Kate Cuschieri

Human Papillomavirus mRNA and p16 Detection as Biomarkers for the Improved Diagnosis of Cervical Neoplasia

Human papillomavirus (HPV) infection of the genital tract is very common and normally follows a benign clinical course; however, in an unfortunate minority of infected individuals, it can cause disease that sometimes leads to cancer. It is accepted that HPV DNA testing has a role in the management of cervical disease both in a prevaccination and postvaccination era; however, to improve the specificity of this approach, there is a requirement to develop and validate tools/assays that can identify women at risk for progressive disease. There is evidence to suggest that detection of viral gene expression both directly and indirectly may constitute a more specific approach for delineating clinically significant infection compared with HPV DNA–based assays. HPV oncogene expression and evidence of its deregulation can be monitored through direct detection of viral mRNA transcripts or through detection of the cellular protein p16. For both approaches, commercial assays have been introduced and numerous studies have been conducted. The present article describes the scientific theory underpinning these approaches, their amenability to routine-diagnostic specimens/settings, and the clinical data that has been garnered through their application thus far. Currently, there is promising data indicating that HPV mRNA and p16 might play an important role in future cervical cancer screening scenarios. Still, large randomized studies are necessary to confirm the preliminary data. Methods: PubMed and OVID were interrogated with search terms “HPV RNA;” “HPV mRNA;” “HPV transcript—detection, testing, and methods;” “p16” AND “cervical cancer;” “p16” AND “CIN;” “p16” AND “histology”; “p16” AND “cytology;” “p16;” and “screening.” (Cancer Epidemiol Biomarkers Prev 2008;17(10):2536–45)

Eurogin Roadmap 2017: triage strategies for the management of HPV-positive women in cervical screening programmes

Cervical cancer screening will rely, increasingly, on HPV testing as a primary screen. The requirement for triage tests which can delineate clinically significant infection is thus prescient. In this EUROGIN 2017 roadmap, justification behind the most evidenced triages is outlined, as are challenges for implementation. Cytology is the triage with the most follow‐up data; the existence of an HR‐HPV‐positive, cytology‐negative group presents a challenge and retesting intervals for this group (and choice of retest) require careful consideration. Furthermore, cytology relies on subjective skills and while adjunctive dual‐staining with p16/Ki67 can mitigate inter‐operator/‐site disparities, clinician‐taken samples are required. Comparatively, genotyping and methylation markers are objective and are applicable to self‐taken samples, offering logistical advantages including in low and middle income settings. However, genotyping may have diminishing returns in immunised populations and type(s) included must balance absolute risk for disease to avoid low specificity. While viral and cellular methylation markers show promise, more prospective data are needed in addition to refinements in automation. Looking forward, systems that detect multiple targets concurrently such as next generation sequencing platforms will inform the development of triage tools. Additionally, multistep triage strategies may be beneficial provided they do not create complex, unmanageable pathways. Inevitably, the balance of risk to cost(s) will be key in decision making, although defining an acceptable risk will likely differ between settings. Finally, given the significant changes to cervical screening and the variety of triage strategies, appropriate education of both health care providers and the public is essential.

The impact of bivalent HPV vaccine on cervical intraepithelial neoplasia by deprivation in Scotland: reducing the gap

Background Cervical cancer disproportionately affects women from lower socioeconomic backgrounds. A human papillomavirus (HPV) vaccination programme was introduced in Scotland in 2008 with uptake being lower and inequitable in a catch-up cohort run for the first threeu2009years of the programme compared with the routine programme. The socioeconomic differences in vaccine uptake have the potential to further increase the inequality gap in regards to cervical disease. Methods Vaccination status was linked to demographic, cytological and colposcopic data, which are routinely collected by the Scottish HPV surveillance system. Incidence rates and relative risk of cervical intraepithelial neoplasia (CIN) 1, 2 and 3 in unvaccinated and vaccinated women were stratified by birth year and deprivation status using Poisson regression. Results Women who received three doses of HPV vaccine have significantly decreased risk of CIN 1, 2 and 3. Vaccine effectiveness was greater in those women from the most deprived backgrounds against CIN 2 and 3 lesions. Compared with the most deprived, unvaccinated women, the relative risk of CIN 3 in fully vaccinated women in the same deprivation group was 0.29 (95% CI 0.2 to 0.43) compared with 0.62 (95% CI 0.4 to 0.97) in vaccinated women in the least-deprived group. Conclusions The HPV vaccine is associated with significant reductions in both low-grade and high-grade CIN for all deprivation categories. However, the effect on high-grade disease was most profound in the most-deprived women. These data are welcoming and allay the concern that inequalities in cervical cancer may persist or increase following the introduction of the vaccine in Scotland.

