Background: Cytological screening with Human Papillomavirus (HPV) triage for equivocal results has been the routine screening procedure for cervical cancer for years worldwide. The dual-marker stain p16/Ki67 (CINtec PLUS) has been shown to offer high sensitivity and specificity in triage of women at risk of developing HPV-related precancerous lesions. We evaluated the utility of CINtec PLUS in women with normal cytology and a positive HPV test, to see if this test can be used as a prognostic biomarker.

Methods: Women of 18 years or older were assembled between January 2018 and December 2022 at two different study sites. These were cytology negative for intra-epithelial neoplasia (NILM) and a positive HPV test. Prognostic value of the CINtec PLUS test for NILM samples and the confounding effect of HPV-subtype, age, university, and follow-up stage were evaluated.

Results: CINtec PLUS was positive in 63 out of 312(20%) NILM/ HPV-positive cases. The positive predictive value (PPV) of CINtec PLUS was 92% for High-Grade Dysplastic Lesion (HSIL), 64% for NILM and 31% for low grade dysplasia. The negative predictive value was 83% for HSIL, 64% for NILM and 83% for low grade dysplasia.

Conclusions: Adding CINtec PLUS with NILM cytology and HPV-positive test can be an important prognostic tool to identify women at risk for a high-grade dysplastic cervical lesion. Importantly, the test can also be used in primary HPV screening programs. However, women with low-grade dysplasia remain at risk for over- and under-treatment.

Cervical cancer, Cytology, HPV • CINtec PLUS, NILM

Cras L, Brock S, Barbe K, Locy H, Broeckx G, et al. (2023) Combined HPV and CINtec PLUS Testing for Triaging Cervical Cancer Screening in a Belgian Cohort. Ann Clin Cytol Pathol 9(1): 1144.

ASC-H: Atypical Squamous Cells, Cannot Rule Out High Grade Squamous Intra-Epithelial Lesion; ASC-US: Atypical Squamous Cells of Undetermined Significance; HPV: Human Papilloma Virus; HR: High-Risk; HSIL: High-Grade Intraepithelial Lesion; NILM: Negative for Intra-Epithelial Neoplasia or Malignancy; LR-HPV: Low-Risk Human Papilloma Virus; LBC: Liquid-Based Cytology; LSIL: Low-Grade Intraepithelial Lesion; PAP-Test: PapanicolaouTest; SCC: Squamous Cell Carcinoma.

Cervical cancer remains a major cause of death worldwide, despite the implementation of primary and secondary prevention strategies [1]. Cervical HPV infections are common, almost all women will get infected in their lifetime [2,3]. The HPV family consists of a group of more than 200 related viruses, divided into Low-Risk (LR) HPVs and High-Risk (HR), oncogenic HPVs. These last have the potential to interfere with the normal function of the cell cycle and cause uncontrolled proliferation, leading to premalignant epithelial lesions. In particular, HPV16+ and HPV18+ strains are responsible for the most HPV-related cervical cancers [3].

Normally, the immune system scans for foreign material. However sometimes the infected cells can escape immune surveillance and evolve into a precancerous lesion [4]. Carcinogenesis is a multi-step process. It takes several years since malignant cells can be in an equilibrium stage with the immune system or can remain dormant [5]. Screening strategies for detection of pre-malignant cervical neoplasia as well as the use of prophylactic vaccines against several HR-HPV subtypes, have proven successful to prevent lesions [6]. Several techniques are being used in population-wide screenings programs to reduce morbidity and mortality from cervical cancer, the most important being cytology and HPV testing [7].

Increasing numbers of cytology negative for intraepithelial neoplasia (NILM) are complemented by HPV testing on demand of the patient or clinician in order to identify more at risk cases. This leads to an increased number of HPV-positive cases, with subsequent referral for colposcopy [8]. These women are at risk for overtreatment in the absence of dysplasia, indicating the importance of refining the triage and management of these women [9]. Therefore, it is important to elucidate, why, when and how an HPV infection becomes oncogenic.

