Tumour Virus Research

The immunological heterogeneity of squamous cell carcinoma and adenocarcinoma of the uterine cervix: a systematic review

Cervical cancer is the fourth most common malignancy in women worldwide and generally driven by persistent infection with high-risk human papillomavirus. Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the two most common histological subtypes, with a relative increase in adenocarcinomas in the last decades. The immunological differences between cervical squamous cell carcinoma and adenocarcinoma remain largely unexplored. Understanding these distinctions is crucial for developing tailored therapies that can improve treatment outcomes for patients with cervical cancer. This systematic review provides an overview of the immunological features of squamous cell carcinoma and adenocarcinoma of the uterine cervix. A systematic search was performed in PubMed, Embase.com, Web of Science, and Cochrane Library. All articles addressing immunological features of squamous cell carcinoma and adenocarcinoma of the uterine cervix were reviewed and included based on predefined inclusion and exclusion criteria. In total, 3207 articles were screened, of which 43 were included. Studies show that cervical squamous cell carcinomas are characterised by a more inflamed tumour microenvironment, but also contain more regulatory T cells and immune checkpoints. In contrast, adenocarcinomas are characterised by lower immune cell infiltration, contributing to its poorer prognosis and more limited response to treatment. The observed differences emphasize the need for further research into subtype-specific differences and distinct therapeutic strategies. For squamous cell carcinomas, future research should focus on combinatorial immune checkpoint blockade, including regulatory T cell-depleting strategies. For adenocarcinomas, oncolytic virotherapy, therapeutic vaccination, and oncogenic signalling interference should be explored.

Precise identification of viral–host integration events in HPV-positive cervical cancers by targeted long-read sequencing

Human papillomaviral (HPV) integrations into host human genome, a frequently observed event in HPV associated cervical cancer, are currently mapped through expensive Whole Genome sequencing (WGS) or RNA sequencing (RNA-seq) methodologies. This study aims to develop a targeted sequencing assay to determine HPV integrations in cervical tumors without the need for WGS or RNA-seq. We employed a library preparation strategy using tiled single primers that bind to HPV genome as a template and possibly extend HPV sequences into adjacent host human genomic sequences resulting in HPV and human chimeric sequences. Using this strategy, we sequenced known HPV integrations in HPV18 positive HeLa and HPV16 positive SiHa cell lines. We further used this method to detect HPV integration sites in four HPV-positive cervical cancer patients and confirmed these integration breakpoints by WGS and Sanger sequencing. Functional impact of HPV integrations was explored through differentially expressed genes within or near topologically associating domain (TAD) boundaries, possibly disrupted by respective integration events in these patients. We found ZFP36L1, CPA3, CPB1 and CXCL8 as some of the differentially expressed genes within disrupted TADs, which are known cancer associated genes. Our approach also reduced the cost of HPV integration detection by 90 % compared to WGS while also minimizing sequencing data volume. We believe that this method captures HPV integrations at significantly reduced costs and lesser sequencing data volume leading to better understanding of disease progression and monitoring cancer treatment.

Transcriptomic profile of the immune genes, oncogenes, and tumor suppressor genes in HPV associated Cervical Intraepithelial Neoplasia 3 (CIN 3) and Cervical Squamous Cell Carcinoma (CSCC): Comparable expressions indicative of invasive potential

Cervical cancer is the fourth most common cancer among women globally, with a woman dying every 2 min. Despite the need to understand the tumor microenvironment (TME) transcriptome of cervical squamous cell carcinoma (CSCC) and cervical intraepithelial neoplasia grade 3 (CIN 3), studies remain limited. This study compares the TME transcriptome of HPV-positive CSCC and CIN 3, analyzing 168 genes involved in tumor cell interactions with inflammatory and immune mediators, transcription, signal transduction, oncogenesis, tumor suppression, angiogenesis, and apoptosis. Co-expressed genes identified in HPV + CSCC and CIN 3 were analyzed using computational biology. Gene Ontology and KEGG enrichment identified relevant biological pathways and cancer hallmarks. Fifty-five co-expressed genes were linked to cancer pathways, inflammatory responses, cell migration, and development. KEGG enrichment highlighted viral protein interactions involving cytokines, IL-17 signaling, and chemokine receptor interactions. These genes were associated with cancer hallmark pathways, including angiogenesis, inflammation, proliferation, genomic instability, invasion, and metastasis. Their similar expression in CSCC and CIN 3 suggests a potential prognostic value and that CIN 3 progression may involve changes in gene expression. We propose the term "CSCC-like carcinoma," indicating CIN 3's increased invasive potential at the molecular level.

