Biomedical subjects

Mark F Evans

Publications and source records attributed to Mark F Evans.

6 recordsLinked to original sources

PubMed2006

Distribution of human papillomavirus types in ThinPrep Papanicolaou tests classified according to the Bethesda 2001 terminology and correlations with patient age and biopsy outcomes.

BACKGROUND: A survey of the distribution of human papillomavirus (HPV) types across the spectrum of cervical cytologic categories defined by the Bethesda 2001 guidelines was conducted with the objective of examining how HPV detection by polymerase chain reaction (PCR) analysis may benefit the management of patients who have abnormal Papanicolaou (Pap) test results. METHODS: DNA samples from women with no intraepithelial lesion or malignancy (NLM) (n = 300 samples); atypical squamous cells of undetermined significance (ASC-US) (n = 200 samples); low-grade squamous intraepithelial lesion (LSIL) (n = 200 samples); atypical squamous cells, cannot rule out high-grade squamous intraepithelial lesion (ASC-H) (n = 200 samples); and high-grade squamous intraepithelial lesion (HSIL) (n = 200 samples) were tested for HPV using a modified general primer (GP)5+/GP6+ PCR assay and dot-blot hybridization with type-specific oligonucleotide probes (PCR assay analytical sensitivity: 1-100 copies of HPV, depending on the HPV type, in a background of 100 ng human DNA). RESULTS: HPV was detected in 27% of NLM samples, in 89.5% of ASC-US samples, in 97.5% of LSIL samples, in 93% of ASC-H samples, and in 96.5% of HSIL samples. Thirty-seven different HPV types were identified in total. One or more of 13 high-risk (HR) HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) were detected in 53% of samples that were diagnosed as ASC-US (59.0% of patients younger than age 30 yrs; 45.5% of patients age 30 yrs and older), in 55.5% of samples that were diagnosed as LSIL (60.0% of patients younger than age 30 yrs; 44.0% of patients age 30 yrs and older), in 80% of samples that were diagnosed as ASC-H, and in 87.5% of samples that were diagnosed as HSIL (P < 0.001). HPV-16 was detected in 17.5% of ASC-US samples, in 15.5% of LSIL samples, in 48.5% of ASC-H samples, and in 49.0% of HSIL samples (P < 0.001). Among abnormal smears, HR HPV was significantly more common in women younger than age 30 years compared with women age 30 years and older (P < 0.002). Follow-up biopsy data were obtained for 359 patients. A "benign" biopsy result was recorded for 47 of 64 women (73.5%) with ASC-US, 30 of 66 women (45.5%) with LSIL, 39 of 87 women (45.0%) with ASC-H, and 26 of 142 women (18.0%) with HSIL and was most common in women age 30 years and older (P < 0.0001). Cervical intraepithelial neoplasia (CIN) Grade I (CIN-I) was found in 14.0% of women with ASC-US, in 39.5% of women with LSIL, in 8.0% of women with ASC-H, and in 7.0% of women with HSIL. CIN-II was diagnosed in 9.5% of women with ASC-US, in 13.5% of women with LSIL, in 19.5% of women with ASC-H, and in 24.0% of women with HSIL. CIN-III was identified in 2 women (3.0%) with ASC-US, in 1 woman (1.5%) with LSIL, in 24 women (27.5%) with ASC-H, and in 71 women (50.0%) with HSIL. CONCLUSIONS: HR HPV testing by PCR of samples diagnosed according to the Bethesda 2001 guidelines may benefit the management of patients with ASC-US or patients with LSIL, especially among women age 30 years and older, by allowing exclusion from referral for biopsy of women who are negative for HR HPV types. However, the small numbers of women who had CIN-III detected after a diagnosis of ASC-US or LSIL limited the assessment of test sensitivity.

Adult↗

PubMed2005

Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus.

