ABSTRACT
BACKGROUND: The US Food and Drug Administration supports innovations to facilitate new indications for high-risk human papillomavirus testing. This report describes the retrospective testing of stored specimens and analysis of existing data to efficiently and cost-effectively support a new indication for the Onclarity human papillomavirus assay (Becton, Dickinson and Company, BD Life Sciences - Integrated Diagnostic Solutions, Sparks, MD). The performance of this index test was compared with that of a predicate test, the cobas human papillomavirus assay (Roche Diagnostics, Indianapolis, IN). Both human papillomavirus assays are based on real-time polymerase chain reaction platforms that detect the presence of 14 high-risk human papillomavirus genotypes. The predicate assay reports human papillomavirus types 16 and 18 as individual results and the other 12 human papillomavirus genotypes as 1 pooled result. The index assay reports 9 independent results (human papillomavirus types 16, 18, 31, 33/58, 35/39/68, 45, 51, 52, and 56/59/66). Both the index and predicate assays are approved by the Food and Drug Administration for cervical cancer screening, but at the time that this study was initiated, the index human papillomavirus assay was not approved for use with cervical specimens collected in PreservCyt (Hologic, Inc, San Diego, CA) liquid-based cytology media. OBJECTIVE: The performance of the index human papillomavirus assay was compared with that of the predicate human papillomavirus assay for the detection of cervical intraepithelial neoplasia grades 2 or greater and 3 or greater (≥CIN2 or ≥CIN3) using PreservCyt liquid-based cytology specimens collected from women aged 21 to 65 years. In addition, the ability of the index test's extended genotyping to stratify ≥CIN2 and ≥CIN3 risks, using these specimens, was evaluated. STUDY DESIGN: The New Mexico HPV Pap Registry was used to select an age- and cytology-stratified random sample of 19,879 women undergoing opportunistic cervical screening and follow-up in routine clinical practice across New Mexico. A subset (n = 4820) of PreservCyt specimens was selected from 19,879 women for paired testing by the index and predicate human papillomavirus assays within age and cytology strata and included women with or without cervical biopsy follow-up. Point estimate differences and ratios were calculated for cervical disease detection and positivity rates, respectively, with 95% confidence intervals to determine statistical significance. The cumulative risk of ≥CIN2 or ≥CIN3, with up to 5-year follow-up, was estimated for the index assay using Kaplan-Meier methods. RESULTS: The 5-year cumulative ≥CIN3 detection rates were 5.6% for the index assay and 4.6% for the predicate assay (difference, 1.0%; 95% confidence interval, 0.5%-1.5%). The ≥CIN3 positivity rates within <1 year were 95.3% for the index assay and 94.5% for the predicate assay (ratio, 1.01; 95% confidence interval, 0.98-1.06). The ≥CIN3 cumulative positivity rates for the index and predicate assays were also similar at 5 years. Among cases of ≥CIN3, the positive agreement rates between the index and predicate assays for human papillomavirus types 16 and 18 were 100.0% (95% confidence interval, 95.0%-100.0%) and 90.9% (95% confidence interval, 62.3%-98.4%), respectively. Human papillomavirus type 16 carried the highest ≥CIN2 or ≥CIN3 risk, followed by human papillomavirus types 18/31/33/58/52/45 and human papillomavirus types 35/56/59/51/56/59/66. CONCLUSION: The index and predicate human papillomavirus assays demonstrated equivalent performance, and extended human papillomavirus genotyping, using the index assay, provided effective ≥CIN2 and ≥CIN3 risk stratification, supporting a new indication for use of the index assay with PreservCyt.
