HPV16 Variants Research Articles

Comparative Analysis of HPV16 Variants in the Untranslated Regulatory Region, L1, and E6 Genes among Vaccinated and Unvaccinated Young Women: Assessing Vaccine Efficacy and Viral Diversity.

HPV16 is occasionally detected in vaccinated women who received the bivalent HPV16/18 vaccine, usually at low viral loads. This study explored potential differences in HPV16 variants between vaccinated and unvaccinated women. HPV16-postive viral loads were detected in 1.9% (17/875) and 13% (162/760) of vaccinated and unvaccinated women, respectively, showcasing the vaccine's high efficacy. The L1, E6, and URR regions of HPV16 were sequenced from genital swabs from 16 vaccinated and 25 unvaccinated women in the HAVANA (HPV Among Vaccinated And Non-vaccinated Adolescents) study. The majority of HPV16 variants from vaccinated and unvaccinated women clustered similarly with sub-lineages A1 and A2. Additionally, a separate cluster within lineage A was found, with the variants sharing the L1-located SNP A753G (synonymous) and the URR-located SNP T340C, which did not occur in the other variants. Furthermore, four variants from vaccinated women had relatively long branches, but were not characterized by specific SNPs. The frequency of G712A in the URR was the only SNP observed to be marginally higher among vaccinated women than unvaccinated women. Non-synonymous SNPs T266A in the FG-loop of L1 and L83V in E6 were common among variants from vaccinated and unvaccinated women, but present in similar frequencies. In conclusion, the detection of HPV16 in vaccinated (and unvaccinated) women seemed to be the result of random circulation within this study population.

HPV16 Genomes: In Silico Analysis of E6 and E7 Oncoproteins in 20 South American Variants.

Human papillomavirus (HPV) is the main risk factor for the development of squamous cell cervical cancer, and E6 oncoprotein and E7 oncoprotein are important components of the viral genome and its oncogenic potential. It is known that different viral variants of HPV16 have different pathology and impact on the development of neoplasia, although few studies have been performed on South American variants. Therefore, the present study aimed to analyze in silico the genomic diversity of HPV16 in 20 complete genome variants of South America in the National Center for Biotechnology Information (NCBI) database. We performed a descriptive study to characterize the polymorphic regions of the E6 and E7 genes in HPV16 variants, using software for genomic data and single nucleotide polymorphism (SNP) analysis and others for phylogenetic analysis. The variants analyzed included six SNPs linked to cancer (A131G, G145T, C335T, T350G, C712A, and T732C) and significant variation (798 nucleotide substitutions). Despite this, the variants showed low genetic diversity. Eighteen variants of unclear significance (VUS) were identified, 10 of which were in the coding E6 regions and 8 in the coding E7 regions. The prevalence of lineage D variants is of concern due to their pathology in cervical cancer and requires more research and epidemiological vigilance regarding their prevalence in the population. The data obtained in this study may contribute to future research on South American variants of HPV16, their pathogenicity, and the development of treatments.

Lineage and sublineage analysis of human papillomavirus types 51 and 59 in Iranian women.

The current study aimed to investigate the sequence variations of HPV 51 and 59 in normal cervical cells and premalignant/malignant lesions of the cervix to know the common variants of HPV 51 and HPV 59 circulating in Iran. To do this, eighty-five samples that were infected by HPV 51 or HPV 59 were investigated using hemi-PCR to amplify the E6 gene followed by sequencing. Our findings indicated that lineages A and B were detected in 80.4% and 19.6% of HPV 51-positive cases, respectively. Among samples infected with HPV 59, 32.2% belonged to lineage A and 67.8% were classified with lineage B. In conclusion, our results showed that lineage A of HPV 51 and lineage B of HPV 59 are more prevalent and distributed in Iran.

HPV16 Phylogenetic Variants in Anogenital and Head and Neck Cancers: State of the Art and Perspectives.