Defining optimal triage strategies for hrHPV screen positive women - an evaluation of HPV 16/18 genotyping, cytology and p16/Ki-67 cyto-immunochemistry.

Background: Several options for the triage of high-risk HPV screen–positive (hrHPV+) women were assessed. Methods: This study incorporated CIN2+ cases and controls, all of whom tested hrHPV+ and whose results of liquid-based cytology (LBC), HPV16/18 genotyping, and p16/Ki67 cytoimmunochemistry were available. Sensitivity and specificity for the CIN2+ of these triage tests were evaluated. Results: Absolute sensitivities of HPV 16/18 typing, LBC, and p16/Ki-67 cytoimmunochemistry for CIN2+ detection were 61.7%, 68.3%, and 85.0% for women with hrHPV+ clinician-taken samples. Respective specificities were 70.5%, 89.1%, and 76.7%. The absolute accuracy of the triage tests was similar for women with a hrHPV+ self-sample. P16/Ki-67 cyto-immunochemistry was significantly more sensitive than LBC although significantly less specific. Conclusions: All three single-test triage options, if positive, exceed the threshold of 20% risk at which colposcopy would be indicated. However, none of them conferred a post-test probability of CIN2+ <2%; which would permit routine recall. P16/Ki-67 cytoimmunochemistry on HPV16/18 negative women had a post-test probability of CIN2+ of 1.7% and 0.6% if also LBC negative. Impact: This is one of the few studies to directly compare the performance of triage strategies of hrHPV+ women, in isolation and combinations. It is the only study assessing triage strategies in women who test hrHPV+ in self-taken vaginal samples. A combined triage option that incorporated HPV 16/18 typing prior to p16/ki-67 cytoimmunochemistry in HPV 16/18–negative women yielded a post-test probability of CIN2+ of >20%, whereas women who tested negative had a probability of CIN2+ of <2%. Cancer Epidemiol Biomarkers Prev; 26(11); 1629–35. ©2017 AACR.

Host chemokine signature as a biomarker for the detection of pre-cancerous cervical lesions

Background The ability to distinguish which hrHPV infections predispose to significant disease is ever more pressing as a result of the increasing move to hrHPV testing for primary cervical screening. A risk-stratifier or “triage” of infection should ideally be objective and suitable for automation given the scale of screening. Results CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL12 emerged as the strongest, candidate biomarkers to detect underlying disease [cervical intraepithelial neoplasia grade 2 or worse (CIN2+)]. For CIN2+, CCL2 had the highest area under the curve (AUC) of 0.722 with a specificity of 82%. A combined biomarker panel of six chemokines CCL2, CCL3, CCL4, CXCL1, CXCL8, and CXCL12 provides a sensitivity of 71% and specificity of 67%. Conclusion The present work demonstrates that the levels of five chemokine-proteins are indicative of underlying disease. We demonstrate technical feasibility and promising clinical performance of a chemokine-based biomarker panel, equivalent to that of other triage options. Further assessment in longitudinal series is now warranted. Methods A panel of 31 chemokines were investigated for expression in routinely taken archived and prospective cervical liquid based cytology (LBC) samples using Human Chemokine Proteomic Array kit. Nine chemokines were further validated using Procartaplex assay on the Luminex platform.