Subject(s)
Papillomavirus Infections , Uterine Cervical Dysplasia , Uterine Cervical Neoplasms , Female , United States/epidemiology , Humans , Uterine Cervical Neoplasms/pathology , Early Detection of Cancer , Retrospective Studies , Uterine Cervical Dysplasia/pathology , Papillomaviridae/genetics , Human papillomavirus 16/genetics , New Mexico , GenotypeABSTRACT
Methylation of host and viral genes is promising for triage of women with high-risk human papillomavirus infections (hrHPV). Using a population-based sample of hrHPV positive women with cervical biopsies within 12 months after cervical screening, the clinical value of the S5 methylation classifier (S5), HPV genotyping and cytology were compared as potential triage tests, for outcomes of cervical intraepithelial neoplasia (CIN) grade 3 or greater (CIN3+), CIN2+ and CIN2, and the area under the curve (AUC) calculated. S5 scores increased with histopathology severity (Ptrend < .001). For CIN3+, the AUC was 0.780 suggesting S5 provides good discrimination between Subject(s)
Papillomavirus Infections
, Uterine Cervical Dysplasia
, Uterine Cervical Neoplasms
, DNA Methylation
, Early Detection of Cancer
, Female
, Human papillomavirus 16/genetics
, Human papillomavirus 18/genetics
, Humans
, Papillomaviridae/genetics
, Papillomavirus Infections/complications
, Papillomavirus Infections/diagnosis
, Papillomavirus Infections/genetics
, Triage
, Uterine Cervical Neoplasms/diagnosis
, Uterine Cervical Neoplasms/genetics
, Uterine Cervical Neoplasms/pathology
, Uterine Cervical Dysplasia/diagnosis
, Uterine Cervical Dysplasia/genetics
, Uterine Cervical Dysplasia/pathology
ABSTRACT
A population of Saccharomyces cerevisiae was cultured for approximately 450 generations in the presence of high glucose to select for genetic variants that grew optimally under these conditions. Using the parental strain BY4741 as the starting population, an evolved culture was obtained after aerobic growth in a high glucose medium for approximately 450 generations. After the evolution period, three single colony isolates were selected for analysis. Next-generation Ion Torrent sequencing was used to evaluate genetic changes. Greater than 100 deletion/insertion changes were found with approximately half of these effecting genes. Additionally, over 180 SNPs were identified with more than one-quarter of these resulting in a nonsynonymous mutation. Affymetrix DNA microarrays and RNseq analysis were used to determine differences in gene expression in the evolved strains compared to the parental strain. It was established that approximately 900 genes demonstrated significantly altered expression in the evolved strains relative to the parental strain. Many of these genes showed similar alterations in their expression in all three evolved strains. Interestingly, genes with altered expression in the three evolved strains included genes with a role in oxidative metabolism. Overall these results are consistent with the physiological observations of optimal growth with glucose as the carbon source. Namely, the decreased ethanol production suggest that the underlying metabolism switched from fermentation to respiration during the selection for optimal growth on glucose.
Subject(s)
Genome, Fungal , Glucose/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/genetics , Ethanol/metabolism , Evolution, Molecular , Gene Expression Profiling , Genomics/methods , Oligonucleotide Array Sequence Analysis , Polymorphism, Single Nucleotide , RNA, Fungal/genetics , RNA, Messenger/analysis , Saccharomyces cerevisiae/metabolism , Sequence Analysis, DNA , Systems Biology/methodsABSTRACT
CONTEXT.: Lower Anogenital Squamous Terminology (LAST) standardization recommended p16INK4a 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 (Ptrend ≤ .001) and within each HPV risk group (Ptrend ≤ .001 except for low-risk HPV [Ptrend < .010]). All abnormal grades of CP-diagnosed biopsies were more likely to test p16 IHC positive with a higher HPV risk group (Ptrend < .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.
Subject(s)
Biomarkers, Tumor/analysis , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Neoplasms/diagnosis , Adult , Cyclin-Dependent Kinase Inhibitor p16/analysis , Female , Humans , Immunohistochemistry , Middle Aged , Papillomavirus Infections/complications , Uterine Cervical Neoplasms/classification , Uterine Cervical Neoplasms/virology , Uterine Cervical Dysplasia/classification , Uterine Cervical Dysplasia/virologyABSTRACT
Here, we report the high-quality draft genome sequence of actinobacterium Kibdelosporangium sp. MJ126-NF4, producer of the type II polyketide azicemicins, obtained using Illumina and PacBio sequencing technologies. The 11.75-Mbp genome contains >11,000 genes and 22 polyketide and nonribosomal peptide natural product gene clusters.
ABSTRACT
We have developed a highly sensitive single-molecule clonal amplification method called dual primer emulsion PCR (DPePCR) for next-generation DNA sequencing. The approach is similar in concept to standard emulsion PCR; however, in DPePCR both primers are attached to the beads, therefore following PCR amplification, both strands of the PCR products are attached to the beads. The ends of each strand can be freed for analysis by restriction digestion of the bridged PCR fragments, which allows efficient paired-end sequencing of fragment libraries.
