Role of HPV 16 variants among cervical samples from Northeastern Brazil

Background: Cervical cancer is the fourth most common type of cancer affecting women globally. In Brazil, it is the third most frequent type of cancer in women and HPV is present in approximately 90% of cases. Evidence suggests that variants of HPV 16 can interfere biologically and etiologically during the development of cervical cancer. Methods: Cervix tumor fragments were collected, their DNA was extracted, and nested PCR was used to detect HPV. Positive samples were sequenced to determine the viral genotype. To characterize the HPV 16 strains, positive samples PCR was used to amplify the LCR and E6 regions of the HPV 16 virus. Results: Data from 120 patients with cervical cancer were analyzed. Most women were between 41 and 54 years of age, had schooling until primary school, a family income between 1 and 2 times the minimum wage and were married/in a consensual union. There was no statistically significant association between HPV or socio-demographic variables and risk factors for cervical cancer (P < 0.05). HPV was present in 88 women (73%). The most prevalent types were HPV 16 (47/54%), HPV 18 (12/13.8%), HPV 35 (6/6.9%) and HPV 45 (5/5.7%). Of the 47 HPV 16 positive cases, variant A (49%) was present in 23 samples, followed by variant D in 20 cases (43%), and variants B and C in 2 cases each (4%).The most prevalent histological type of HPV 16 tumors was epidermoid carcinoma, followed by adenocarcinoma. There was a statistically significant association between HPV 16 variants and the tumors’ histological types (P < 0.001). Conclusions: Knowledge of HPV 16 variants will provide data on their influence on the pathological and oncogenic aspects of cervical lesions.


Introduction
Cervical cancer is the fourth most common cancer type affecting women worldwide, with more than 265,000 estimated deaths annually and more than 80% of cases occurring in developing countries [1,2]. According to INCA, cervical cancer is the third most common cancer type in the Brazilian female population. They estimate that there will be 16,370 new cervical cancer cases for the biennium from 2018-2019 [3].
It is estimated that in the state of Maranhão there will be around 1,090 new cervical cancer cases for 2018, with240 of them the capital, São Luís, with a gross rate of incidence of 30.55 cases per 100,000 inhabitants [3].
Human Papillomavirus (HPV) is the main etiological factor for cervical cancer and is associated with the development of diseases ranging from benign warts to invasive cancer [4]. Although approximately 80% of women acquire HPV infection by the age of 50, less than 1% of persistent infections progress to invasive cervical cancer [5].
The reason why only some uterine cervical lesions associated with high-risk genotypes progress to invasive cancer remains unknown. Evidence suggests that variants of the same HPV type can interact biologically and etiologically during cancer development [6,7].
The HPV16 is the most carcinogenic HPV type followed by HPV18 and most infections are asymptomatic [8]. A few studies have been conducted in Brazil on HPV 16 variants. This type of study has never been conducted in Maranhão. Interest in this topic has been growing in recent years, with variation in carcinoma prognosis at different stages of the disease depending on the viral variant found attracting special interest. Even in research carried out other places in the world, many of these questions remain unclear and many of the findings are contradictory, making it necessary to develop new and continuous approaches to this topic.
Despite of the relationship between different HPV type and the development of cancer being well established, evidence suggests that genetic variations between the same viral type may influence infection potential, viral persistence, the development of precursor lesions and the progression to invasive cancer [9][10][11][12]. Due to the high prevalence of HPV 16 in cases of cervical cancer, the association between HPV 16 and cancer has been studied at the level of intratype variants, and several lines of study have attributed a higher risk of invasive cervical cancer to some HPV 16 strains.
Studies on HPV variants have been developed with the objective of understanding their association with pathological and oncogenic aspects of cervical lesions. Factors influencing HPV infection in cervical cancer are not completely clear, but it is believed that HPV 16 variants playa fundamental role in cervical carcinogenesis and are currently recognized as an important marker for research on viral transmission, persistence and carcinogenicity [5]. Variation in these aspects may contribute to disparities in cervical cancer incidence.

Type of Study
This is descriptive, prospective, and transversal study.