Clinical indications for host-cell DNA methylation markers in cervical screening and management of cervical intraepithelial neoplasia: A review

DNA methylation of host-cell genes is an epigenetic process that regulates gene expression and is associated with cervical cancer development. Studies on the natural history of cervical intraepithelial neoplasia (CIN) and the molecular alterations associated with cervical carcinogenesis led to the identification of several host-cell DNA methylation markers. Over the past years, various studies on methylation markers have shown promising results in terms of diagnostic and prognostic value to improve cervical cancer screening and management of CIN. This review provides an overview of the clinical indications of host-cell DNA methylation markers in cervical screening and management of CIN, and outlines avenues for further applications.

Analysis of the progression of cervical cancer in a low-and-middle-income country: From pre-malignancy to invasive disease

To better understand cervical cancer progression, we analyzed RNA from 262 biopsies from women referred for colposcopy. We determined the HPV type and analyzed the expression of 51 genes. HPV31 was significantly more prevalent in precancer than stage 1 cancer and invasive cancer (p < 0.0001), and HPV16 increased in invasive disease (p < 0.0001). CCNE1, MELTF, and ULBP2 were significantly increased in HPV16-positive compared to HPV31 precancers, while NECTIN2 and HLA-E expression decreased. Markers of the innate immune system, DNA repair genes, and cell cycle genes are significantly increased during cancer progression (p = 0.0001). In contrast, the TP53 and RB1 tumor suppressor gene expression is significantly decreased in cancer cells. The T cell markers CD28 and FLT3LG expression decreased in cancer while FOXP3, IDO1, and ULBP2 expression increased. There is a significantly higher survival rate in individuals with increased expression of CD28 (p = 0.0005), FOXP3 (p = 0.0002), IDO1 (p = 0.038), FLT3LG (p = 0.026), APOBEC3B (p = 0.0011), and RUNX3 (p = 0.019), and a significantly lower survival rate in individuals with increased expression of ULBP2 (p = 0.035). These results will help us elucidate the molecular factors influencing the progression of cervical precancer to cancer. Understanding the risk of progression of specific HPV types and sublineages may aid in the triage of positive patients, and better knowledge of the immune response may aid in developing and applying immunotherapies.

The oncogenic role of the NSD histone methyltransferases in head and neck and cervical cancers

Understanding the role of NSD proteins in virus-induced cancers could reveal new therapeutic strategies. Targeting NSD proteins may not only disrupt the epigenetic changes triggered by viruses but also help restore normal cellular function. For instance, developing NSD inhibitors could counteract abnormal histone modifications caused by viral infections and slow cancer progression. Our review on the NSD protein family emphasizes its critical role in epigenetic regulation and cancer progression, also in virus-induced cancers. As research on the molecular mechanisms of NSD proteins advances, these proteins are emerging as promising candidates for targeted cancer therapies, particularly in cancers driven by histone modifications and transcriptional dysregulation.

Association of two genomic variants with HPV type-specific risk of cervical cancer

Human papillomavirus infection is integral to developing invasive cervical cancer in the majority of patients. In a recent genome-wide association study, rs9357152 and rs4243652 have been associated with seropositivity for HPV16 or HPV18, respectively. It is unknown whether these variants also associate with cervical cancer triggered by either HPV16 or HPV18. We investigate whether the two HPV susceptibility variants show association with type-specific cervical cancer in a genetic case-control study with cases stratified by HPV16 or HPV18, respectively. We further tested whether rs9357152 modulates gene expression of any of 36 genes at the human leukocyte antigen locus in 256 cervical tissues. rs9357152 was associated with invasive HPV16-positive cervical cancer (OR 1.33, 95%CI 1.03-1.70, p = 0.03), and rs4243652 was associated with HPV18-positive adenocarcinomas (OR 2.96, 95%CI 1.18-7.41, p = 0.02). These associations remained borderline significant after testing against different sets of controls. rs9357152 was found to be an eQTL for HLA-DRB1 in HPV-positive cervical tissues (p We find evidence that HPV seropositivity variants at chromosome 6 and 14 may modulate type-specific cervical cancer risk. rs9357152 may exert its effect through regulating HLA-DRB1 induction in the presence of HPV. In regard of multiple testing, these results need to be confirmed in larger studies.