HPV16 is responsible for approximately 60% and 90% of global HPV-induced cervical and oropharyngeal cancers, respectively. HPV16 intratype variants have been identified by HPV genome sequencing and classified into four phylogenetic lineages (A-D). Our understanding of HPV16 variants mostly derives from epidemiological studies on cervical cancer (CC) in which HPV16 B, C, and D lineages (previously named "non-European" variants) were mainly associated with high-grade cervical lesions and cancer. Although a predominance of HPV16 lineage A (previously named "European variants") has been observed in head and neck squamous cell carcinoma (HNSCC), epidemiological and in vitro biological studies are still limited for this tumor site. Next Generation Sequencing (NGS) of the entire HPV genome has deepened our knowledge of the prevalence and distribution of HPV variants in CC and HNSCC. Research on cervical cancer has shown that certain HPV16 sublineages, such as D2, D3, A3, and A4, are associated with an increased risk of cervical cancer, and sublineages A4, D2, and D3 are linked to a higher risk of developing adenocarcinomas. Additionally, lineage C and sublineages D2 or D3 of HPV16 show an elevated risk of developing premalignant cervical lesions. However, it is still crucial to conduct large-scale studies on HPV16 variants in different HPV-related tumor sites to deeply evaluate their association with disease development and outcomes. This review discusses the current knowledge and updates on HPV16 phylogenetic variants distribution in HPV-driven anogenital and head and neck cancers.

Distribution of human papillomavirus among Vietnamese women with cervical cancer and unusual genetic variability of HPV16

HPV16, with typical mutations that differ in geographical distribution and carcinogenic potency, has implications for cervical cancer screening, clinical diagnosis, and treatment. DNASTAR and MEGA were used to identify HPV16 variants and construct a phylogenetic tree. The most prevalent HPV genotypes were HPV16 (63.9%), HPV18 (26.7%), and other HPV (6.9%). HPV16 alterations were found in all E6, E7, and L1 genes, including 15 missense and 18 synonymous mutations. Missense mutations include R10G, Q14H, D25E, H78Y, L83V (E6); M29V, R35K, L78R, L95P (E7); H73Y, T176 N, N178T, T317P, T386S, L472F/I (L1). HPV16 sublineages include A1 (17.2%), A2 (0.9%), A3 (56.0%), A4 (19.0%), D1 (4.3%), and D3 (2.6%). Although several mutations in the oncoproteins E6, E7, and L1 have been detected, mutations known to be associated with cervical cancer risk, such as D25E and L83V, occur at a relatively low frequency. This suggests that HPV16 mutations are associated with cervical cancer through a complicated mechanism.

HPV16 Intratypic Variants in Head and Neck Cancers: A North American Perspective.

Human papillomavirus (HPV) is the major causative agent for cervical and many head and neck cancers (HNCs). HPVs randomly acquire single nucleotide polymorphisms (SNPs) that may become established via positive selection. Within an HPV type, viral isolates differing by <2% in the L1 region are termed "variants" and classified based on combinations of SNPs. Studies in cervical cancer demonstrate clear differences between HPV16 intratypic variants in terms of persistence of infection, tumor histology, cancer risk, and death. Much less is known about the frequency of HPV16 variants in HNC, and their effects on clinical outcomes. We combined HPV16 positive (HPV16+) HNC samples from a local Southwestern Ontario, Canada cohort with those from the Cancer Genome Atlas to create a larger North American cohort of 149 cases with clinical data and determined the distribution of intratypic variants and their impact on clinical outcomes. Most isolates were lineage A, sublineage A1, or A2, with roughly half exhibiting the T350G polymorphism in E6. Univariable analysis identified significant differences between 350T and 350G intratypic variants in clinical T, N, and O staging, as well as disease-free survival. Multivariable analysis failed to identify any clinical factor as a statistically significant covariate for disease-free survival differences between 350T and 350G. Significant differences in several measures of B-cell mediated immune response were also observed between 350T and 350G intratypic variants. We suggest that HPV genetic variation may be associated with HNC clinical characteristics and may have prognostic value.

Decoding the Genetic and Structural Features of HPV16 E5 Oncogene in Cervical Cancer Isolates from Pakistan: A Pilot Study.

