HPV 18 Variants in Women with Cervical Cancer in Northeast Brasil

Background: Human Papillomavirus (HPV) is the main etiological factor for the development of cervical cancer. HPV 18 is the second most frequent, accounting for up to 65% of all cases. HPV intratypic variation may inuence the potential for progression to invasive cancer.The aim of this study was to evaluate the prevalence of human papillomavirus 18 intratypic variants in cervical cancer samples from women in the Northeast of Maranhão. Methods: The study was composed of 118 women over 18 years of age with a diagnosis of cervical cancer. Tumor fragments were collected and subjected to DNA extraction and Polymerase Chain Reaction (PCR) for HPV detection using the PGMY09/11 and GP+5/6 primers. Positive samples were submitted to automated sequencing for viral genotyping. To determine the HPV 18 lineages, positive samples were submitted to PCR, using specic primers to amplify the LCR and E6 regions of HPV 18 virus. Results: HPV was present in 88 women (73.3%), and HPV 16 was the most prevalent (48/54%), followed by HPV 18 (12/13.6%). Histologically, squamous cell carcinoma was predominant (79.1%). Among the HPV 18 variants identied, 10 (80%) belonged to lineage A, and sublineages A1, A2, A3, and A4. HPV 18 B variant (2/29%) was also detected, with the sublineages B1 and B2. In this study, the C variant was not found. There was no statistically signicant association between the HPV 18 lineages found and sociodemographic and lifestyle variables (p > 0.05). Conclusions: Women with cervical cancer are associated with a higher frequency of HPV 16 and 18 in the Northeast region of Brazil, with a high of the lineage A among women with HPV 18. no and


Background
Cervical cancer is the fourth most common type of cancer among women in the world (1). Each year, 527,624 women are diagnosed with cervical cancer and 265,672 die from this disease (1). According to INCA estimates, 16,370 new cases of cervical cancer were estimated in Brazil for each year of the biennium 2018-2019, with an approximate risk of 15.43 cases per 100,000 women (2). Most cases of cervical cancer occur in Africa, Latin America and Asia, where for every 9 cases of cancer, at least 1 is cervical cancer (3). In developed countries such as those in North America and Europe, the incidence of cervical cancer is less than 9 per 100,000 inhabitants (4). In developing countries, less than 50% of women affected by cervical cancer survive for more than 5 years compared to 66% in developed countries (5). This type of malignant neoplasm presents a variety of histological types, with squamous cell carcinoma accounting for up to 95% of cases, followed by adenocarcinoma and adenosquamous carcinoma (6).
Human papillomavirus (HPV) infection is the main etiologic factor for cervical cancer, with more than 200 types already identi ed and classi ed according to their oncogenic potential (7). Among subtypes, HPV 18 is the second most frequent, accounting for up to 65% of all cases of invasive cervical cancer worldwide (8).
Studies have reported that HPV 18 is closely associated with the development of glandular lesions and adenocarcinoma (ADC) compared to squamous cell carcinoma (SCC) (8). This nding indicates that HPV 18 shares phylogenetic traits that favor the development of adenocarcinoma compared to other HPV genotypes (8).
HPV intratypic variants show different distribution and speci city in various parts of the globe. These variants are characterized based on the isolation of the same viral type and have a difference of less than 10% in the nucleotide sequence of the L1 region of the virus (9). These differences promote changes in pathogenicity and oncogenic potential between variants of the same viral type (10). Studies suggest that different HPV 18 variants co-evolved with the three major human phylogenetic branches, the African, Caucasian and Asian. In view of this, the variants were classi ed into three distinct lineages, the African (AF), the European (E), and Asian-Amerindian (As/Ai) (8,10). Currently, a new nomenclature classi es HPV 18 variants in three lineages: A (including the lineages previously classi ed as As/Ai and E), B and C (previously classi ed as lineage Af) (8).
Genetic variations in the same viral type may in uence the potential for infection, viral persistence, development of precursor lesions, and progression to invasive cancer (9,12). Compared to studies conducted with HPV 16, few works have described the frequency of invasive cervical cancer associated with HPV 18 infection. It is therefore necessary to develop researches to understand their association with pathological and oncogenic aspects of cervical lesions (8,9). The objective of this study was to evaluate the prevalence of human papillomavirus (HPV 18) intratypic variants in cervical cancer samples in women assisted in the Northeast of Maranhão.

