No abstract

Women with high‐grade cervical intraepithelial neoplasia (HGCIN) frequently present with multiple cervical lesions and multiple concomitant Human papillomavirus (HPV) genotype infections. To elucidate HPV genotype attribution in different regions on the cervix, we performed molecular mapping of cervical disease in women with HGCIN. Thirteen subjects referred to colposcopy for abnormal cervical cancer screening results were included. A cervical smear and biopsies from 4 different areas on the cervix were collected. HPV genotyping using Linear Array (for cytology) or SPF10 LiPA25 (for histology) were performed in 13 smears, 52 whole sections from biopsies and 138 tissue regions isolated with laser capture microdissection (LCM). Twelve subjects had a diagnosis of CIN3 and one subject had a diagnosis of CIN2 based on the worst histology found in 4 biopsies. Eight of the 13 smears (62%) showed multiple genotype infections. Four of 13 women (31%) had multiple HPV infections in their biopsies. After performing LCM‐PCR, only one woman (8%) had two different carcinogenic HPV types in morphologically distinct, but colliding HGCIN lesions. HPV16 was identified as the causal type in all women with HPV16 in cytology. A large proportion of other HPV types found in cervical smears were not detected at the tissue level. Using tissue‐based genotyping and LCM‐PCR analysis, we were able to attribute an individual HPV type to each area of CIN lesions. We demonstrate that HPV16 is even more etiologically dominant than previously thought, based on various genotype attribution models.

Recent studies have shown that CADM1/MAL methylation levels in cervical scrapes increase with severity and duration of the underlying cervical intraepithelial neoplasia (CIN) lesion. Multiple lesions of different histological grades and duration are frequently present on the cervix. To gain more insight into the possible epigenetic heterogeneity and its consequences for the methylation status in cervical scrapes, we performed an exploratory study of CADM1/MAL methylation in different grades of CIN lesions present in women with multiple cervical biopsies. CADM1-M18 and MAL-M1 methylation was assessed using a standardised, multiplex, quantitative methylation specific PCR on 178 biopsies with various grades of CIN in 65 women, and in their corresponding cervical scrapes. CADM1/MAL methylation positivity increased with disease severity, from 5.5% in normal biopsies to 63.3% and 100% in biopsies with CIN3 and cervical cancer, respectively. In the majority (8/9) of women where besides a CIN2/3 lesion a biopsy from normal cervical tissue was present, the CIN2/3 biopsy was CADM1/MAL methylation positive and the normal biopsy was CADM1/MAL methylation negative. A good concordance (78%) was found between CADM1/MAL methylation results on the scrapes and the biopsy with the worst diagnosis, particularly between samples of women with CIN3 and cervical cancer (92% and 100% concordance, respectively). Thus, in women with multiple cervical biopsies, CADM1/MAL methylation increases with severity of the lesion and is lesion-specific. CADM1/MAL methylation status in cervical scrapes appears to be representative of the worst underlying lesion, particularly for CIN3 and cervical cancer.

Grading cervical intraepithelial neoplasia (CIN) determines clinical management of women after abnormal cytology with potential for overdiagnosis and overtreatment. We studied a novel biomarker of human papillomavirus (HPV) life-cycle completion (panHPVE4), in combination with the minichromosome maintenance (MCM) protein cell-cycle marker and the p16INK4a transformation marker, to improve CIN diagnosis and categorization. Scoring these biomarkers alongside CIN grading by 3 pathologists was performed on 114 cervical specimens with high-risk (HR) HPV. Interobserver agreement for histopathology was moderate (κ=0.43 for CIN1/negative, 0.54 for CIN2/≤CIN1, and 0.36 for CIN3). Agreement was good or excellent for biomarker scoring (E4: κ=0.896; 95% confidence interval [CI]: 0.763-0.969; p16INK4a : κ=0.798; 95% CI: 0.712-0.884; MCM: κ=0.894; 95% CI: NC (this quantity cannot be calculated). Biomarker expression was studied by immunofluorescence and immunohistochemistry and was correlated with 104 final CIN diagnoses after histologic review. All 25 histologically negative specimens were p16INK4a and panHPVE4 negative, although 9 were MCM-positive. There were variable extents of p16INK4a positivity in 11/11 CIN1 and extensive panHPVE4 staining in 9/11. Ten CIN2 lesions expressed panHPVE4 and p16INK4a, and 13 CIN2 expressed only p16INK4a. CIN3 showed extensive p16INK4a positivity with no/minimal panHPVE4 staining. PanHPVE4, unlike MCM, distinguished CIN1 from negative. PanHPVE4 with p16INK4a separated CIN2/3 showing only expression of p16INK4a, indicating transforming HR-HPV E7 expression, from CIN1/2 showing completion of HR-HPV life cycle by E4 expression and variable p16INK4a expression. PanHPVE4 and p16INK4a staining are complementary markers that could provide simple, reliable support for diagnosing CIN. Their value in distinguishing CIN1/2 that supports HR-HPV life-cycle completion (and which might ultimately regress) from purely transforming CIN2/3 needing treatment warrants further research.

Summary Objectives Because current guidelines recognise high‐grade anal squamous intraepithelial lesions (HSILs) and low‐grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16INK4a (p16) and E4 in low‐risk human papillomavirus (lrHPV) and high‐risk (hr)HPV‐associated SILs as a potential basis for more selective treatment. Methods Immunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV‐positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10‐PCR‐DEIA‐LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0–4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0–2) to mark HPV production and life cycle completion. Results There were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade. Conclusions Combined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a ‘wait and see’ policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high‐risk populations, all patients with high‐grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 INK4a (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified. Moreover, p16/E4 staining can separate high‐risk HPV‐associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.