The human Papillomavirus twilight zone – Latency, immune control and subclinical infection

The incorporation of HPV DNA testing into cervical screening programs has shown that many HPV-positive women are cytologically normal, with HPV-positivity fluctuating throughout life. Such results suggest that papillomaviruses may persist in a latent state after disease clearance, with sporadic recurrence. It appears that virus latency represents a narrow slot in a wider spectrum of subclinical and possibly productive infections. Clinical studies, and animal model infection studies, suggested a key role for host immune surveillance in maintaining such asymptomatic infections, and although infections may also be cleared, most studies have used the term 'clearance' to describe a situation where the presence of HPV DNA falls below the clinical detection level. Given our knowledge of papillomavirus immune evasion strategies and the restricted pattern of viral gene expression required for 'basal cell' persistence, the term 'apparent clearance' and 'subclinical persistence' of infection may better summarise our understanding. Subclinical infection also encompasses the lag phase, which occurs between infection and lesion development. This is dependent on infection titre, with multifocal infections developing more rapidly to disease. These concepts can usefully influence patient management where HPV-positivity occurs sometime after the onset of sexual activity, and where vertical transmission is suspected despite a lag period.

Characterization of HPV subtypes in invasive cervical cancer in Botswana patients using a pan-pathogen microarray technology

Human papillomavirus (HPV) plays a significant role in the development of cervical cancers in the setting of co-infection with HIV. Botswana has a high prevalence of HIV and cervical cancer. In this study, we investigated the distribution of HPV subtypes in cervical cancer biopsy samples from patients in Botswana using a highly sensitive pan-pathogen microarray technology, PathoChip, to detect both high- (HR-HPV) and low-risk HPV (LR-HPV) subtypes in women living with HIV (WLWH) and women living without HIV. We analyzed samples from 168 patients, of which 73% (n = 123) were WLWH with a median CD4 count of 479.5 cells/μL. Five HR-HPV subtypes were detected in the cohort: HPV 16, 18, 26, 34, and 53. The most prevalent subtypes were HPV 26 (96%) and HPV 34 (92%); 86% of WLWH (n = 106) had co-infection with four or more HR-HPV subtypes compared to 67% (n = 30) of women without HIV (p 200 cells/μL and HIV-negative patients. Although the majority of cervical cancer specimens in this cohort were determined to have multiple HPV infections, the most prevalent HR-HPV subtypes (HPV 26 and HPV34) found in these cervical cancer samples are not covered in the current HPV vaccines. Though no conclusions can be made on the direct carcinogenicity of these subtypes the results do underlie the need for continued screening for prevention of cervical cancer.

Opportunities to advance cervical cancer prevention and care

Cervical cancer (CaCx) is a major public health issue, with over 600,000 women diagnosed annually. CaCx kills someone every 90 s, mostly in low- and middle-income countries. There are effective yet imperfect mechanisms to prevent CaCx. Since human papillomavirus (HPV) infections cause most CaCx, they can be prevented by vaccination. Screening methodologies can identify premalignant lesions and allow interventions before a CaCx develops. However, these tools are less feasible in resource-poor environments. Additionally, current screening modalities cannot triage lesions based on their relative risk of progression, which results in overtreatment. CaCx care relies heavily on genotoxic agents that cause severe side effects. This review discusses ways that recent technological advancements could be leveraged to improve CaCx care and prevention.

DNA methylation at individual CpG-sites of EPB41L3, HTERT and FAM19A4 are useful for detection of cervical high-grade squamous intraepithelial lesions (HSIL) or worse: Analysis of individual CpG-sites outperforms averaging