Subject(s)
DNA Primers/metabolism , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Emulsions , Reproducibility of Results , Streptococcus pyogenes/geneticsABSTRACT
While HPV 16 variant lineages have been well characterized, the knowledge about HPV 18 variants is limited. In this study, HPV 18 nucleotide variations in the E2 hinge region were characterized by sequence analysis in 47 control and 51 tumor specimens. Fifty of these specimens were randomly selected for sequencing of an LCR-E6 segment and 20 samples representative of LCR-E6 and E2 sequence variants were examined across the L1 region. A total of 2770 nucleotides per HPV 18 variant genome were considered in this study. HPV 18 variant nucleotides were linked among all gene segments analyzed and grouped into three main branches: Asian-American (AA), European (E), and African (Af). These three branches were equally distributed among controls and cases and when stratified by Hispanic and non-Hispanic ethnicities. Among invasive cervical cancer cases, no significant differences in the three HPV variant branches were observed among ethnic groups or when stratified by histopathology (squamous vs. adenocarcinoma). The Af branch showed the greatest nucleotide variability when compared to the HPV 18 reference sequence and was more closely related to HPV 45 than either AA or E branches. Our data also characterize nucleotide and amino acid variations in the L1 capsid gene among HPV 18 variants, which may be relevant to vaccine strategies and subsequent studies of naturally occurring HPV 18 variants. Several novel HPV 18 nucleotide variations were identified in this study.
Subject(s)
Capsid Proteins/genetics , DNA-Binding Proteins/genetics , Genetic Variation , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Base Sequence , Case-Control Studies , DNA, Viral/genetics , Female , Genetic Linkage , Humans , Molecular Sequence Data , Papillomaviridae/classification , Papillomaviridae/isolation & purification , Papillomavirus Infections/virology , Phylogeny , Sequence Homology, Nucleic Acid , United States , Uterine Cervical Neoplasms/virology , Viral ProteinsABSTRACT
Genital human papillomaviruses (HPV) are considered to be one of the main risk factors for the development of cervical cancer. The P97 promoter at the E6-proximal end of the long control region (LCR) regulates the transcription of viral genes, especially the oncogenes E6 and E7. The LCR contains binding sites of several viral and cellular transcription factors, which either activate or repress the P97 promoter. Intratype variants of HPV-16 belong to six geographically clustered phylogenetic groups distributed all over the world. These variants exhibit differences in E6 protein activities and in tumour progression in vivo. Seven HPV-16 variants were investigated by sequencing the entire LCR (nt 7060-124) and by comparing the transcriptional activities of their P97 promoters. Previously unknown nucleotide variations were identified in all LCRs investigated. In luciferase assays, 3.3- and 2.8-fold increases in P97 promoter activity were detected in the Asian American c and North American 1 variants when compared with the European reference clone. The African variants 1a and 2a exhibited P97 promoter activities comparable to the European reference clone. After recombining different LCR fragments, the region responsible for enhanced transcription in the Asian American c and North American 1 variants could be attributed to the E6-proximal end of the LCR (nt 7619-124).
Subject(s)
Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Promoter Regions, Genetic , Repressor Proteins , Base Sequence , Humans , Oncogene Proteins, Viral/chemistry , Tumor Cells, CulturedABSTRACT
A comparison of two PCR-based human papillomavirus (HPV) DNA detection and genotyping systems (PGMY LBA and SPF(10) LiPA) was conducted in two laboratories. Both systems are based on broad-spectrum PCR for the detection of HPV DNA, followed by reverse hybridization with type-specific probes. A total of 400 selected cervical scrape specimens in PreservCyt solution (55% normal cytology, 18% atypical squamous cells of unknown significance, 14.8% low-grade squamous intraepithelial lesions [SIL], and 12.5% high-grade SIL) were tested for the presence of HPV DNA. In this selected group of specimens, the overall agreement between the two methods for the detection of any HPV DNA was high (kappa = 0.859). When the 20 common HPV genotypes identified by both methods were considered (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 56, 58, 59, 66, and 68), compatible genotype-specific results were observed in 96.5% of the samples, even when multiple HPV genotypes were present. However, for some specific HPV genotypes, there were significant differences in HPV detection by the two methods. PGMY LBA detected more HPV type 42 (P = 0.002), HPV type 56 (P = 0.039), and HPV type 59 (P < 0.001), whereas SPF(10) LiPA detected more HPV type 31 (P < 0.001) and HPV type 52 (P = 0.031). For the remaining genotypes, including HPV types 16 and 18, the results obtained by the two methods were not significantly different. In general, both genotyping methods are highly suitable for clinical and epidemiological studies.