Period and Location of study
The study was conducted from January 2016 to December 2017, at the High Complexity Care Unit

Population and Sample
The study population was composed of 120 women with a diagnosis of cervical cancer, who were treated at the previously mentioned hospitals. was added to the sample and it was again homogenized in the vortex.
The mixture was transferred to a tube containing a silica column (QIAamp MinElute) and centrifuged at 8,000 rpm for 2 minutes. After centrifugation, the column was repositioned in a new collector tube, and the filtrate was discarded.
The column was then washed successively with 500 µL of wash buffer 1 and wash buffer 2 centrifuging at 8,000 rpm for 2 minutes between washes. The column was then incubated with 100 µL of AE Buffer at room temperature for 5 minutes before being centrifuged at 14,000 rpm for 4 minutes to obtain the DNA. The samples were identified and stored at -20ºC for later use in PCR reactions.

DNA Quantification
The extracted DNA was quantified by reading absorbance in a Nanovue spectrophotometer (GE) at a wave length of 260nm. The purity of the DNA was verified by reading at 280nm to detect possible protein contamination. The samples were considered pure when the A260/A280 ratio was between 1.6 and 1.9.

Detection of HPV DNA by Nested PCR
HPV DNA in the cervical tumor samples was identified by Nested PCR using a Veriti 96 Well Thermal Cycler (Applied Biosystems, Thermo Scientific, California, USA). This technique consists of amplifying the DNA of interest in two rounds and using specific primers for round. With two rounds of PCR, the first amplification is more general and the second reaction, which uses the Amplicon produced in the first round as a template, leads to amplification of smaller and more specific sequences of viral DNA.
In the first-round, the primers PGMY 09 and 11 (Table 1) were used to amplify 450 bp sequences of the L1 region of viral DNA; in the second round, the primers GP +5 and GP+6 were used to amplify 190bp sequences of the L1 region of the viral DNA [12]. We used samples known to be positive as a positive control and water as a negative control.
To minimize the possibility of external contamination, the reactions were prepared in a laminar flow chapel exposed to ultraviolet light for approximately 15 minutes, along with all plastic material used. Table 1. Primer sequences used for the Nested PCR reaction to identify HPV DNA. (Invitrogen) [15].

Visualization of amplified products
5 µL aliquots of each reaction with loading buffer (Sigma-Aldrich, USA) and 0.1% Gel Red dye were visualized by electrophoresis in 1.5% agarose gel in TBE 1X buffer.

Purification of PCR products
The PCR products were purified with a Genelute PCR Clean up Kit according to the manufacturer's protocol (Sigma-Aldrich, USA). Briefly, 500 µL of capture buffer was added to 100 µL of PCR product in each microtube. The material was centrifuged and 2 µL of purified DNA was run in a 2% agarose gel to evaluate the purified sample.

Automatic sequencing
HPV genotypes were determined by automated sequencing of the PCR products using a1000 The Chromas program was used to obtain electropherograms of the HPV DNA sequences present in the samples. To identify the HPV type the nucleotide sequences were compared to the Genbank Nucleotide Sequence Database using the BLAST program (NCBI).

Statistical analyses
Descriptive statistical analysis was performed using the Stata program (version 14.0). The x 2 (Chisquare) test was used to verify the association between HPV and sociodemographic and clinical variables, and P values ≤ 0.05 were considered statistically significant. The values corresponding to don't know/did not respond were excluded from the association analysis.

Phylogenic Analysis
The phylogenetic tree of the HPV 16 strains was constructed from the 1,300bp sequences from the E6 and LCR regions using the "neighbor joining" method with p-distance (obtained with pairwise deletion)

Sociodemographic and clinical data
Among the women diagnosed with cervical cancer, HPV was present in 88 (73.3%). The majority of these women were in the age group 40 to 49 years of age (34/28.33%), and self-declared with mixed-race (84/70%), had schooling up to elementary school (51/42.50%), had a family income between 1 and 2 times the minimum wage (66/55%) and were married or in a consensual union (62/51.67%).
There was no statistically significant association between the sociodemographic variables and the presence of HPV (p < 0.05) ( Table 2).    1).