Study design
This is a descriptive, cross-sectional study. The research sample was composed of 118 women older than 18 years with diagnosis of cervical cancer, treated at a reference oncology hospital in the state of Maranhão between January 2016 and June 2017. This study was approved by the Research Ethics Committee of the Federal University of Maranhão -UFMA under number 1.289.419/2015. The women who agreed to participate in the study signed an Informed Consent Form (ICF). Questionnaires were used to collect the sociodemographic information of the patients.
Sample collection, DNA extraction, PCR for HPV detection Fresh tumor biopsies were collected and stored in Later RNA at -80 °C. DNA extraction was performed using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The presence of HPV DNA was detected by the nested polymerase chain reaction (PCR) technique, with a rst round of reactions with the PGMY09/11 primers, and a second with the GP + 5/6 primers, amplifying the sequence of the L1 region of the virus genome.
Sequencing to identify HPV types PCR products from positive samples were puri ed using the DNA and Gel Band Puri cation kit (GE Healthcare) and sequenced using the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) on ABI Prism3130XL Genetic Analyzer (Applied Biosystems). Chromatograms of the sequences obtained were initially analyzed and edited in the 4Peaks software (Nucleobytes, Amsterdam, Netherlands). The sequences were then submitted to the online BLASTn software (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/ ) for the identi cation of HPV types.

Identi cation of HPV 18 variants
Two pairs of initiators were used to amplify the entire CSF region and the E6 gene, speci c for HPV 18. The reaction was performed in 25 ul of mix containing 1X PCR Buffer, 2.5 mM MgCl2, 0.2 µM of each DNTP, 100 µmol/L of each primer, 50-100 ng of DNA, and 2.5U of Platinum Taq DNA Polymerase (Life Technologies). Ampli cation was performed as follows: 95 °C for 10 min; followed by 40 cycles of 1 min at 95 °C, 1 min at annealing temperature, and 1 min at 72 °C, followed by a nal extension at 72 °C for 15 min.
Consensus sequences were aligned according to lineage-speci c reference sequences suggested by Burk (2013) using the Mega software (version 6, www.megasoftware.net). Then, in the step of PCR reactions, the products generated are subjected to the same puri cation protocols and direct sequencing for identi cation of HPV type.
Editing, sequence assembly, and phylogenetic analysis HPV 18 lineages were identi ed according to high-quality electropherograms of the LCR and E6 sequences and characterized according to lineage-distinctive single-nucleotide variations (SNVs) of HPV 18 CSF and E6 sequences. Sequence alignment was performed using the MEGA software 5.2. Phylogenetic analysis of HPV 18 sequences was done using the Maximum Likelihood method in the PHYML software and the GTR model. Bootstrap values were estimated after 1000 replications to evaluate the support of the internal branches. Phylogenetic reconstruction was performed using 1300 bp sequence data from the LCR and E6 regions, including reference sequences suggested by Burk et al (13).