Many anogenital cancers are caused by high-risk HPV. The most common subtype is HPV16, which is prevalent in the world, including Pakistan. Various amino acid residues in HPV16 E5 are associated with high cell cycle progression and proliferation. Lack of studies on HPV16E5 in Pakistan prompted the current study. This is the first report on the occurrence of pathogenic E5 variant of HPV16 in tissue sections obtained from invasive cervical cancerous patients in Pakistan. A subset of 11 samples from HPV-positive biopsies were subjected to E5 gene amplification using PCR and analyzed using bioinformatics programs. The bioinformatics analysis detected mutations causing structural variations, which potentially contribute to the oncogenic properties of proteins. The two-point mutations, C3979A and G4042A, observed in isolate 11 caused the substitution of isoleucine for leucine and valine at positions 44 and 65 in E5 protein. The rest of the isolates had Leu44Val65 amino acids. Intratypic variations and phylogenetic analysis revealed that the majority of the isolates were closely clustered with European-Asian lineage. Moreover, C3979A and G4042A contributed to higher degree of interactions with host receptors, i.e. EGFR. This is the first study reporting HPV16 variants in a Pakistani population based on variations in the E5 region. Our findings indicate that isolate 11 has a strong interaction with the intracellular domain of EGFR, which may enhance the generation of downstream signals. Since this was a pilot study to explore E5 gene mutation, future studies with large samples are absolutely needed.

E7 Oncogene HPV58 Variants Detected in Northeast Brazil: Genetic and Functional Analysis.

Cervical cancer is associated with persistent infections by high-risk Human Papillomavirus (HPV) types that may have nucleotide polymorphisms and, consequently, different oncogenic potentials. Therefore, this study aimed to evaluate the genetic variability and structural effects of the E7 oncogene of HPV58 in cervical scraping samples from Brazilian women. The study was developed with patients from hospitals in the metropolitan area of Recife, PE, Brazil. The most frequent HPV types were, in descending order of abundance, HPV16, 31, and 58. Phylogenetic analysis demonstrated that the isolates were classified into sublineages A2, C1, and D2. Two positively selected mutations were found in E7: 63G and 64T. The mutations G41R, G63D, and T64A in the E7 protein reduced the stability of the protein structure. Utilizing an NF-kB reporter assay, we observed a decrease in the NK-kB pathway activity with the HPV58-E7 variant 54S compared to the WT E7. The other detected E7 HPV58 variants presented similar NF-kB pathway activity compared to the WT E7. In this study, it was possible to identify mutations that may interfere with the molecular interaction between the viral oncoproteins and host proteins.

Impact of HPV-16 Lineages Infection in Response to Radio-Chemotherapy in Cervical Cancer.

HPV is strongly related to cervical cancer. HPV lineages can contribute to a response to cervical cancer therapy. The aim of this research was to estimate the frequency of human papillomavirus (HPV)-16 lineages in specimens of cervical cancer, relate the pathological factors in these variants, and assess their response to treatment with radical chemoradiotherapy. Samples of cervical cancer were collected from women who were referred to a reference cancer hospital to test the presence of human papillomavirus-type DNA. The standard protocol of this service consisted of cisplatin-based chemotherapy of 40 mg/m2, plus conventional pelvic irradiation in doses of 45-50.4 Gy and high dose-rate brachytherapy of 28-30 Gy to Point A. The response to chemotherapy was evaluated after three months in patients with the HPV-16 lineage. HPV DNA was detected in 104 (88.1%) of the 118 patients. HPV-16 was present in 63 patients (53%). Lineages of HPV-16 were identified in 57 patients and comprised 33 instances of (57.8%) lineage A, 2 instances of lineage B (3.5%), 2 instances of lineage C (3.5%), and 20 instances of (35.0%) lineage D. The median age of the patients was 48.4 years (range 25-85 years). Squamous cell carcinoma was detected 48 times (84.2%). Adenocarcinoma was more likely to occur in lineage D, as three of the four cases occurred in this lineage. A total of 11 patients with the HPV-16 variant were treated with chemoradiotherapy. After three months, it was observed that nine of the eleven patients (81.8%) achieved a complete response, five with the lineage A type, two with the lineage C type, and two with the lineage D type. The two cases of partial response and disease progression, one of each, occurred in lineage A. In addition to the small number of patients and HPV variants, we noticed a better response in patients with the HPV-16 lineage A. Increasing the sample size could be helpful to better assess the impact of HPV variants on cervical cancer treatment.

Human Papillomavirus 16 Lineage A Variants Associated With Persistent Genital Infections in Men: The HPV Infection in Men (HIM) Study.