Histological types of analyzed tumors
The most prevalent types of tumor histological types were epidermoid carcinoma (EC) with a total of 95 cases (79.1%) and 11 (9.1%) cases of adenocarcinomas. Of the 120 women in this study, 45 (37.5%) had stage IIIB tumors, in cases where HPV 16 was present, stage III was predominant with 23 samples.

Discussion
Cervical cancer is associated with low socioeconomic indices, presenting a higher prevalence in regions with high poverty, high illiteracy rates and precarious hygiene habits [13,14].
The virus was highly prevalent (88/120 -73%) in the tumor samples examined. The prevalence of HPV in invasive cervical cancer samples can range from 70% to 100%, which may be associated with different techniques used to detect the virus [15][16][17].
HPV 16 is the most prevalent in cervical cancer worldwide, followed by HPV 18. However, the frequency of HPV types may vary according to the geographic region of the population under analysis.
Other studies point out that types16, 18, 31, and 45as the four most prevalent HPV types in South and Central America [18,19].
Studies have sought to evaluate the role of intratype variants of HPV16 and HPV18 in the persistence of viral infection, the risk of cervical intraepithelial neoplasia development, and the development of invasive cervical cancer [11,20].
For both HPV 16 and HPV 18, the distribution of variants worldwide is influenced by geographic and ethnic factors.
In a case-control study conducted by Hang et al. (2016) [5], we attempted to evaluate the association between HPV 16 variants and the risk of cervical cancer in 298 women with HPV 16 in China and found that variant A was predominant.
In the study conducted by Volpini et al., (2017) [21] in Brazil on 24 women positive for HPV 16 A study by Villa et al. (2000) [22], also in Brazil, examined the geographic differences in intratype variations of HPV 16 and their associations with the development of cervical cancer precursor lesions.
Variant A (54%) was the most frequent, followed by variant D in 22% of the cases. It also emphasizes the strong association between persistence and the presence of the non-European variants B, C and D when compared to the European prototype A.
In the present study in São Luís do Maranhão, the most prevalent strain in the population studied was variant A for HPV 16, followed by variant D.
Studies also indicate a variation in the distribution of HPV types among histological types. HPV 16 has been associated with squamous cell carcinoma and HPV 18 has a higher prevalence in adenocarcinoma than HPV 16. However, few studies have attempted to identify associations between HPV 16 variants and the histologic type of cervical tumors [23,24] In the study by Hang et al. (2016) [5], it was observed among the cases of women with cervical cancer infected with HPV 16, 289 (97%) had a pattern of squamous cell carcinoma, followed by adenocarcinoma (2.3%) and adenosquamous carcinoma (0.7%). The data also showed an increase in the prevalence of A1 -3 in Europe (67.9 to 97% for all histological types), variant D in South-Central America (61.5 to 63%, for adenocarcinoma and adenosquamous carcinoma), A4 in Asia (from 11.5 to 27.6% for all types) and variants B and C for Africa (from 28 to 66.7% and from 12.3 to 37.5% for ECC and ADC) [27] These results show a differential association between HPV 16 lineages and the histological features of cervical tumors in different geographic regions. Molecular differences among viral lineages and interactions with tumors may be one of the factors associated with the observed variation in prevalence.
There have also been studies on socioeconomic aspects. A study by Wang et al. (2015) [28] reported that women who received Pap smear test tended to have higher education levels, corroborating the results of other studies, and that this may be associated with a lack of information and delays in searching for treatment. In a study conducted by Manga et al. (2015) [29] on 209 women who sought care for cervical cancer screening, the mean age was 39.6 years old, and only 15% of the women were not literate, 88% were married and 48% had a paid activity.
In this study conducted in São Luís-MA, it was shown that variant A was the most prevalent variant in HPV 16 patients, corroborating data from the literature that shows a higher prevalence of variant A in mixed populations, as is the case of the Brazilian population.
Further studies are needed to understand the origin and progression of cancer, as well as the relationship between HPV 16 and its variants with the development of lesion, a cervical cancer precursor. The data presented here may help the development of future epidemiological studies on HPV 16 variants, as well as in the creation of strategies to combat the types that remain circulating, and which were not included in the currently available vaccines against HPV.  Phylogenetic tree of HPV 16 with lineages.