BACKGROUND: The GP5+/GP6+ PCR assay is a well-established HPV detection technique. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. METHODS: Firstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng-100 ng) diluted in a background of C-33A DNA (100 ng-2 microg). Secondly, the detection of small quantities (15ag-1.5pg) of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56) diluted in C-33A DNA was investigated. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors) were tested for HPV. Six different PCR protocols were assessed. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. RESULTS: HPV detection sensitivity was dependent on the total amount of DNA in a PCR. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 microg or 1 microg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (approximately 28 copies HPV-16) in 500 ng or 100 ng background DNA. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 microg or 1 microg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. HPV recombinant plasmids were each detected with high (albeit varying) sensitivity by a touchdown protocol. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA) than by a touchdown approach (15fg detection limit). HPV-52 was not amplified by the standard protocol at the dilutions tested. Seventeen different HPV types were demonstrated in 47/65 (72%) abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. Twenty-one different HPV types were recorded in 111/114 (97%) vaginal lesions. Multiple infections were also detectable using a touchdown approach. Of 26 breast tumors, 5 (19%) tested HPV positive by the standard assay and 15/26 (58%) using a touchdown protocol. CONCLUSION: Touchdown modification of the GP5+/GP6+ PCR assay enables the detection of HPV undetected under regular assay conditions. The use of standardized DNA quantities in a PCR rather than standard sample volumes containing arbitrary amounts of DNA is supported. A touchdown approach may be beneficial as an analytical test for the re-evaluation of (apparently) HPV negative abnormal cervical cytological or histological samples, and for investigating the association of HPV with disease conditions at diverse organ sites. The clinical utility of a touchdown approach for HPV detection requires further investigation as increased assay analytical sensitivity may not necessarily equate with improved clinical sensitivity or specificity.

Journal Article↗

PubMed2005

p16INK4A immunoexpression and HPV in situ hybridization signal patterns: potential markers of high-grade cervical intraepithelial neoplasia.

Integration of human papillomavirus (HPV) into the cell genome is considered to be an important event in the progression of cervical neoplasia. p16, also a useful biomarker of cervical intraepithelial neoplasia (CIN), shows increased immunoexpression with worsening grades of CIN. This study examines the correlation between p16 immunoexpression, grade of CIN, HPV type, and HPV in situ hybridization diffuse and punctate signal patterns (linked to episomal and integrated viral particles, respectively) in 44 cervical biopsies/LEEP excisions classified as CIN 1 and CIN 2/3. In 22 of 25 (88%) CIN 1 lesions, p16 immunoexpression was confined to the lower half of the epithelium, with sporadic to focal staining in 11 of 25 cases (44%). In CIN 2/3 lesions, 15 of 17 (88.2%) showed diffuse, two-thirds to full-thickness staining of the epithelium. High-risk HPV types were found in 20 (80%) CIN 1 lesions and 17 (100%) CIN 2/3 lesions. Punctate signals were detected in only 3 (13.6%) of high-risk HPV-positive CIN 1 lesions and in 17 of 17 (100%) CIN 2/3 lesions (P<0.001). p16 immunoexpression and the presence of punctate signal on HPV in situ hybridization correlated with the degree of cervical neoplasia (P<0.001). However, 3 cases of CIN 1 demonstrating punctate signals did not demonstrate a comparable CIN 2/3 p16 staining pattern. Similarly, two CIN 1 lesions with comparable CIN 2/3 p16 staining showed no evidence of viral integration. Both increased p16 immunoexpression and punctate signal correlate with CIN 2/3 grade, supporting the use of either, or both, tests to confirm CIN 2/3. Strong p16 immunostaining in CIN 1 appears independent of HPV punctate signal type.

Biomarkers, Tumor↗

PubMed2004

Human papillomavirus integration: detection by in situ hybridization and potential clinical application.

Human papillomaviruses (HPVs) are accepted as a necessary cause of cervical neoplasia. However, the benefits of testing simply for high-risk HPV types are limited because of their high prevalence in intraepithelial lesions of all grades, the majority of which regress if left untreated. One factor considered to be of key importance for the progression of intraepithelial lesions to invasive disease is integration of HPV into the host cell genome. Although questions remain about the prevalence of integration amongst pre-invasive lesions, sensitive in situ hybridization techniques utilizing tyramide reagents may aid determination of the significance of HPV infection by enabling routine detection of both high-risk HPV and its physical status. This will provide important data relevant not only to our understanding of the biology of HPV-associated neoplasia, but also potentially to clinical testing for HPV.

