BACKGROUNDA 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.METHODSDNA 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).RESULTSHPV 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.CONCLUSIONSHR 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. Cancer 2006. © 2006 American Cancer Society.

Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.

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.

No abstract

Human papillomavirus (HPV) infection, most commonly genotype 16 of the alpha-9 family, is implicated in the etiology of a subset of oropharyngeal squamous cell carcinomas (OPSC) worldwide. Data are scarce regarding OPSC in South Africans, and three prior studies suggest no significant etiologic role for HPV. We aimed to investigate for evidence of HPV etiology in OPSCs from black South Africans by polymerase chain reaction (PCR) methodologies with determination of HPV subtype by sequencing, in situ hybridization (ISH), and p16INK4a immunohistochemistry (IHC), as a surrogate marker for an HPV-driven tumor. It was hypothesized that HPV-driven tumors would be positive by PCR plus IHC and/or ISH whereas OPSCs with HPV background infections (HPV-passenger) would be positive by PCR alone. Formalin-fixed, paraffin embedded tissues from 51 OPSCs collected between 2005 and 2010 from 41 patients were analyzed for HPV by GP5?6? PCR (targeting the HPV L1 region), pU-1M/pU- 2R PCR (targeting the HPV E6/E7 region) and HPV-31 specific PCR (targeting the E5 region), chromogenic ISH, and p16INK4a IHC. All cases positive by PCR were subject to sequencing to determine HPV genotype. The patient mean age was 58.0 years and 88 % were male. Of the 51 evaluable tumors, 48 (94.1 %) were positive for HPV DNA by PCR: 25 (49.1 %) met criteria for an HPV-driven tumor, 23 (45.1 %) for HPV-passenger, and 3 (5.9 %) were HPV unrelated. Sequencing of the PCR-positive cases revealed the following genotypes: combined HPV-16 and 31 (41.7 %), HPV-31 (25.0 %), HPV-16 (22.9 %), combined HPV-16 and 18 (6.3 %), and a single case each of HPV 18 and HPV 33. Studies via ISH were negative in all cases. In accordance with worldwide trends but contrary to prior South African data, HPV likely plays an etiologic role in a significant subset (at least 49.1 %) of OPSC in black South Africans. We found that the alpha-9 HPV family, particularly HPV-16 and 31 either in combination or separately, to predominate in our sample tumors. The use of multiple PCR primers increased sensitivity of viral detection, and a HPV-31 specific primer confirmed the presence of this genotype in many samples. Further studies including HPV E6/E7 mRNA assays are needed to better elucidate the pathogenic role of HPV in black South African OPSCs.