Statistical analysis
Descriptive statistical analysis was performed in the Stata software (version 14.0), and data were presented in the form of gures and tables. The x 2 (Chi-square) test was used to check the association between HPV and sociodemographic and clinical variables; p values ≤ 0.05 were considered statistically signi cant. The Fisher Exact test was used to verify the association between HPV18 and clinical variables; p values ≤ 0.05 were considered statistically signi cant.
Regarding risk factors for HPV infection, the women reported being non-smokers (57,6%), having more than 3 sexual partners in a lifetime (67,8%), having more than 3 pregnancies (40,6%), knowing the purpose of the Papanicolaou test in CC prevention (73.7%), and not having family history of cervical cancer (80,5%). There was no statistically signi cant association between the presence of HPV and sociodemographic and lifestyle variables among cervical cancer women (p > 0.05). Characterization of HPV 18 positive women Table 2 report the sociodemographic data of HPV 18 positive women. The pro le of women with HPV 18 was age between 40 and 49 years (54.5%), married or common-law married (75%), with schooling up to complete or incomplete elementary education (50%), and monthly income less than 1/2 minimum wage (83.3%).
Most of HPV 18 positive women reported being non-white skin color, being non-smokers (58.3%), having less than 3 sexual partners in a lifetime (75%), and having more than 3 pregnancies (58.3%). There was no statistically signi cant association between the HPV 18 lineages found and sociodemographic and lifestyle variables among cervical cancer women (p > 0.05).    (18,19). The HPV 16 and 18 variants have been extensively studied to analyze their role in the development of cervical intraepithelial neoplasms and progression to invasive cervical cancer, taking into account the geographical and ethnic in uence in the distribution of variants worldwide (8,11,14). Speci c variations of HPV 18 may present high oncogenic potential and in uence the variation of distribution of cervical cancer in different regions (20).
Few studies with HPV 18 variants have been carried out until present, preventing comparisons of the present ndings with other studies and regions. Some studies reported that the lineage A of HPV 16 and HPV 18 are more prevalent in European populations, while the lineage D of HPV 18 predominates in populations with Amerindian ancestry. The lineages B and C are more prevalent in the African continent (14,21). Most studies have been conducted in women with cervical intraepithelial lesions, also showing a high prevalence of the lineage A. Previous studies reported a high risk of cervical intraepithelial lesions in women with European or Asian HPV 18 variants. They also report that infection by European HPV 18 variants are more likely to persist. However, a study by Villa et al. (2000) indicated that infection by non-European variants presents a greater chance of persistence of infection and association with pre-invasive lesions (20,22). Thus, more research is needed to understand which factors may be associated with virus lineages in the persistence and progression of cervical lesions.
The population of Maranhão is made up of people of different ancestry, with up to 75% African ancestry, besides Amerindian and indigenous (23). Due to the high contribution of African offspring, it was expected that there would be a greater distribution of HPV 18 B and C lineages in the population of Maranhão. The low prevalence of these lineages may be associated with intense ethnic mixture, with individuals presenting varying proportions of several ancestors, or with the level of carcinogenic potential in the studied population (14,21,24). Other studies in Brazil were carried out in order to investigate the HPV 16 lineages in Brazilian regions, but this is the rst study to identify the HPV 18 lineages in the population of Maranhão.
In a study conducted in Brazil by Vidal et al. (2016), a cohort of 71 women with cervical cancer infected with HPV 18 had a higher prevalence of lineage A followed by lineage B; lineage C was not found either (14). Similarly, the sublineage B1 (46 samples) prevailed in the variant B (11) .
Studies also indicate a variation in the distribution of HPV types, among them histological types. HPV 16 has been associated with SCC, whereas HPV 18 has a higher prevalence in ADC (26).
In this study, there was no statistically signi cant association between HPV presence and sociodemographic variables in women with cervical cancer. A similar result was obtained in the study by Chen et al. (2015), who sought to analyze the distribution of HPV 18 variants according to histological type (SCC or ADC). HPV 18 sublineages were compared in 453 cases of cancer and 236 controls, as well as in 81 cases of ADC and 160 cases of SCC. However, no statistically signi cant difference was found in the distribution of HPV 18 variants between cases and controls or between ADC and SCC at both general and regional level (11). The study of Arias-Pulido et al. (2005) with 15 cases of ADC and 10 of SCC did not nd statistically signi cant differences in the distribution of HPV 18 variants among the patients analyzed. These results indicate that the high prevalence of HPV 18 in ADC is due to the speci c tropism of HPV 18 variants for glandular cells (11,27,28).

Strengths And Limitations
The limiting factor in this study was the low number of women infected with HPV 18 and the lack of a control group to compare women with HPV 18 and cervical cancer and those with HPV 18 but without cervical cancer. This was the rst study conducted in the Northeast of Brazil to analyze the distribution of HPV 18 variants in women with cervical cancer who were not vaccinated against HPV. This population is resident in one of the regions with lowest socioeconomic and educational levels, with di cult access to health and education services.

Conclusions
In conclusion, the results showed that women with cervical cancer are associated with a higher frequency of HPV 16 and 18 in the Northeast region of Brazil, with a high prevalence of lineage A among women with HPV 18. Information about the diversity of HPV may contribute to the development of diagnostics and therapeutics approaches in order to reduce the number of cervical cancers.
The data from this study provide important information regarding the distribution of HPV in this population and will provide a basis for future studies on the distribution of HPV after the implementation of the HPV vaccination system and the impacts on the number of cervical cancer cases in Brazil. Availability of data and materials

Abbreviations
All data generated or analysed during this study are included in this published article.

Competing interests
The authors report no con ict of interest.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Authors' contributions GRBS, APAC, ZSB, FCBV and MDSBN participated in interpretation of data, drafted and critically revised the manuscript. RSL, FVF, ZSB, LMOB, MCB, ECF, MAMM and MDSBN contributed to study design, interpretation of data, and critically revised the manuscript. MACNS, APAC, LHLC and FRBS analyzed and assisted in interpretation of the data and assisted in drafting the manuscript. WEMF, DFS, MCB and ECF contributed to interpretation of data and critically revised the manuscript. All authors read and approved the nal manuscript