 1539-44 The performance of three line blot assays (LBA) - Linear Array HPV genotyping assay (LA; Roche Diagnostics), INNO-LiPA HPV Genotyping Extra (LiPA; Innogenetics) and the Reverse Hybridization assay (RH; Qiagen) was evaluated using quantitated whole genomic HPV plasmids (types 6, 11, 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, 59, 68b) as well as epidemiologic samples. In a plasmid titration series LiPA and RH did not detect 50 international units (IU) of HPV 18 in the presence of 5 x 10(4) or more IU HPV16. HPV DNA (1-6 types) in the plasmid challenges at 50 (IU) or genome equivalents (GE) were identified with an accuracy of 99.9% by LA, 97.3% by LiPA and 95.4% by RH with positive reproducibility of 99.8% (Kappa=0.992), 88.2% (Kappa=0.928) and 88.1% (Kappa=0.926) respectively. Two instances of mis-typing occured with LiPA. Of the 120 epidemiologic samples 76 were positive for high-risk types by LA, 90 by LiPA and 69 by RH with a positive reproducibility of 87.3% (Kappa=0.925), 83.9% (Kappa=0.899) and 90.2% (Kappa=0.942) respectively. Although all the assays had good concordance in the clinical samples, the greater accuracy and specificity in the plasmid panel suggest that the LA assay has an advantage for internationally comparable genotyping studies. |
The HPV vaccine impact monitoring project (HPV-IMPACT): assessing early evidence of vaccination impact on HPV-associated cervical cancer precursor lesions.
Hariri S , Unger ER , Powell SE , Bauer HM , Bennett NM , Bloch KC , Niccolai LM , Schafer S , Markowitz LE . Cancer Causes Control 2011 23 (2) 281-8 The following paper describes a collaboration between the Centers for Disease Control and Prevention and five Emerging Infections Program sites to develop a comprehensive population-based approach to monitoring human papillomavirus (HPV) vaccine impact on cervical cancer precursors and associated HPV genotypes. The process of establishing this novel monitoring system is described, and development details such as enumeration of sources for reporting cervical intraepithelial neoplasia 2/3 and adenocarcinoma in situ, approaches to case ascertainment, electronic reporting, and HPV typing are outlined. Implementation of a feasible and sustainable surveillance system for HPV-associated cervical precancers will enable evaluation of the direct impact of HPV vaccination. |
Oral sampling and human papillomavirus genotyping in HIV-infected patients.
Steinau M , Reddy D , Sumbry A , Reznik D , Gunthel CJ , Del Rio C , Lennox JL , Unger ER , Nguyen ML . J Oral Pathol Med 2011 41 (4) 288-91 BACKGROUND: Oral human papillomavirus (HPV) is associated with several health complications especially in combination with HIV infections. Screening may be useful, but methodologies and results have varied widely in previous studies. We conducted a pilot study in an HIV-positive population to evaluate HPV detection in four different oral sample types. METHODS: Upon enrollment, an oral-rinse (OR) sample was collected in 10 ml saline. Additional samples of the buccal mucosa, tonsils, and oral lesion if present were collected with cytology brushes. DNA was extracted using LC-MagNAPure, and the Linear Array HPV genotyping Assay (Roche) was used for HPV genotyping. RESULTS: In samples from 100 HIV-positive participants, HPV was detected in 39 (%) of the oral rinses, 13 (%) mucosal and 11 (12.9%) tonsil brushings. Of seven lesion brushings collected, four were HPV positive. All participants with HPV detected in mucosal, tonsil, or lesion brushings were also positive in the OR sample. Among the rinse samples, 27 different genotypes were detected with HPV84 (n = 6), HPV55 (n = 5), and HPV83 (n = 5) being the most common. Multiple infections were detected in 17 samples (range 2-9, mean 1.9 types). As potential cofactors, only receptive oral sex was significantly associated with HPV (P = 0.018, odds ratio 2.9, 95% CI 1.2-6.9). CONCLUSION: Sampling is a significant factor for oral prevalence studies. Oral rinse provides the best representation for HPV in the oral cavity. To evaluate associated cofactors other than receptive oral sex, larger studies with case-control design are necessary. J Oral Pathol Med (2011) |
Convergent genomic studies identify association of GRIK2 and NPAS2 with chronic fatigue syndrome.
Smith AK , Fang H , Whistler T , Unger ER , Rajeevan MS . Neuropsychobiology 2011 64 (4) 183-94 BACKGROUND: There is no consistent evidence of specific gene(s) or molecular pathways that contribute to the pathogenesis, therapeutic intervention or diagnosis of chronic fatigue syndrome (CFS). While multiple studies support a role for genetic variation in CFS, genome-wide efforts to identify associated loci remain unexplored. We employed a novel convergent functional genomics approach that incorporates the findings from single-nucleotide polymorphism (SNP) and mRNA expression studies to identify associations between CFS and novel candidate genes for further investigation. METHODS: We evaluated 116,204 SNPs in 40 CFS and 40 nonfatigued control subjects along with mRNA expression of 20,160 genes in a subset of these subjects (35 CFS subjects and 27 controls) derived from a population-based study. RESULTS: Sixty-five SNPs were nominally associated with CFS (p < 0.001), and 165 genes were differentially expressed (≥4-fold; p ≤ 0.05) in peripheral blood mononuclear cells of CFS subjects. Two genes, glutamate receptor, ionotropic, kinase 2 (GRIK2) and neuronal PAS domain protein 2 (NPAS2), were identified by both SNP and gene expression analyses. Subjects with the G allele of rs2247215 (GRIK2) were more likely to have CFS (p = 0.0005), and CFS subjects showed decreased GRIK2 expression (10-fold; p = 0.015). Subjects with the T allele of rs356653 (NPAS2) were more likely to have CFS (p = 0.0007), and NPAS2 expression was increased (10-fold; p = 0.027) in those with CFS. CONCLUSION: Using an integrated genomic strategy, this study suggests a possible role for genes involved in glutamatergic neurotransmission and circadian rhythm in CFS and supports further study of novel candidate genes in independent populations of CFS subjects. |
Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues.
