Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV‐proofer and consensus PCR: A 2‐year follow‐up of women with ASCUS or LSIL pap smear

Abstract

Key words: HPV; human papillomavirus; DNA; mRNA; PreTect HPV-Proofer; NASBA; PCR; ASCUS; LSIL Cytological cervical cancer screening programs have been successful in reducing the incidence of cervical cancer, even though a single conventional Papanicolaou (Pap) smear is only moderately accurate and does not achieve concurrent high sensitivity and specificity. 1,2 The management of women with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) is problematic because only a small proportion will progress to cervical intraepithelial neoplasia (CIN) 3 and invasive cervical carcinoma (ICC). Histologically verified CIN has been found in 10 -60% of women with an ASCUS diagnosis, with CIN2/3 present in more than 5%. 3-11 Pap smear follow-up of women with an ASCUS smear fails to identify all women at higher risk of CIN2ϩ, suggesting that cervical cancer screening programs might benefit from implementing new diagnostic tests in the triage of women with equivocal Pap smears. 12 Infection with high-risk (HR) types of HPV is necessary for the development of ICC 13-16 and the expression of the E6/E7 oncogenes is necessary for conversion to and maintenance of malignancy in cervical tissue. [17][18][19] Therefore, detection of the E6/E7 mRNA of HR-HPV types might serve as a better risk evaluation factor than mere DNA detection for the development of high-grade squamous intraepithelial lesion (HSIL) and ICC. 20 The combination of cytology and HPV testing seems to save additional life at a reasonable cost compared to Pap testing alone. 21,22 Detection of E6/E7 mRNA can be achieved by using the commercial PreTect HPV-Proofer assay (NorChip AS, Klokkarstua, Norway), that utilizes nucleic acid sequence based amplification (NASBA).The aim of our study was to assess whether a positive HPV mRNA or DNA test at the time of an ASCUS or LSIL Pap-smear identifies women diagnosed with a histological CIN2ϩ after 2 years of follow-up. Material and methods Study subjectsThe study subjects comprise a subgroup from 4,136 women older than 30 years of age that visited a selection of gynecologists in Oslo, Norway, and have been tested in 2001 for the presence of HPV DNA by Gp5ϩ/6ϩ consensus PCR and E6/E7 transcripts by real-time multiplex NASBA (PreTect HPV-Proofer, NorChip AS) in addition to cytology. 35 PreTect HPV-Proofer detects mRNA from HPV types 16, 18, 31, 33 and 45, whereas Gp5ϩ/6ϩ consensus PCR detects HPV DNA from the L1 region in Ͼ20 HPV types. We included all women with an index Pap smear diagnosis of ASCUS or LSIL (n ϭ 77). The index Pap smear refers to the smear taken together with the HPV testing. Information on Pap smears in the 10-year period before the inclusion in our study was obtained from CRN registers. Former abnormal smears mean any smear that is not normal or unsatisfactory and has been taken before the index smear in 2001. Follow-upSeventy-seven women were followed up for 24 months in the registers of the Cancer Registry of Norway (CRN) with subsequent Pap smears...

Type‐specific human papillomavirus oncogene messenger RNA levels correlate with the severity of cervical neoplasia

“…E6 type-specific PCR was used to validate multiple infections. Quantitative real-time reverse transcriptase (QRT-PCR) and real-time PCR used to measure mRNA levels and DNA viral loads of 6 HPV oncogenic types (HPV 16,18,31,33, 52 and 58) in 720 liquid-based cytology samples. The HPV DNA and RNA measurements were correlated with cervical lesions diagnosed by histopathologic examination.…”

“…The HPV types 16,18,31,33 and 45 are reportedly the most prevalent types associated with cervical cancer worldwide. 1,5 The most common oncogenic HPV type in preinvasive and cervical cancer is HPV 16, which is detectable in 21% of women with low-grade squamous intraepithelial lesions (LSILs), 6 in more than 50% of women with cervical intraepithelial neoplasia grade 3 (CIN 3) and in women with cervical cancer in America and Europe.…”

“…[8][9][10][11][12][13][14][15] On the other hand, the occurrence of infection with HPV types 52 or 58 is relatively uncommon in cervical cancer in the Americas, Europe, Africa and Southeast Asia. 1 The presence of HPV types 16,18,31,33 and 45 E6 and E7 mRNA is found in the majority of cervical carcinomas. 16 However, the importance and prevalence of HPV 52 and 58 transcripts in normal populations and preinvasive and cervical cancers in different geographic areas such as Taiwan and other Asian countries have not been explored.…”

“…Among HPV DNA-positive women, it is most important to identify those with increased risk of having or developing precancerous lesions or cervical carcinoma by measuring DNA viral loads 17 or high-risk (HR) HPV transcription, 18 or by detecting integrated HPV for cervical carcinogenesis. Integration is a proposed mechanism for the progression from cervical intraepithelial neoplasia (CIN) to carcinoma.…”

“…21,22 Only a small proportion of HPV DNA-positive women with a normal, atypical squamous cell of uncertain significance or LSIL diagnosis have detectable mRNA expression. 18 HPV DNA testing will not discriminate between active, latent and transient infections. Moreover, recent data indicates that the risk of conversion from an acute infection to a transforming infection is substantially different for individual HR types.…”