Ijkenberg and McMenamin, et al investigated the use of CINtec PLUS on NILM cytology samples [6,10]. The CINtec PLUS test uses two markers, p16 and Ki67, and has been shown to have increased total sensitivity (66% in Belgium) compared to cytology and HPV testing in detecting cervical dysplasia [9,10]. In normal cells, p16 has an anti-proliferative effect and arrests the cell cycle after mitosis. In cells infected by HR-HPV which has become oncogenic, cell cycle arrest does not occur. This inhibits a negative feedback control mechanism, resulting in over-expression of p16. Ki67 is a proliferation marker that can be detected in the nuclei of dividing cells and gives insight in the activation of the cell cycle [11,12]. Simultaneous detection of these biomarkers is indicative for an aberration in the cell cycle and oncogenic, HPV-related transformation [13].

In the summer of 2022, the federal Minister of Health announced plans for a new screening program, based on HRHPV testing, so called HPV primary screening. In such a program, the triaging problem of HPV positive NILM samples, will still be present and may even become more poignant. This study aims to investigate the prognostic value of the CINtec PLUS test in women with NILM cytology and HPV-positivity and its possible clinical implementation in the current screening programme.

The study was approved by the Ethics Committee of the University Hospital Brussels (B.U.N. 1432022000100 / EC number: EC-2022-134.

Study Population

Women were recruited via the Departments of Pathology at the University Hospital of Antwerp (UZ Antwerp) and the University Hospital of Brussels (UZ Brussel). 152 cases from cervical screening (Brussels) and 160 cases from both cervical screening and follow up cervical cytology (Antwerp) were included in the cohort. Both populations included women aged 18 years and above. Retrospectively, consecutive series of cytology samples that were diagnosed as NILM (negative for intraepithelial lesion or malignancy) with a positive additional HPV test for high-risk HPV subtypes, requested by either the clinician or the patients, were included in the cohort. Data from follow up cytology and/ or biopsy were collected. The follow-up period was 6 months to 1 year after the initial screening result.

In the UZA, on clinical request, a CINtec PLUS test was performed on HPV-positive cases following the manufacturer’s protocol (see below). HPV-positive cases with NILM cytology from the UZ Brussels, were sent for CINtec PLUS cytology analysis at the pathology lab in UZ Antwerp. In the UZ Brussels series, the CINtec data were not used in a clinical setting. All samples were pseudo-anonymized prior to enrollment in the study. Women with a history of previous gynecologic tumour, gynecologic radiotherapy or any kind of therapy in the pelvic region, were not included in the cohort. All samples with an abnormal/ borderline cytology and non-representative samples were excluded.

Cytology

In both labs, cytology testing was performed using the ThinPrep technique with the T5000 Processor (Hologic®). This system is used to process liquid -based cytology samples into monolayer slides. After completing the ThinPrep technique a PAP stain (polychromatic cytological stain) is performed on the Sakura® stainer. Cytology samples were analysed by a qualified and trained cytologist and cytopathologist and interpreted according to the Bethesda System.

HPV Testing

In UZB, HPV testing was performed using the Cobas 4800 HPV test (Roche®) which is a CE-IVD labeled, qualitative in vitro test for the detection of HPV in patient samples. The assay uses amplification of target DNA by PCR and nucleic acid hybridisation to detect 14 HR HPV types in one assay. This test specifically identifies HPV 16 and HPV 18, while simultaneously detecting ‘other high-risk HPV subtypes’ (31,33,35,39,45,51,52,56,58,59,6 6,68) at clinically relevant level of infection.

In UZA testing for HR-HPV was performed with the Cepheid HPV Xpert test. This is a comparable technique to the Cobas test, but it tests separately for the 14 different HR-HPV types. For this study, results were grouped like the Cobas results, to enable comparisons.

CINtec PLUS Immunocytochemistry

CINtec PLUS (Roche®) combines two biomarkers: a mouse monoclonal antibody p16 (p16INK4a) and proliferation biomarker, a rabbit monoclonal antibody Ki67. The kit includes a DAB brown detection chemistry for p16 and a fast red detection chemistry for Ki67. The brown color for p16 will stain in the cytoplasm and nucleus or only in the cytoplasm and the red color for Ki67 will stain only in the nucleus [10]. If simultaneously expressed, the markers indicate the effect of carcinogenically transforming HPV infections [3]. The test was performed on the Ventana BenchMark ULTRA IHC/ISH system (Roche®).

The CINtec PLUS test was considered positive when at least one dual-stained cervical cell was present (brown cytoplasmic staining for p16 and red nuclear staining for Ki67). The test was considered negative when staining was completely absent or if only one biomarker was present (brown cytoplasmic staining for p16 or red nuclear staining for Ki67) [6,10].