Disease Progression↗

PubMed2003

Optimization of biotinyl-tyramide-based in situ hybridization for sensitive background-free applications on formalin-fixed, paraffin-embedded tissue specimens.

BACKGROUND: Over the past five years in situ hybridization techniques employing tyramide amplification reagents have been developed and promise the potential detection of low/single-copy nucleic acid sequences. However the increased sensitivity that tyramide amplification brings about may also lead to problems of background staining that confound data interpretation. METHODS: In this study those factors enabling background-free biotinyl-tyramide based in situ hybridization assay of formalin-fixed paraffin-embedded tissues have been examined. SiHa, HeLa and CaSki cell lines known to contain HPV integrated into the cell genome, and archival cervical pre-invasive lesions and carcinomas have been successfully assessed using biotinylated HPV and centromeric probes. RESULTS: The single most important factor both for sensitivity and clean background was a tissue unmasking regimen that included treatment with 10 mM sodium citrate pH 6.0 at 95 degrees C followed by digestion with pepsin/0.2 M HCl. Concentrations both of probe and primary streptavidin-peroxidase conjugate and pH of hybridization mix and stringency washes were also critical for sensitivity. Certain probes were more associated with background staining than others. This problem was not related to probe purity or size. In these instances composition of hybridization mix solution was especially critical to avoid background. 3-amino-9-ethylcarbazole was preferred over 3,3'-diaminobenzidene as a chromogen because background was cleaner and the 1-2 copies of HPV16 integrated in SiHa cells were readily demonstrable. HPV detection on metaphase spreads prepared from SiHa cells was only successful when a fluorescent detection method was combined with tyramide reagent. 'Punctate' and 'diffuse' signal patterns were identified amongst tissues consistent with the former representing integration and 'diffuse' representing episomal HPV. Only punctate signals were detected amongst the cell lines and were common amongst high-grade pre-invasive lesions and carcinomas. However it remains to be determined why single/low-copy episomal HPV in basal/parabasal cells of low-grade lesions is not also detectable using tyramide-based techniques and whether every punctate signal represents integration. CONCLUSIONS: A tyramide-based in situ hybridization methodology has been established that enables sensitive, background-free assay of clinical specimens. As punctate signals characterize HPV in high-grade cervical lesions the method may have potential for clinical applications.

Journal Article↗

PubMed2002

Biotinyl-tyramide-based in situ hybridization signal patterns distinguish human papillomavirus type and grade of cervical intraepithelial neoplasia.

In this study, the prevalence of human papillomavirus integration in cervical intraepithelial neoplasia Grades I, II, and III has been investigated using a highly sensitive biotinyl-tyramide-based in situ hybridization methodology. This method is able to demonstrate integrated viral DNA by punctate signals within the nucleus and episomal viral DNA by a diffuse signal throughout the nucleus. Fifteen viral types were identified by General Primer 5+/6+ polymerase chain reaction assay among 26 Grade I and 22 Grade II/III lesions. High-risk human papillomavirus (Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) was found in 20 (77%) Grade I and in 22 (100%) Grade II/III lesions (P =.025). Human papillomavirus Type 16 was identified in 2 (7%) Grade I and in 15 (68%) Grade II/III samples (P <.0001) and was distinguished from other high-risk types by its demonstration in both Grade I and Grade II/III lesions as frequent punctate signals, detectable at all levels of the epithelium including the basal layer. In contrast, punctate signals, when detected among Grade I lesions that were positive for other high-risk types, did not involve the basal layer and were restricted to occasional cells in the superficial layers. However, Grade II/III lesions positive for high-risk types other than human papillomavirus Type 16 demonstrated frequent punctate signals throughout the epithelium. Overall, punctate signals were detected in 22 (100%) high-risk human papillomavirus-positive Grade II/III lesions and in 5 (25%) high-risk positive Grade I lesions (P <.0001). These data are consistent with human papillomavirus Type 16 possessing a high potential for integration, which may explain its frequent association with cervical intraepithelial neoplasia Grade III and carcinomas. Acquisition of the punctate correlate, especially in the basal layer, is also indicated as important in the development of Grade II/III lesions. The data illustrate the unique potential of biotinyl-tyramide-based in situ hybridization to address key issues concerning the biology of cervical intraepithelial neoplasia.

Biotin↗