Human papillomavirus (HPV) 16 non-A lineage variants have higher carcinogenic potential for cervical cancer. HPV-16 variants natural history among males is not established. We evaluated HPV-16 variants prevalence and persistence in the external genitalia of men enrolled in the prospective HPV Infection in Men (HIM) Study. The HIM Study included men from the United States, Brazil, and Mexico. HPV-16 variants were distinguished using polymerase chain reaction sequencing. The prevalence of HPV-16 variants was assessed, and associations with infection persistence were estimated. We characterized the HPV-16 variants for 1700 genital swab samples from 753 men and 22 external genital lesions in 17 men. The prevalence of HPV-16 lineages differed by country and marital status (P < .001). Overall, 90.9% of participants harbored lineage A variants. The prevalence of non-A lineages was heterogenous among countries. HPV-16 lineage A variants were associated with a 2.69-fold increased risk of long-term persistent infections compared with non-A lineages. All high-grade penile intraepithelial neoplasia harbored lineage A variants and occurred in the context of long-term persistent infections with the same variants. The prevalence and persistence of HPV-16 variants observed at the male external genitalia suggest differences in the natural history of these variants between men and women, which may be associated with intrinsic differences in the infected genital epithelia.

Association of HPV genotype and phylogenic clade with oropharyngeal cancer outcomes.

6058 Background: Human papillomavirus (HPV) is a causative agent of oropharyngeal squamous cell carcinoma (HPVOPC). There are multiple different HPV genotypes based on variations in the nucleotide sequence of the L1 capsid, with HPV16 being most common in HPVOPC (85%). HPV genotypes can be further classified into phylogenic clades, including alpha-9 (A9) and alpha-7 (A7). HPVOPC is associated with a more favorable prognosis than HPV-negative disease, but the impact of specific HPV genotype and phylogenic clade on patient outcomes is not well understood and has profound implications for de-escalation studies in HPVOPC. Methods: A retrospective cohort study was conducted at a large multicenter health system. Patients diagnosed with non-metastatic HPVOPC or unknown primary from 2012-2021 and treated with curative intent were included. Low-risk HPV types (6, 11, etc) and local recurrences were excluded. HPV molecular typing was prospectively performed according to institutional protocol. Cox regressions were used to measure the effect of HPV genotype (16 versus non-16) on overall survival (OS) and event-free survival (EFS). Gender, smoking status, alcohol use, ECOG performance status, T stage, N stage, and subsite (oropharynx versus known primary) were covariates. A secondary survival analysis according to high-risk HPV clade (A9 versus A7) was also performed. Results: The total cohort included 520 patients, the majority of whom were HPV16 (86.9%, n = 452), A9 clade (97.1%, n = 505), oropharynx primary (94.8%, n = 493), male (86.9%, n = 452), white (72.5%, n = 377), non-Hispanic (74.2%, n = 386), non-active smokers (88.3%, n = 459), ECOG 0 (75.6%, n = 393), and N2 stage (65.2%, n = 339). The most common non-16 HPV genotypes were 18 (n = 7), 33 (n = 21), and 35 (n = 29). HPV genotype was not associated with OS (HR = 1.64, 95% CI 0.85-3.16, p= 0.14) or EFS (HR = 1.15, 95% CI 0.64-2.07, p= 0.65). A7 clade was associated with a worse OS compared to A9 in the univariate analysis (HR = 3.43, 95% CI 1.07-10.97, p= 0.04), but not in the multivariate analysis (HR = 1.67, 95% CI 0.45-6.17, p = 0.44). Finally, HPV clade was not associated with EFS (HR = 1.38, 95% CI 0.34-5.60, p= 0.66). Conclusions: While HPV genotype was not associated with survival differences in patients with HPVOPC, HPV A7 clade only predicted worse OS in univariate analysis. Although the total cohort size was large, this analysis was limited by small numbers of subjects with non-16 HPV genotypes and non-A9 clade, which is reflective of the predominance of HPV16 in HPVOPC. Thus, a pooled multicenter collaboration is needed to further address this question. Moreover, future studies should investigate whether outcomes may be related to intratypic HPV16 variants that affect oncoprotein function and/or immunogenicity. Detection of a subgroup of HPVOPC with possible inferior outcomes has important implications for studying treatment de-escalation in HPVOPC.