Dual-stained cytology was performed on residual cellular material out of the LBC vial collected at the initial screening visit [14]. Interpretation of the dual stain marker was highly reproducible with minimal training [15]. Samples were analysed by two trained pathologists.

Statistics and Analysis

A Linear Discriminant Analysis (LDA) was used for statistical analysis performed by the software programme SPSS v. 27. For this purpose, outcome of follow-up smears was divided into 4 variables (NILM, dysplasia, HSIL and no-follow up). Age, hospital (UZA or UZB) and HPV-subtype were considered confounders whereas CINtec PLUS was considered as the test to predict the outcome variable. Positive Predictive Value (PPV), Negative Predictive Value (NPV), false positives and false negatives were calculated. For each of the 4 different outcome variables a separate LDA was performed.

Overview of Cohort and Population Characteristics

The average age for the UZ Antwerp cohort is 49.4 years (range 24-82y) and for UZ Brussels 43 years (range 21-70y). Both cohorts had similar screening and HPV test results. HPV16+ was found in 62 cases (19.9%), and had follow up in 56 cases, 3 of which yielded high grade dysplasia. HPV18+ was present in 17 cases (5.4%). 14 of those had follow up, only 1 of which contained high grade dysplasia. 255 cases contained HPVother+ (81.7%). Of the 214 which received follow up, 10 were high grade lesions. In total 191(61.2%) of all follow up results were NILM. 12(3.8%) of all follow up results were high grade (Table 1).

CINtec PLUS test was positive in 63 women and was negative in the remaining 249 women. There were no significant differences between both cohorts in CINtec results. There were no nonconclusive results, all tests were analysable and interpretable by pathologists. In the CINtec positive group, follow-up smears were NILM in 38.1%, 34.9% contained a low-grade lesion and 17.5% a HSIL. In 9.5% there was no follow-up. In the CINtec negative group, follow-up smears were NILM in 67%, 16.9% contained a low-grade lesion, 1 case in 249 was a HSIL. 15.7% or 39 cases had no follow-up (Figures 1,2)

Figure 1: Overview of the study cohorts in relation to CINtec PLUS testing and clinical follow-up Results.

Figure 2: Image of a positive p16Ink4a and Ki67 dual-stain Immunocytochemistry.

(Table 1). In all, of 12 high-grade lesions found, only 1 was CINtec negative. This case contained HPVother+.

Follow-Up Cytology Stage and Hpv Subtype Did Not Show Correlation

The relationship of the CINtec PLUS test and of HPV subtype variables is represented in (Table 1). Within the CINtec positive samples with HSIL, HPV16+ was present in 4.8% of the cases, HPV18+ in 1.6% and HPVother+ in 14.3%. The low-grade dysplastic lesions showed HPV16+ in 7.9%, HPV18+ 1.6% and HPVother+ 25.4%. Finally, the HPV subtype distribution in the NILM smears was HPV16+ 9.5%, HPV18+ 4.8%, HPVother+ 30.2%. In contrast, CINtec PLUS negative samples with a HSIL follow up smear were not present for both HPV16+ and HPV18+ and was only in one case positive for HPVother subtype (0.4%). The low-grade dysplastic lesions showed HPV16+ 3.6%, HPV18+ 0.4% and for HPVother+ 14.1%. Finally, 13.3% had HPV16+ NILM smears, HPV18+ 3.2% and HPVother+ 53.8% (Table 1). These findings were not statistically significant.

Combination of HPV and CINtec PLUS testing proves valuable to detect lesions evolving to HSIL

The clinical performance of CINtec PLUS triage shows a high Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of 92% and 83% for HSIL respectively, regardless of age or HPV subtypes. CINtec PLUS test resulted in an accuracy of 84% for the HSIL. This is illustrated by the Receiver Operating Characteristic (ROC) curve (Figure 3),

Figure 3: Diagnostic performance for detecting HSIL is shown with a receiver operating characteristic curve.

were sensitivity is plotted against 1-specificity. The obtained Area under the Curve (AUC) provides the value for the accuracy; in this case for HSIL. The lowgrade cervical lesion category showed a low PPV and high NPV value of 31% and 83%, respectively, resulting in an accuracy of 73%. The follow-up smears with NILM showed a PPV and NPV value of both 64% (Table 2) (Figure 3).