Global methylation analysis of gene promoters is promising for detection of high-grade squamous intraepithelial lesions or worse (HSIL+) in high-risk human papillomavirus (hrHPV)-positive women. However, diagnostic performance of methylation data at individual CpG-sites is limited. We explored methylation for predicting HSIL+ in self- and clinician-collected samples from Papua New Guinea. Methylation of EPB41L3 (1-6 CpG-sites), hTERT (1-10 CpG-sites) and FAM19A4 (1-5 CpG-sites) was assessed through pyrosequencing from 44 HPV+ samples (4 cancers, 19 HSIL, 4 low-grade squamous intraepithelial lesions (LSIL), 17 normal). New primers were designed for FAM19A4 directed to the first exon region not explored previously. In clinician-collected samples, methylation at CpG-sites 4 and 5 of EPB41L3 were the best HSIL predictors (AUC >0.83) and CpG-site 4 for cancer (0.925). Combination of EPB41L3 sites 2/4 plus FAM19A4 site 1 were the best HSIL+ markers [100% sensitivity, 63.2% specificity]. Methylation at CpG-site 5 of FAM19A4 was the best HSIL predictor (0.67) in self-collected samples, and CpG-sites 1 and 3 of FAM19A4 for cancer (0.77). Combined, FAM19A4 site 1 plus HPV 16/18 detection yielded sensitivity of 82.6% and specificity of 61.9%. In conclusion, methylation at individual CpG-sites of EPB41L3 and FAM19A4 outperformed global analysis and improved HSIL+ detection, warranting further investigation.

Detection of high-risk HPV in FFPE specimens of various tumours using the BD Onclarity™ HPV Assay

Analysis of high-risk HPV status on formalin-fixed paraffin-embedded (FFPE) tissue material is valuable for cervical-, head and neck-, anogenital- and other types of cancer, but commercial HPV assays have been developed specifically for cervix swab cells. We evaluated the BD Onclarity™ HPV Assay for the detection of high-risk HPV on an assortment of relevant FFPE tissues with known HPV status. Detection of high-risk HPV types using the BD Onclarity™ HPV Assay in FFPE specimens was easy and accurate.

Evaluating the performance of the Xpert HPV assay in detecting HPV positive cases in Morocco

Recently, the World Health Organization recommended integrating HPV testing into cervical cancer screening programs globally. This study aimed to compare the GeneXpert assay with PCR-sequencing for HPV detection and genotyping to assess the feasibility of incorporating HPV molecular testing into cervical cancer screening. A total of 1000 women aged 30 or 40 from rural and urban areas across four regions in Morocco with high sexually transmitted infection prevalence were recruited. After excluding 21 invalid tests, DNA testing on the remaining 979 samples showed an HPV prevalence of 4.0 % (39/979) by PCR and 5.0 % (49/979) by Xpert, with an overall prevalence of 5.7 % (56/979) when combining both techniques. The concordance rate between the tests was 97.5 %. Notably, the Xpert HPV assay was highly efficient in detecting HPV, with nearly all identified HPVs being high-risk oncogenic types, predominantly HPV16, 18, 31, 35, and 45. The Xpert HPV assay has demonstrated excellent analytical performance, making it a reliable option for HPV detection in vaginal and cervical swabs. Its integration into primary cervical cancer screening programs could significantly enhance the early detection of HPV-positive cases, thereby strengthening the screening framework and potentially reducing both the incidence and mortality of cervical cancer. Future studies should focus on confirming these results and exploring the utility of this method in conjunction with other diagnostic tools such as visual inspection with acetic acid (VIA) for a comprehensive assessment of its effectiveness in real-world settings.

Cervical cytology and HPV distribution in Cape Verde: A snapshot of a country taken during its first HPV nation-wide vaccination campaign

Cervical cancer ranks as the third most common female cancer in Cape Verde and is the leading cause of cancer-related deaths among women in the country. While Human Papillomavirus (HPV) vaccination, which started in 2021, is anticipated to significantly reduce disease incidence, cervical screening remains crucial for non-vaccinated women. We retrospectively reviewed gynecologic cytology exams and HPV tests performed in Cape Verde between 2017 and April 2023 and processed at IMP Diagnostics. For this study, we considered 13035 women with cytology examinations performed and, 2013 of these, also with an HPV molecular test. Cytology diagnostics comprised 83 % NILM cases; 12 % ASC-US; 2.7 % LSIL; 1.2 % ASC-H; 0.5 % HSIL and 0.1 % SCC. In 505 (25.1 %) high-risk HPV infection was detected. Prevalence of HPV infection varied with age, peaking at young ages - ≤24 years old (55.5 %) and 25-35-year-old women (31.5 %) - and the lowest after 66 years old (9.7 %). Herein we present a comprehensive study regarding Cape Verde's cervical cytology and HPV distribution, aiming to provide a snapshot of the country's cervical cytology results and HPV distribution in recent years. Moreover, these data may contribute to establish a baseline to assess, in the future, the vaccination impact in the country.