Steinau M , Patel SS , Unger ER . J Mol Diagn 2011 13 (4) 377-81 DNA from archived FFPE can be used for papillomavirus genotyping, but potential problems include paraffin as a physical barrier, DNA cross-linking, and PCR inhibitors. To address these complications, we combined a commercially available DNA isolation kit (Qiagen DNeasy) with a heat treatment and evaluated the resulting DNA with regards to HPV typing. DNA was extracted from 10-mum sections from 150 FFPE cancer samples. One protocol followed the manufacturer's recommendation, including paraffin removal by xylene and tissue lysis at 56 degrees C. A second section was directly incubated at 120 degrees C and subsequently lysed at 65 degrees C. After spin-column purification, both extracts were tested with a linear array HPV genotyping assay. Additionally, cellular DNA yield, HPV16 DNA copies, and PCR inhibitors were assessed by real-time qPCR assays. Inadequate linear array HPV genotyping assay results were significantly more frequent (P = 0.0003) in xylene-treated (29/150, 19.3%) than in heat-treated extracts (8/150, 5.3%). HPV detection also differed, with 94/150 (62.7%) and 110/150 (73.3%) positive results, respectively (P = 0.0026). The heat method also yielded more PCR-amplifiable cellular DNA (8.2-fold; P < 0.001) and HPV16 copies (6.5-fold; P = 0.009), although PCR inhibitors also had a greater effect (P = 0.035). Aggressive heat treatment demonstrated an advantage over traditional xylene purification protocols, resulting in higher DNA yields and increased sensitivity for HPV testing. |
Impact of HPV assay on observed population prevalence.
Unger ER , Steinau MS , Lin JM , Patel SS , Swan DC . Diagn Mol Pathol 2011 20 (2) 101-4 Type-specific surveillance of human papillomavirus (HPV) has been proposed as an early indicator of vaccine impact. Longitudinal comparison of HPV typing results requires stable assays with high type-specific reproducibility. Assays are evolving and the impact of even minor changes in the assay format may be difficult to anticipate. We initiated a population-based study of HPV with the prototype line blot (PLB) assay. These reagents were replaced by the research use only Linear Array (LA) HPV Genotyping kit. The assays are similar in principle and earlier comparisons found increased sensitivity and detection of more types per sample with LA; however, in samples from women with cervical abnormalities, the overall concordance was good. Slight changes in sensitivity may be more significant in samples from a general population with lower viral loads in the samples. Residual extracts from 3001 self-collected vaginal swabs from women in the general US population originally tested with PLB were retested with LA. With LA, all the samples were hybridized. PLB hybridization was restricted to samples with probable amplicon in gel electrophoresis. For HPV detection, the agreement between the 2 assays was 78.6% (kappa=0.55) with a positive concordance of 52.8%. However, this masks the observation that repeat testing with LA led to the detection of HPV in nearly twice as many samples. Agreement improves if comparison was restricted to the samples hybridized. These results emphasize that assay comparisons should consider the clinical-epidemiologic context of sample collection. Studies designed to examine temporal trends in type-specific prevalence should archive residual material to permit retesting if assays change. |
Functional genomics of serotonin receptor 2A (HTR2A): interaction of polymorphism, methylation, expression and disease association.
Falkenberg VR , Gurbaxani BM , Unger ER , Rajeevan MS . Neuromolecular Med 2010 13 (1) 66-76 Serotonergic neurotransmission plays a key role in the pathophysiology of neuropsychiatric illnesses. The functional significance of a promoter polymorphism, -1438G/A (rs6311), in one of the major genes of this system (serotonin receptor 2A, HTR2A) remains poorly understood in the context of epigenetic factors, transcription factors and endocrine influences. We used functional and structural equation modeling (SEM) approaches to assess the contributions of the polymorphism (rs6311), DNA methylation and clinical variables to HTR2A expression in chronic fatigue syndrome (CFS) subjects from a population-based study. HTR2A was up-regulated in CFS through allele-specific expression modulated by transcription factors at critical sites in its promoter: an E47 binding site at position -1,438, (created by the A-allele of rs6311 polymorphism), a glucocorticoid receptor (GR) binding site encompassing a CpG at position -1,420, and Sp1 binding at CpG methylation site -1,224. Methylation at -1,420 was strongly correlated with methylation at -1,439, a CpG site that is dependent upon the G-allele of rs6311 at position -1,438. SEM revealed a strong negative interaction between E47 and GR binding (in conjunction with cortisol level) on HTR2A expression. This study suggests that the promoter polymorphism (rs6311) can affect both transcription factor binding and promoter methylation, and this along with an individual's stress response can impact the rate of HTR2A transcription in a genotype and methylation-dependent manner. This study can serve as an example for deciphering the molecular determinants of transcriptional regulation of major genes of medical importance by integrating functional genomics and SEM approaches. Confirmation in an independent study population is required. |
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