CINtec PLUS positivity correlated significantly (p -value of < 0.001) with a follow-up cytology result of HSIL, while negative CINtec PLUS negativity was significantly related with a NILM follow-up cytology (p < 0.001). In contrast, low-grade lesions and the non-follow-up lesions could not be discriminated based on CINtec PLUS positivity or negativity. Regarding the HPV subtypes, no significant differences between HPV 16 / 18 / other were detected.

This retrospective interuniversity study aimed to evaluate the clinical utility and prognostic value of CINtec PLUS immunocytochemistry in a primary screening population of 312 HPV-positive women with NILM cytology.

Current primary screening in Belgium uses cervical cytology. Sensitivity of this technique is relatively low, as it is a morphology-based technique, and rather labor-intensive even when automated [16]. Nevertheless, a cervical smear is very specific for the detection of HSIL [14]. Additional HPV testing in triage is known to significantly increase the sensitivity for the detection of HSIL but has low specificity [10,14,16]. This is especially important in young women under 30 years of age with a relatively high prevalence of often subclinical HPV infections, creating a risk for over-treatment with colposcopy [6]. In our study, there were only 45 cases (14%) without follow up, even though they had no cytological lesion. 191(72%) of all follow up results were NILM. This illustrates the risk of over-treatment.

In previous studies the sensitivity of dual-stained immunocytochemistry reached more than 90% of the sensitivity level of HPV testing. At the same time, specificity is significantly higher as compared to HPV testing, reducing the clinical falsepositive HPV results and colposcopy referrals by almost 50% [10]. This is confirmed in this Flemish study, where 11 out of 12 HSILs in follow -up had a positive CINtec PLUS result. This could not have been predicted just by cytology and HPV screening results.

Previous studies showed that CINtec PLUS positivity is associated with HSIL or invasive carcinoma and that simultaneous detection of the tumour suppressor protein p16 and the proliferation marker Ki67 is associated with oncogenic HPV-associated transformation in the cervical cells [17]. In September 2020, the FDA approved the expanded use of CINtec PLUS as a triage of HPV positive women, to clarify further clinical management. Our study in NILM samples, confirms this observation as CINtec PLUS shows high PPV (92%) and high NPV (83%) for high grade dysplasia (HSIL). These results are promising to improve clinical follow-up for HPV positive women with positive CINtec PLUS results.

In addition, for follow-up cytology resulting in NILM, the CINtec PLUS test shows a PPV of 64% and an NPV of 64%. These results confirm previous findings in the literature with a PPV of 66% [6]. In concordance with previous results, our data suggest that a CINtec PLUS test is a valuable tool to avoid overtreatment in women with NILM and recommend a normal screening interval to them while in women possibly harbouring a high-grade lesion, the CINtec PLUS is a valuable tool to avoid under treatment and identify these lesions faster [6,18].

However, low-grade lesions are more difficult to differentiate by CINtec PLUS. Results in borderline or low-grade lesions (ASCUS, LSIL) show low PPV (31%) and high NPV (83%). This is understandable in the light of oncogenic transformation. Only a subset of these lesions is really dysplastic. Because of this, women with such lesions are at risk for overtreatment [6,19]. There is a reduced sensitivity for low-grade lesions, in particular for women < 30 years. This needs to be more carefully considered in individual risk assessments of women. Much more research is needed in this area. However, CINtec PLUS was found to be more specific than HPV for such lesions and reduced the number of colposcopic referrals for women with LSIL [20,21].

A recent study has shown that hyper methylation of CpG islands in proximity to the human gene methylation panel (specific for CIN3 and cervical cancer) (DLX1, ITGA4, RXFP3, SOX17 and ZNF671) correlated with the presence of both low-grade and high-grade cervical lesions [22]. This GynTect study shows an excellent specificity for SCC, CIN3 and even for CIN1/2 and found a similar PPV (74.5%) value as for CINtec PLUS (69.2%) although they highlight that it did not allow differentiation between lesions prone to progression and lesions that might persist or regress. Although this can be an interesting alternative for the future, this molecular test has its boundaries as it requires more financial resources, trained lab staff for technical performance and proper analysis. In comparison CINtec PLUS is a relatively low-cost test which is easily implemented and can be interpreted in the same lab as the cytologic evaluation [7].

