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 the National Institute of Cancer (INCA), 16,370 new cases of cervical cancer were estimated to occur each year in Brazil in 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 nine cases of cancer, at least one is cervical cancer.3 In developed North American and European countries, the incidence of cervical cancer is less than nine per 100,000 inhabitants.4 In developing countries, less than 50% of women affected by cervical cancer survive for more than five 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 identified and classified 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 finding 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 specificity 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, namely African, Caucasian, and Asian. In view of this, the variants were classified into three distinct lineages, the African (AF), the European (E), and Asian-Amerindian (As/Ai).8,10 Currently, a new nomenclature classifies HPV 18 variants in three lineages: A (including the lineages previously classified as As/Ai and E), B, and C (previously classified as lineage Af).8 In addition to classification in variants (up to 10% divergence), there is the classification into sublineages, when a divergence of 0.5 to 1% occurs between the same variant. For HPV 18, the sublineages are classified into: variant A (A1, A2, A3, A4 and A5), variant B (B1, B2 and B3), and variant C (has no sublineages).11

Genetic variations in the same viral type may influence 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 reports 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 HPV 18 intratypic variants in cervical cancer samples in women assisted in the Northeast of Maranhão state, Brazil.

There was a high prevalence of HPV in cervical tumor samples of women resident in the Northeast of Brazil in this study. HPV 16 and 18 were the most common subtypes, similar to other studies carried out in Brazil.14-17

Regarding HPV 18 infected women, our data show high prevalence of lineage A, followed by lineage B, while lineage C was not found. Regarding the sublineages, the A1, A2, A3 and A4 were documented for lineage A. In turn, the sublineages B1 and B2 were found for the lineage B. Moreover, our data show that cervical cancer women infected with HPV 18 had similar sociodemographic data as women infected with other virus types, mainly low-level of education, low-income, and residence in the countryside. We have also showed that there was no statistically significant association between HPV presence and sociodemographic variables in women with cervical cancer.

Regarding the characteristics of cervical tumors, squamous cell carcinoma was predominant in both HPV 18 and other HPV-types infected women, as well as in HPV 18 lineage A. Regarding staging and tumor size, both HPV 18 lineage A and B tumor samples were primarily staging 2 and 3, with tumor size up to 7 cm.

The prevalence of HPV in cervical tumor samples may range from 70 to 100% depending on the technique used for virus detection. Other studies have indicated that the four most prevalent types of HPV in South and Central America are HPV 16, 18, 31, and 45.18,19 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 influence in the distribution of variants worldwide.8,11,14 Specific variations of HPV 18 may present high oncogenic potential and influence 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 findings 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. It has also been reported that infection by European HPV 18 variants were more likely to become persistent. However, a study by Villa et al. (2000) indicated that infection by non-European variants presented a greater chance of persistence of infection and association with pre-invasive lesions.20,22 Thus, more research is needed to understand which virus lineages may be associated with persistence and progression of cervical lesions.

The population of Maranhão is made up of people of different genetic background, 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 ancestries, or with the level of carcinogenic potential in the studied population.14,21,24 Other studies in Brazil were carried out to investigate the HPV 16 lineages in Brazilian regions, but this is the first 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 A study carried out by Xu et al. (2018) with 138 women diagnosed with HPV 18 infection sought to identify the variants present in the population of Taizhou, China. The study concluded that all HPV 18 variants found belonged to the A lineage, while B and C lineages were not found in that population.8

In a study conducted in the Netherlands by King et al. (2016) with 835 male and female subjects, HPV 18 was identified in 108 individuals. Of these, 86% belonged to variant A, 14% belonged to variant B. The (non-European) variant C was also identified in 15 samples, most of which were taken from non-Dutch individuals.25 In a study by Chen et al. (2015) with 711 HPV 18 positive samples from 39 countries, there was predominance of variant A in all regions, except in sub-Saharan Africa where variant B prevailed. Variant C occurred only in Africa. In samples belonging to variant A, the sublineage A1 (248 samples) was predominant. Similarly, the sublineage B1 (46 samples) prevailed in the variant B.11

In Brazil, HPV vaccine was incorporated into the National Immunization Program (NIP) in 2014.26 The target population are girls aged 9 to 14 years and boys aged 11 to 14 years.27 In the state of Maranhão, only 70.8% of girls and 47.5% of boys were vaccinated.28

Studies also indicate a variation in the distribution of HPV types associated with histological types. HPV 16 has been associated with SCC, whereas HPV 18 has a higher prevalence in ADC.29 Nonetheless, in this research SCC was more prevalent among HPV 18 infected patients. Prevalence studies have shown that 60–70% of SCC cases occurred in HPV16 or HPV18 infected women.30

In this study, there was no statistically significant association between HPV presence and sociodemographic variables in women with cervical cancer. A similar result was obtained 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 significant 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 have not found statistically significant 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 specific tropism of HPV 18 variants for glandular cells.11,31,32

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 first 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 difficult access to health and education services.

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, and with a high prevalence of lineage A among women infected with HPV 18. Information about the diversity of HPV may contribute to the development of diagnostic and therapeutic 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 HPV vaccination and how it impacts on the number of cervical cancer cases in Brazil.

ADC, adenocarcinoma; HPV, human papillomavirus; ICF, Informed Consent Form; SCC, squamous cell carcinoma; SNV, single-nucleotide variations; PCR, polymerase chain reaction; UMT, undifferentiated malignant tumor.

This project was approved by the Research Ethics Committee of the Federal University of Maranhão CEP-UFMA), under Consolidated Opinion N° 1.289.419/2015. Women that accepted to participate by signing an Informed Consent Form.

Women that accepted to participate by signing an Informed Consent Form, giving their consent for publication.

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

This research was supported by Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Estado do Maranhão (FAPEMA) (BEPP 10/2015).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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 final manuscript