Validation of EUROArray HPV test using the VALGENT framework

BACKGROUNDnRobust clinical and analytical validation of human papillomavirus (HPV) tests is a pre-requisite for their use in cervical cancer screening given the transience of most high-risk HPV infections.nnnOBJECTIVESnTo evaluate the EUROArray HPV test (PCR-based full HPV genotyping test) using the international validation of the VALGENT framework, which offers an opportunity to determine analytical and clinical performance according to internationally accepted performance metrics.nnnSTUDY DESIGNnA total of 1300 consecutive and 300 abnormal cervical samples derived from the Slovenian screening programme were tested with the EUROArray HPV test. Clinical performance for the detection of cervical intraepithelial neoplasia grade 2 and above (CIN2+) was performed and compared to a standard comparator test (Hybrid Capture 2). Intra- and inter-laboratory reproducibility of the assay was performed in a subset of 500 samples.nnnRESULTSnThe relative clinical sensitivity and specificity of EUROArray HPV vs HC2 was 0.93 (95% Confidence Interval (CI), 0.88-0.99; P non-inferiority(ni)u2009=u20090.1413) and 1.01 (95% CI, 0.99-1.02; Pniu2009=u20090.0001), respectively. Application of an a-posteriori cut-off for HPV16 led to relative values of 0.98 (95% CI, 0.92-1.03; Pniu2009=u20090.0076) and 1.00 (95% CI, 0.97-1.03; Pniu2009=u20090.007), respectively. The assay showed excellent intra- and inter-laboratory reproducibility (concordance ≥ 94%, Kappas ≥0.85).nnnCONCLUSIONnAt the predefined cut-off, EUROArray HPV was less sensitive than HC2 for the detection of CIN2+. However, when an optimised cut-off was applied, EUROArray HPV fulfilled international criteria for its use in cervical cancer screening.

Human Papillomaviruses (HPVs)

HPVs are epitheliotropic viruses with double-stranded DNA genomes and eight coding genes defined as “early” or “late” depending on when they are expressed. Over 200 HPVs have been identified with 13 types considered oncogenic or high risk (HR). HPV type 16 confers the greatest risk, being responsible for around 60% of cervical cancers. HPV infection is common with a global point prevalence of around 10% and most infections are transient. The life cycle of HPV is inextricably linked with squamous epithelial differentiation and, during a productive infection, involves tightly regulated sequential gene expression at the separate epithelial layers before particle release. Some persistent infections can lead to cancer; in this scenario the productive life cycle is not completed, and deregulated expression of early oncoproteins E6 and E7 stimulates uncontrolled cellular proliferation while abrogating tumor suppressor function.

Evaluation of a Novel Single-Tube Method for Extended Genotyping of Human Papillomavirus

ABSTRACT The use of high-risk human papillomavirus (HPV) testing for surveillance and clinical applications is increasing globally, and it is important that tests are evaluated to ensure they are fit for this purpose. In this study, the performance of a new HPV genotyping test, the Papilloplex high-risk HPV (HR-HPV) test, was compared to two well-established genotyping tests. Preliminary clinical performance was also ascertained for the detection of CIN2+ in a disease-enriched retrospective cohort. A panel of 500 cervical liquid-based cytology samples with known clinical outcomes were tested by the Papilloplex HR-HPV test. Analytical concordance was compared to two assays: a Linear Array (LA) HPV genotyping test and an Optiplex HPV genotyping test. The initial clinical performance for the detection for CIN2+ samples was performed and compared to that of two clinically validated HPV tests: a RealTime High-Risk HPV test (RealTime) and a Hybrid Capture 2 HPV test (HC2). High agreement for HR-HPV was observed between the Papilloplex and LA and Optiplex HPV tests (97 and 95%, respectively), with kappa values for HPV16 and HPV18 being 0.90 and 0.81 compared to the LA and 0.70 and 0.82 compared to the Optiplex test. The sensitivity, specificity, positive predictive value, and negative predictive value of the Papilloplex test for the detection of CIN2+ were 92, 54, 33, and 96%, respectively, and very similar to the values observed with RealTime and HC2. The Papilloplex HR-HPV test demonstrated a analytical performance similar to those of the two HPV genotyping tests at the HR-HPV level and the type-specific level. The preliminary data on clinical performance look encouraging, although further longitudinal studies within screening populations are required to confirm these findings.