Diversity of cervicovaginal human papillomavirus (HPV) genotypes and naturally occurring E6/E7 DNA polymorphisms of HPV-16 in Ghana

Human papillomavirus (HPV) E6 and E7 oncogene expression is essential for cervical carcinogenesis. Evidence exists that E6/E7 variants may have different transforming activities while the risk of HPV-16 variants (A/D) differs by race/ethnicity. We determined the type-specific diversity of HPV infection in women with high grade cervical disease or cervical cancer in Ghana and investigated naturally occurring E6/E7 DNA variants in this population. HPV genotyping was carried out on 207 cervical swab samples collected from women referred to a gynaecology clinic at two teaching hospitals in Ghana. HPV-16, HPV-18 and HPV-45 were detected in 41.9%, 23.3% and 16.3% of cases respectively. HPV-16 E6/E7 DNA sequencing was performed in 36 samples. Thirty samples contained E6/E7 variants of the HPV-16-B/C lineage. 21/36 samples were of the HPV-16C1 sublineage variant and all contained the E7 A647G(N29S) single nucleotide polymorphism (SNP). This study reveals the diversity of E6/E7 DNA and the dominance of HPV16 B/C variants in cervicovaginal HPV infection in Ghana. Type-specific HPV diversity analysis indicates that most Ghanaian cervical disease cases are vaccine preventable. The study provides an important baseline from which for the impact of vaccine and antivirals on clinically relevant HPV infection and associated disease can be measured.

Pre-Vaccination Human Papillomavirus Genotypes and HPV16 Variants among Women Aged 25 Years or Less with Cervical Cancer.

In 2007, Australia introduced a national human papillomavirus (HPV) vaccination program. In 2017, the onset of cervical screening changed from 18 to 25 years of age, utilising human papillomavirus (HPV) nucleic acid testing. The objective of the study is to describe the HPV genotypes and HPV16 variants in biopsies from women ≤ 25 years of age with cervical carcinoma (CC) (cases), compared with those aged >25 years (controls), in a pre-vaccination cohort. HPV genotyping of archival paraffin blocks (n = 96) was performed using the INNO-LiPA HPV Genotyping assay. HPV16-positive samples were analysed for variants by type-specific PCR spanning L1, E2 and E6 regions. HPV16 was the commonest genotype in cases (54.5%, 12/22) and controls (66.7%, 46/69) (p = 0.30), followed by HPV18 (36.3%, 8/22 vs. 17.3% 12/69, respectively) (p = 0.08). Furthermore, 90% (20/22) of cases and 84.1% (58/69) of controls were positive for HPV16 or 18 (p = 0.42); 100% (22/22) of cases and 95.7% (66/69) of controls had at least one genotype targeted by the nonavalent vaccine (p = 0.3). The majority of HPV16 variants (87.3%, 48/55) were of European lineage. The proportion of unique nucleotide substitutions was significantly higher in cases (83.3%, 10/12) compared with controls (34.1%, 15/44), (p < 0.003, χ2, OR 9.7, 95%CI 1.7-97.7). Virological factors may account for the differences in CCs observed in younger compared with older women. All CCs in young women in this study had preventable 9vHPV types, which is important messaging for health provider adherence to new cervical screening guidelines.

Characterization of Human Papillomavirus 16 from Kinshasa (Democratic Republic of the Congo)-Implications for Pathogenicity and Vaccine Effectiveness.

Human Papillomavirus (HPV) type 16 is the main etiological agent of cervical cancer worldwide. Mutations within the virus genome may lead to an increased risk of cancer development and decreased vaccine response, but there is a lack of information about strains circulating in Sub-Saharan Africa. Endocervical cytology samples were collected from 480 women attending a voluntary cervical cancer screening program at Monkole Hospital and four outpatient centers in Kinshasa, Democratic Republic of the Congo (DRC). The prevalence of HPV infection was 18.8% and the most prevalent high-risk types were HPV16 (12.2%) followed by HPV52 (8.8%) and HPV33/HPV35 (7.8% each). HPV16 strains were characterized: 57.1% were classified as C lineage; two samples (28.6%) as A1 and one sample belonged to B1 lineage. HPV33, HPV35, HPV16, and HPV58 were the most frequent types associated with low-grade intraepithelial lesion while high-grade squamous intraepithelial lesions were predominantly associated with HPV16. Several L1 mutations (T266A, S282P, T353P, and N181T) were common in Kinshasa, and their potential effect on vaccine-induced neutralization, especially the presence of S282P, should be further investigated. Long control region (LCR) variability was high with frequent mutations like G7193T, G7521A, and G145T that could promote malignancy of these HPV16 strains. This study provides a helpful basis for understanding HPV16 variants circulating in Kinshasa and the potential association between mutations of LCR region and malignancy and of L1 and vaccine activity.