Differences in integration frequencies and APOBEC3 profiles of five high-risk HPV types adheres to phylogeny

Persistent infection with Human Papillomavirus (HPV) is responsible for almost all cases of cervical cancers, and HPV16 and HPV18 associated with the majority of these. These types differ in the proportion of viral minor nucleotide variants (MNVs) caused by APOBEC3 mutagenesis as well as integration frequencies. Whether these traits extend to other types remains uncertain. This study aimed to investigate and compare genomic variability and chromosomal integration in the two phylogenetically distinct Alpha-7 and Alpha-9 clades of carcinogenic HPV types. The TaME-seq protocol was employed to sequence cervical cell samples positive for HPV31, HPV33 or HPV45 and combine these with data from a previous study on HPV16 and HPV18. APOBEC3 mutation signatures were found in Alpha-9 (HPV16/31/33) but not in Alpha-7 (HPV18/45). HPV45 had significantly more MNVs compared to the other types. Alpha-7 had higher integration frequency compared to Alpha-9. An increase in integration frequency with increased diagnostic severity was found for Alpha-7. The results highlight important differences and broaden our understanding of the molecular mechanisms behind cervical cancer induced by high-risk HPV types from the Alpha-7 and Alpha-9 clades.

Women with a positive high-risk human papillomavirus (HPV) test remain at increased risk of HPV infection and cervical precancer ≥15 years later

Little is known about the long-term association between high-risk human papillomavirus (hrHPV) test results in women participating in a hrHPV-based cervical cancer screening program. To address this question, we collected data of 2217 women who participated in the POBASCAM hrHPV-based screening trial (enrolment 1999/2002) and also attended the Dutch hrHPV-based screening program between January 2017 and March 2018. Among 143 women who tested hrHPV-positive in 1999/2002, 45 (31.5%) had ≥ CIN2 or hysterectomy before 2017 and 17 (11.9%) tested hrHPV-positive at the 2017/2018 screen. In comparison, among 2074 women who tested hrHPV-negative in 1999/2002, 10 (0.5%) had ≥ CIN2 or hysterectomy before 2017 and 119 (5.7%) tested hrHPV-positive at the 2017/2018 screen. It follows that in the group of women who were not treated for ≥ CIN2 or had a hysterectomy in between the two screens 15 years apart (N = 2162), women who were hrHPV-positive in 1999/2002 had a higher risk of being hrHPV-positive in 2017/2018 than those who were hrHPV-negative in 1999/2002 (OR 3.4, 95% CI 1.8-6.1). A similar association was found at the genotype level for genotype-concordant results (5.1, 1.0-11.3) and for genotype non-concordant results (3.7, 1.6-6.7). Women who were hrHPV-positive in 2017/2018 had a higher risk of CIN3 after a hrHPV-positive result in 1999/2002 than after a hrHPV-negative result (5.8, 1.0-27.8). In conclusion, a positive hrHPV result in screening gives a long-term increased risk of a hrHPV-positive result, also for different genotypes, and a long-term increased risk of CIN3. This supports the concept of risk-stratification in hrHPV-based cervical cancer screening where previous hrHPV results are included in screening recommendations.

Cervical cancer therapies: Current challenges and future perspectives

Cervical cancer is the fourth most common female cancer worldwide and results in over 300 000 deaths globally. The causative agent of cervical cancer is persistent infection with high-risk subtypes of the human papillomavirus and the E5, E6 and E7 viral oncoproteins cooperate with host factors to induce and maintain the malignant phenotype. Cervical cancer is a largely preventable disease and early-stage detection is associated with significantly improved survival rates. Indeed, in high-income countries with established vaccination and screening programs it is a rare disease. However, the disease is a killer for women in low- and middle-income countries who, due to limited resources, often present with advanced and untreatable disease. Treatment options include surgical interventions, chemotherapy and/or radiotherapy either alone or in combination. This review describes the initiation and progression of cervical cancer and discusses in depth the advantages and challenges faced by current cervical cancer therapies, followed by a discussion of promising and efficacious new therapies to treat cervical cancer including immunotherapies, targeted therapies, combination therapies, and genetic treatment approaches.