Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.

The implementation of high-risk human papillomavirus testing (hrHPV testing) as a screening method in substitute for cytology has evoked the need for more sensitive and less objective tests for the triage of HPV-positive women. In a cohort of 1763 HPV-positive women, the potential of immunocytochemical p16 and Ki-67 dual staining as compared to cytology, alone or in combination with HPV partial genotyping, was tested for triage of women attending a cervical cancer screening program. Performance was measured using sensitivity, specificity, and positive and negative predictive values. Comparisons were assessed using logistic regression models and the McNemar test. Dual staining was evaluated in a prospectively collected study cohort of 1763 HPV-screened women. For triage of CIN2+ and CIN3+, NPV and sensitivity, 91.8% and 94.2% versus 87.9% and 89.7%, respectively, were significantly higher using dual staining together with HPV 16/18 positive, as compared to cytology (p < 0.001). The specificities, however, were lower for dual staining as compared to cytology. Conclusions: Dual staining is safer for decision-making regarding HPV-positive women’s need for follow-up with colposcopy and biopsy, as compared to cytology.

The purpose of this study was to establish a gene signature that may predict CIN3 regression and that may aid in selecting patients who may safely refrain from conization. Oncomine mRNA data including 398 immune-related genes from 21 lesions with confirmed regression and 28 with persistent CIN3 were compared. L1000 mRNA data from a cervical cancer cohort was available for validation (n = 239). Transcriptomic analyses identified TDO2 (p = 0.004), CCL5 (p < 0.001), CCL3 (p = 0.04), CD38 (p = 0.02), and PRF1 (p = 0.005) as upregulated, and LCK downregulated (p = 0.01) in CIN3 regression as compared to persistent CIN3 lesions. From these, a gene signature predicting CIN3 regression with a sensitivity of 91% (AUC = 0.85) was established. Transcriptomic analyses revealed proliferation as significantly linked to persistent CIN3. Within the cancer cohort, high regression signature score associated with immune activation by Gene Set enrichment Analyses (GSEA) and immune cell infiltration by histopathological evaluation (p < 0.001). Low signature score was associated with poor survival (p = 0.007) and large tumors (p = 0.01). In conclusion, the proposed six-gene signature predicts CIN regression and favorable cervical cancer prognosis and points to common drivers in precursors and cervical cancer lesions.

Approximately 20–40% of high-grade Cervical Intraepithelial Neoplasia (CIN) regresses spontaneously, but the natural prognosis of an individual lesion is unpredictable. Gain of the chromosomal 3q region, which contains the human telomerase RNA gene on 3q26, is found in CIN lesions and cervical carcinoma and shows correlation with disease grade. The aim of this study is to assess whether 3q26 gain as a single genetic marker can predict the natural prognosis of high-grade CIN, by performing a review of the literature and pilot study. A literature review was conducted. Additionally, we performed a pilot study in 19 patients with histologically confirmed high-grade CIN lesions who were followed for a mean of 115 days, after which loop excision was performed. Fluorescent in situ hybridization analysis was performed on the initial diagnostic biopsies to determine gain of 3q26. Eight studies were included in the literature overview, with a total of 407 patients. Of these, only 22 patients had high-grade lesions. All studies found an association between 3q26 gain and disease prognosis. Positive predictive values (PPV) ranged from 50 to 93%, negative predictive values (NPV) ranged from 75 to 100%. Only five out of 155 patients (3.2%) without 3q26 gain showed disease persistence or progression. In our pilot study on 3q26 gain in high-grade CIN, the PPV of 3q26 gain for disease persistence was 67%, the NPV 100%. All four patients without 3q26 gain showed disease regression. In conclusion, the absence of 3q26 gain in diagnostic biopsies may be applied to identify high-grade CIN lesions with a high probability of disease regression.

Human papilloma virus (HPV) infections vary in their oncogenic potential, and whether an infection progresses to cervical intraepithelial neoplasia (CIN) also depends on the immune response. Therefore, the aim of the present study was to explore biomarkers related to the immune system and cell proliferation, in combination with HPV classified as having high (HOP) or low oncogenic potential (LOP), that can possibly guide a more accurate identification of women following cervical cancer screening programmes in need for immediate follow-up with a biopsy. A next-generation sequencing transcriptomic immune profile analysis applied to 28 persistent CIN3 lesions and 14 normal biopsies identified four genes, the immune markers ARG1 and HLA-DQB2 and the tumour markers CDKN2A and KRT7, as possible markers for differentiating between CIN3 and normal tissue. To validate these findings, analysis of the relative gene expression of these markers by use of reverse transcriptase real-time quantitative polymerase chain reaction was performed in an independent cohort of 264 (82 normal, 64 CIN1, and 118 CIN2/CIN3) biopsies, and the data were combined with information on the HOP- or LOP-HPV identified in the biopsies. Statistical analysis was performed with receiver operating characteristic curves, reporting area under the curve (AUC) with 95% confidence intervals (CIs), and logistic regression. Statistically significantly higher median expression levels of CDKN2A (p < 0.001) and KRT7 (p = 0.045) and significantly lower expression of ARG1 (p = 0.012) were found in biopsies with HOP-HPV infections, with no difference detected for HLA-DQB2 (p = 0.82). Models using expression levels of CDKN2A (AUC, 0.91; 95% CI, 0.86–0.95), KRT7 (0.86, 0.81–0.91), or ARG1 (0.78, 0.70–0.85) together with HOP/LOP-HPV class were significantly better than HPV class alone (0.72, 0.66–0.79) in discriminating CIN2/3 versus CIN1 (p < 0.001, p < 0.001, and p = 0.014, respectively).

Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV); however, factors such as HPV genotype and individual immune response may also contribute to its development. The loop electrosurgical excision procedure (LEEP) is a treatment for high-grade cervical intraepithelial neoplasia (CIN), as approximately 30% of these cases may progress to cancer. However, 20–40% of cases will regress spontaneously. HPV16 infection constitutes the highest risk for progression to cervical cancer and a lower probability of regression. Knowledge regarding the regression of lesions caused by other high-risk genotypes alone or in association with biomarker expression and lesion length has been limited. In the present study, the regression rates of high-grade squamous intraepithelial lesions were calculated. Twenty-one percent of the 161 women diagnosed with CIN2-3 on colposcopy-directed biopsies exhibited regression (defined as CIN1 or less) in the subsequent cone excisions. The mean interval between biopsy and treatment was 113 days (range of 71–171). High-grade lesions of the squamous epithelium caused by HPV16, together with lesions caused by HPV31, 52 and 58, showed significantly lower regression rates (HR 0.54, 0.22–0.75; low-regression group) than lesions caused by HPV18, 33, 35, 39, and 45 (HR 2.85, 1.54–5.28; high-regression group). A multivariate analysis of HPV genotypes, epithelial expressions of pRb and p53, immune cell proportions in the stroma (CD4/CD25 and CD4/CD8), and lesion lengths correctly predicted regression in 78% (Harrell’s C). A Harrell’s C value of 82% for the low-regression group indicates that different HPV genotypes or groups, together with divergent patterns of tumor suppressors, immune cells, and lesion size, can give prognostic information regarding the outcome of CIN2-3.