Characterization of the Human Papillomavirus 16 Oncogenes in K14HPV16 Mice: Sublineage A1 Drives Multi-Organ Carcinogenesis.

The study of human papillomavirus (HPV)-induced carcinogenesis uses multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene promoter to drive the expression of all HPV early oncogenes. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model. This information can be considered of great importance to further enhance this K14HPV16 model as an essential research tool and optimize its use for basic and translational studies. Our study evaluated HPV DNA from 17 samples isolated from 4 animals, both wild-type (n = 2) and HPV16-transgenic mice (n = 2). Total DNA was extracted from tissues and the detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by next-generation sequencing techniques. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). A comparative genome analysis of K14HPV16 samples revealed three mutations to the human papillomaviruses type 16 sublineage A1 representative strain. Knowledge of the HPV 16 variant is fundamental, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer.

CarcinoHPVPred: An ensemble of machine learning models for HPV carcinogenicity prediction using genomic data.

Human papillomavirus (HPV) infections often show no symptoms but sometimes lead to either warts or carcinoma based on the HPV genotype. The relationship between HPV infections and cervical cancer have been well studied in the past two decades. However, distinguishing carcinogenic HPV variants from non-carcinogenic ones remains a major challenge in clinical genetic testing of HPV-induced cancer samples. All of the published HPV carcinogenicity prediction methods are neither publically available nor tested with two-thirds of available HPV variants. The nucleotide composition-based studies are the simplest and most precise methods of characterizing new genomes. Hence, there is a need for machine learning models which can predict the carcinogenic nature of newly discovered HPV based on their genomic composition. We developed a standalone and web tool, CarcinoHPVPred (h t t p :// test5.bicpu.edu.in/CarcinoHPVPred.php), for predicting the phenotype of HPV with a range of a high accuracy between 94% - 100%. This tool consists of machine learning models build upon genomic features of two genes namely E2 and E6. Overall, the accurate and early prediction of carcinogenic nature of HPV can be performed with this only available tool of its kind till date.

Epidemiology and Molecular Biology of HPV Variants in Cervical Cancer: The State of the Art in Mexico.

Cervical cancer (CC) continues to be a major public health problem in Mexico, ranking second among cancers in women. A persistent infection with human papillomaviruses (HPV) is the main risk factor for CC development. In addition, a significant fraction of other cancers including those of the anus, oropharynx, and penis are also related to HPV infection. In CC, HPV-16 is the most prevalent high-risk HPV type, followed by HPV-18, both being responsible for 70% of cases. HPV intratype variant lineages differ in nucleotide sequences by 1–10%, while sublineages differ by 0.5–1%. Several studies have postulated that the nucleotide changes that occur between HPV intratype variants are reflected in functional differences and in pathogenicity. Moreover, it has been demonstrated that HPV-16 and -18 intratype variants differentially affect molecular processes in infected cells, changing their biological behavior that finally impacts in the clinical outcome of patients. Mexico has participated in providing knowledge on the geographical distribution of intratype variants of the most prevalent HPVs in premalignant lesions of the cervix and cervical cancer, as well as in other HPV-related tumors. In addition, functional studies have been carried out to assess the cellular effects of intratype variations in HPV proteins. This review addresses the state of the art on the epidemiology of HPV-16 and HPV-18 intratype variants in the Mexican population, as well as their association with persistence, precancer and cervical cancer, and functional aspects related to their biological behavior.

