**7. HPV E6/E7 mRNA screening methods**

Several recent studies have clearly shown that testing for HPV mRNA instead of HPV DNA can be clinically useful [70, 93, 94]. The most relevant transcripts for diagnostic purposes are those encoding viral oncoproteins E6 and E7. The detection of viral mRNA can be done by reverse

transcriptase real time PCR [95] or by nucleic acid sequence-based amplification (NASBA). For the latter, three commercially available assays that detect E6/E7 transcripts are currently available. PreTect HPV-Proofer (HPV-Proofer; NorChip, Klokkarstua, Norway) is an assay based on NASBA technology, which allows qualitative determination of E6/E7 mRNA tran‐ scriptsofthefivemostfrequentlyidentifiedhr-HPVtypesincervicalcancerworldwide:HPV-16, HPV-18, HPV-31, HPV-33 and HPV-45 [96]. The NucliSENS EasyQ HPV V1 assay (NucliS‐ ENS; bioMérieux) was launched in 2007 and is based on the original HPV-Proofer assay, except for the proprietary hardware platform and the software for NASBA measurements and data analysis[97].APTIMAHPVAssay(APTIMA;Gen-Probe,SanDiego,CA,USA)isatranscriptionmediated amplification-based assay, which allows the detection of E6/E7 mRNA transcripts of 14HPVtypes:HPV-16,HPV-18,HPV-31,HPV-33,HPV-35,HPV-39,HPV-45,HPV-51,HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68. The assay generates a qualitative result for the presence/absenceof14targetedHPVsanddoesnotallowtheexactdeterminationofHPVtype(s) presentinaclinical specimen[98].APTIMAyieldedsimilar sensitivityforCIN2+comparedwith hc2,Amplicor andLinearArray(95.2vs 99.6%, 98.9%and98.2%,respectively),but a significant‐ ly higher specificity (42.2 vs 28.4%, 21.7% and 32.8%, respectively) [55]. In a comparative evaluation of APTIMA and hc2 on PreservCyt specimens collected from 800 women referred to colposcopy, APTIMA showed comparable sensitivity to hc2 for the detection of CIN2+ (91 vs 95%), as well as CIN3+ (98 vs 99%), but had higher clinical specificity (>55 vs 47% for CIN2+; 53 vs 44% for CIN3+) [99]. APTIMA had the best sensitivity/specificity balance measured by AUC (areaunderROCcurve) comparisontest(significantforCIN2+),andthe colposcopyreferralrate (9.2%) comparable to that of liquid citology (8.7%) [100].

cervical surface and (ii) the level of viral production in the area of infection. Viral load has been suggested to be a potential biomarker for cervical intraepithelial neoplasia grade 2 (CIN2) or greater, but currently there is no consistent evidence that a one-time measurement of viral load is a useful marker of prevalent disease or disease progression [101]. A widespread productive infection might be associated with high viral load, while a small incipient CIN3 with low-level virus production might be associated with low viral load. Furthermore, viral load in a cytological sample is subject to sampling variation in which there are varying proportions of lesional cells, normal epithelial cells, inflammatory exudate, and blood. A further complication in using viral load to predict neoplasia of CIN2 or greater is the high prevalence of multiple carcinogenic HPV infections detected in cervical samples. The current paradigm is that cervical lesions clonally expand following infection with one specific genotype (one virus-one lesion concept). On the cervical surface, multiple independent infections or lesions may occur that are caused by different genotypes. Without specific genotyping conducted *in situ*, assigning a causal HPV genotype to a specific lesion can only be based on assumptions [102]. HPV16 is the only genotype for which there is some indication that viral load may predict viral persis‐

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Quantitating HPV viral load seems to be a rational strategy of identifying women at risk for persistent HPV infection and progression to high-grade dysplasia. Accordingly, a high viral load could represent many cells with few virions each or a few cells containing many virions. An inaccurate description of the viral biology and the possible implications for the host could result from this discrepancy. Recently, studies have focused on longitudinal observations of viral load to predict viral clearance or lesion progression [106, 107]. Initial data indicate that repeated measurements can improve prediction of persistence or clearance, but these data are, so far, limited to HPV16 only. Although signal intensities from HC2 or various endpoint PCRbased assays have been proposed and partly used as surrogates for viral load, these approaches have limitations [108, 109]. HC2 only gives aggregate signal strength for a pool of 13 carcino‐ genic types, and commercial genotyping assays, such as the Roche Linear Array (LA) or Innogenetics InnoLiPA (line probe assay), do not formally report quantitative results. Over‐ estimation of the presence of oncogenic HPV may result. Despite these caveats, the develop‐ ment of HPV viral load assays that may reliably be used as an adjunct screening tool to identify women at increased risk of progression to CIN 2+ and cervical cancer remains a promising tool in cervical cancer screening. Recently, Wentzensen et al [110] measuring signal intensities on LA HPV genotyping strips provides quantitative information comparable to viral load measurements based on Q-PCR. This approach offers the potential for viral load assessment for 37 types in parallel, simplifying conducting repeated measurements of viral load in epidemiologic studies and addressing the problems of multiple HPV genotype infections in

Screening for HPV integration into the host genome is a subcategory of HPV diagnostics. HPV integration is a key molecular event in the transition from an innocuous HPV infection to one that has oncogenic potential. Human papilloma virus integration results in increased expres‐ sion of the viral E6 and E7 proteins. Increased expression of these proteins ultimately results in the disruption of host cell proteins, p53 and retinoblastoma protein [111]. Tests that detect

tence and progression to precancer [103-105].

studies of HPV load.