Findings

Our findings regarding predictive value and accuracy of CINtec PLUS in HPV-positive NILM women are in correlation with previous studies that have investigated these features in different populations [23-26]. A limitation of our study is a short followup period of 48 months, registered from the beginning of 2018 to the end of 2022, while the development of a dysplastic lesion can be highly variable and can often take more time. One study noted that CIN2/CIN3 lesions had a short-term progression rate of ± 30% [7]. This implies that women who currently have NILM cytology and positive CINtec, can still develop HSIL after the reported follow-up period. For these women, an extended followup period with CINtec PLUS testing may be required, something we could not determine at this point. On the other hand, another study found out that CIN2 lesions showed an overall regression rate of 50% [27].

In our study, the outcome of follow-up smears was divided into 4 groups (NILM, dysplasia, HSIL and no follow-up). The disadvantage of working with an all-encompassing term dysplasia for LSIL, ASC-US and ASC-H is that samples with overt reactive changes that were interpreted as ASC-US could end up under the heading of dysplasia. In this way, a bias would be created that could affect PPV, NPV and accuracy.

In contrast with large cohort studies, our study did not observe any significant differences between outcome and HPV subtype and age which can be due to our small population. Multiple other studies with separate HPV16 and HPV18 testing noted a correlation with the probability of developing an HSIL, especially in older women [28,29]. Moreover HPV16-positive women show a higher absolute risk for cervical pre-cancer in contrast with other oncogenic HPV types, which may have clinical implications [6,30,31]. Our study population contains quite a high number of non-16/18 hrHPV types. The reason for this is unclear. There is a group of African as well as eastern European women includes, which may have influenced the type distribution. Since the study was not designed to evaluate such differences, we do not know if this could be an explanation. It could also be a legitimate shift of the HPV distribution, as part of the population already vaccinated for HPV16/18. A third possibility is, that the HPV tests used in this study picked up more non-16/18 types, co-infections etc. Variations in specificity for the respective types could explain this. However, we used standardized, clinically approved tests, which are applied in daily practice [32,33]. Therefore we don’t expect these tests to deviate much from expected performance.

Glandular lesions were not taken into consideration as our cohort did not include any glandular lesions in follow-up. The value of CINtec PLUS in women with glandular lesions was evaluated elsewhere and demonstrated good prognostic results [34-37].

Notable in this study is the number of women with no follow-up smear (n = 46, 14.8%). This may be due to the initial negative smear which causes women in Belgium to be followed in a 3-year follow-up schedule. Currently HPV infections without a concurrent dysplastic lesion are not covered by the screening guidelines. Therefore, it is unclear what management such cases need. On the other hand, it is important to properly inform the female population to reduce barriers about screening and improve screening compliance [38-40].

At the moment immunocytochemistry tests such as CINtec PLUS are not reimbursed by the RIZIV/INAMI for cervical screening purposes. This may be a reason why there is only little information on the use of this test in the Belgian/ Flemish screening programme. If the accuracy of CINtec PLUS to identify high grade dysplasia could be confirmed in this population, this could lead to more efficient (faster) and cost-effective screening. Less repeat testing and less colposcopies would be needed to confirm the presence of dysplasia.

In the nearby future Belgium may follow other countries and introduce a cervical screening programme with HPV testing as primary screening modality [41-43]. In such a programme triage of HPV positive cases will still be necessary, to avoid an excess of colposcopy follow up. This could be done with either cytology, CINtec or a combination of tests to provide a strategy to identify those women who need follow up.

This study investigated the added value of a CINtec PLUS test in 312 women with a NILM and HPV-positivity. We note a significant added value for women who are at risk of developing HSIL. However, the risk of over- and under treatment of women with low-grade dysplasia remains real. We have shown that CINtec PLUS is not only a valuable tool for equivocal screening results but also has added value for initial NILM cytology. The same holds true in a setting with HPV primary screening.

SS: Conceptualisation, Funding acquisition, Supervision, Writing-review and editing. LC: Project administration, Investigation, Methodology, Resources, Data analysis, Writing manuscript and editing. SB: Data interpretation. HL: Data interpretation. KB: Data analysis and Writing-review. GB: Data collection and Writing-review.

Statement of Ethics

A written informed consent was not required and the retrospective analysis was undertaken after approval from the Institutional Ethics Committee on human research UZ Brussel/ VUB (BUN: 1432022000100/EC number 2022-134).

Funding Sources

This study was supported by a research grant from Roche providing CINtec PLUS test kits for the study. The study was investigator initiated and conducted independently.

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