The Genomic Landscape of Vulvovaginal Squamous Cell Carcinoma (048)

The Genomic Landscape of Vulvovaginal Squamous Cell Carcinoma (048)

Transcriptomic immune profiling for cervical squamous cell carcinoma: Does HPV type matter? (170)

<b>Objectives:</b> Immunotherapy (IO) has shown initial promise in treating metastatic or recurrent cervical cancer. However, response rates have been modest, and PD-L1 has shown limited value as a predictive marker. Recently-presented data exploring transcriptomic immune profiling (TIP) in cervical cancer suggested that <i>(a)</i> far more immune-relevant signals in cervical cancer can be evaluated and <i>(b)</i> squamous cell carcinoma (SCC) has increased immunogenicity than adenocarcinoma. Given the near-universal HPV+ status of cervical SCC, we sought to explore whether TIP signals vary with respect to HPV variants. <b>Methods:</b> Cervical SCC tumors were analyzed using next-generation sequencing and whole-exome sequencing (NGS: NextSeq, 45 or 592 Genes and NovaSEQ, WES), immunohistochemistry (IHC), and WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was tested by IHC (positive: CPS ≥1). Microsatellite instability (MSI) was tested by FA, IHC, and NGS. Tumor mutational burden (TMB) was measured by totaling all somatic mutations per tumor (TMB-h: ≥10 mutations/MB). Immune cell infiltration was calculated by QuantiSeq. HPV16/18 status was determined using WES. Significance was tested by Chi-square and Wilcoxon rank-sum test and adjusted for multiple comparisons (q<0.05). <b>Results:</b> Total 89 cervical SCC tumors with available HPV16/18 status were included: 63 (71%) were HPV16+ or 18+ while 26 (29%) were non-HPV16/18. HPV16/18+ had significantly fewer <i>TP53</i> alterations compared to non-HPV16/18 tumors (1.7% vs 33%, q=0.01). There was a trend toward increased regulatory T cell infiltration in HPV16/18+ tumors (2.2% vs 1.9%) (p=0.02; q=0.2) accompanied with a trend towards increased immune checkpoint (IC) gene expression: CD86 (1.4-fold, p=0.05/q=0.5), <i>IFNG</i> (2.2-fold, p=0.03; q=0.3) and <i>IDO1</i> (2.5-fold, p=0.03; q=0.3). Additionally, there was a trend towards increased T cell inflammation (32% vs 19%, p=0.16) in HPV16/18+ tumors (Table 1). Among prevalent mutations/pathways in this dataset, there were no significant differences in PI3K (42% vs 42%), Telomerase Maintenance (12% vs 15%) or RTK RAS (22% vs 27%) pathway alterations (q=1). There were no significant differences in IO-related biomarkers: PD-L1 (92% vs 82%), dMMR/MSI-H (4.8% vs 3.7%) or TMB (25% vs 19%) (q=1). No significant genomic, transcriptomic or immune differences were seen between HPV16+ and HPV18+ tumors. <b>Conclusions:</b> Individual TIP markers were not significantly different with respect to high-risk HPV types for cervical SCC. Besides <i>TP53</i> alterations, major cancer-relevant mutations/pathways were not significantly different for the HPV16/18+ versus non-HPV16/18 groups. The HPV16/18+ group had trends of increased regulatory T cell infiltration and IC gene expression. These data are hypothesis-generating for the concept that HPV16/18+ cervical SCC tumors might have differentially modulated immune activity compared to non-HPV16/18 cervical SCC tumors.

The E6 Oncoprotein of HPV16 AA-c Variant Regulates Cell Migration through the MINCR/miR-28-5p/RAP1B Axis.

The E6 oncoprotein of HPV16 variants differentially alters the transcription of the genes involved in migration and non-coding RNAs such as lncRNAs. The role of the lncRNA MINCR in cervical cancer and its relationship with variants of oncogenic HPV remain unknown. Therefore, the objective of this study was to analyze the effect of the E6 oncoprotein of the AA-c variant of HPV16 in cell migration through the MINCR/miR-28-5p/RAP1B axis. To explore the functional role of MINCR in CC, we used an in vitro model of C33-A cells with exogenous expression of the E6 oncoprotein of the AA-c variant of HPV16. Interfering RNAs performed MINCR silencing, and the expression of miR-28-5p and RAP1B mRNA was analyzed by RT-qPCR. We found that C33-A/AA-c cells expressed MINCR 8-fold higher compared to the control cells. There is an inverse correlation between the expression of miR-28-5p and RAP1B in C33-A/AA-c cells. Our results suggest that MINCR might regulate the expression of RAP1B through the inhibition of miR-28-5p in CC cells expressing the E6 oncoprotein of HPV16 AA-c. We report, for the first time, that the MINCR/miR-28-5p/RAP1B axis positively regulates cell migration in CC-derived cells that express the E6 oncoprotein of the AA-c variant of HPV16.