Journal of Medical Virology 88:144–152 (2016)
Prevalence of Human Papillomavirus Types in Cervical Cancerous and Precancerous Lesions of Ecuadorian Women Lorena Mejıa,1 Diana Mu~ noz,1 Gabriel Trueba,1 Leopoldo Tinoco,2 and Sonia Zapata1* 1
Instituto de Microbiologıa, Universidad San Francisco de Quito, Quito, Ecuador Unidad de Ginecologıa y Colposcopıa, Hospital SOLCA, Quito, Ecuador
2
Human Papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of uterine cancer. In Ecuador there is limited information about HPV types (and variants) in cancerous lesions; however, identifying the type-specific HPV prevalence in cervical lesions of women living in Ecuador is important to better predict the impact of HPV prophylactic vaccination in this country. We studied the prevalence of HPV types in cervical cancerous or precancerous lesions from 164 Ecuadorian women and found that 86.0% were HPV positive. The most common types were HPV16 (41.8%) and HPV58 (30.5%). Interestingly, HPV18 was detected only in 2.8% of the HPV-positive samples. Fifteen DNA sequences (genes E6 and L1) from 16 samples positive for HPV16 belonged to the European lineage, considered one of the least carcinogenic lineages, and 1 (6.25%) to the Asian-American lineage. Similar analysis in 12 HPV58 positive samples showed that 10 (83.3%) sequences grouped in sublineage A2, which belongs to the oldest HPV58 lineage, 1 belonged to A3 and 1 to lineage C. This study suggests that the currently used HPV vaccines (bivalent and tetravalent) may have lower effectiveness in Ecuador than in other geographic locations where HPV18 is more prevalent. J. Med. Virol. 88:144–152, 2016. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
HPV16 variants; HPV58 variants; cervical cancer; Ecuador
INTRODUCTION Human Papillomavirus (HPV) is responsible for 100% of cervical cancer cases [Walboomers et al., 1999; Khan et al., 2005] which is the fourth most important cause of cancer associated deaths in women worldwide [Bruni et al., 2014b]; in Ecuador, it C 2015 WILEY PERIODICALS, INC.
is the leading cause of cancer deaths in women of all ages [Bruni et al., 2014a]. Human Papillomavirus is a non-enveloped, double stranded circular DNA virus, protected by an icosahedral protein capsid [Zheng and Baker, 2006], which belongs to the Papillomaviridae family. Approximately 40 types (classified in the Alphapapillomavirus genus) can infect mucosal tissue in the anogenital area [Dunne and Markowitz, 2006] and each has different associations with cancer. Viral types known as “low-risk” can cause condyloma acuminata, respiratory papillomatosis, and low-grade cervical intraepithelial lesions; those classified as “high-risk” can cause squamous and glandular highgrade intraepithelial lesions and cancer (cervical, anal, vulvar, vaginal, penile and oropharyngeal) [Jong 2004; Dunne and Markowitz 2006; Doorbar et al., 2012]. However, 90% of high-risk HPV infections are cleared within two years by host immune responses [Dunne and Markowitz 2006; WHO, 2010]. Types HPV16 and HPV18 are the two most commonly associated with cancerous lesions in the cervix around the world (70% of cases) [Dunne and Markowitz, 2006] and these types are covered by the vaccines used in many countries including Ecuador [WHO 2013; Ministerio de Salud P ublica, 2014b]. Identification of genome variants could provide important information about the virulence, distribution and spread of the virus in specific geographic regions. High-risk HPV types have diverged into lineages and sublineages (1.5–10% and 0.5–1.4% of nucleotide divergence, respectively) with varying
Grant sponsor: Universidad San Francisco de Quito Disclaimer: The authors declare no conflicts of interest. Correspondence to: Sonia Zapata, Instituto de Microbiologıa, Universidad San Francisco de Quito, Diego de Robles s/n y Pampite, 170157 Quito - Ecuador. E-mail: [email protected] Accepted 22 June 2015 DOI 10.1002/jmv.24310 Published online 16 July 2015 in Wiley Online Library (wileyonlinelibrary.com).
HPV Types on Cervical Cancer in Ecuadorian Women
carcinogenicity [Schiffman et al., 2010; Chen et al., 2011]. Furthermore, variants of HPV16 and HPV18, are associated with specific geographic locations and even to ethnic populations, whereas in others, such as HPV58, the association is less clear. To better forecast the impact of HPV prophylactic immunization in Ecuador, we analyzed the prevalence of HPV types in precancerous and cancerous lesions from the genital tract of 164 Ecuadorian women. HPV lineages and sublineages were identified in samples infected with either HPV16 or HPV58, the most frequent types in this study. MATERIALS AND METHODS Clinical Samples Each cervical precancerous or cancer biopsy was obtained by a gynecologist from patients that underwent colposcopy in the SOLCA (Sociedad de Lucha contra el C ancer) Hospital in Quito, from October to December 2011 and from May 2012 to June 2013. This hospital is a national referral hospital for cancer patients. Informed consent was obtained from all the participants, and the study was approved by Universidad San Francisco de Quito (USFQ) bio-ethics committee. Pathological tissue was collected by biopsy and split in two parts; one part was sent to SOLCA’s histopathology laboratory and the other was preserved in 70% ethanol at 20˚C for further processing. All of the samples with high-grade intraepithelial neoplasia (CIN II and CIN III) and cancer were subjected to DNA extraction and amplification of HPV sequences. Nucleic Acid Extraction Total nucleic acids were isolated by a modified version of the CTAB method described elsewhere [Saghai-Maroof et al., 1984]. Biopsies (in 75% ethanol) were washed twice in 1X PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4.7H2O, 1.4 mM KH2PO4, pH 7). Each sample was split in half, one part was kept in a freezer at 20˚C and the other was cut into small pieces with a sterile scalpel and digested with 20 ml proteinase K (20 mg/ml) in 700 ml CTAB solution (2% CTAB weight/vol, 1.4 M NaCl, 20 mM EDTA pH 8, 100 mM TrisHCl pH 8) for 2 hr at 65˚C with occasional mixing (by inverting the 1.5 ml tubes). Chloroform/isoamyl alcohol (24:1 vol/vol, 700 ml) was added and the solution was vigorously mixed to form an emulsion that was centrifuged at 13,300g for 5 min at room temperature. The aqueous phase (500 ml) was removed and placed into a new tube with 1 ml of cold ethanol 100%, mixed by threeto-five inversions and kept at 20˚C overnight. After centrifugation at 16,110g for 10 min, the supernatant was discarded followed by washing it with 1 ml of 70% ethanol and centrifugation at 16,110g for 10 min. The supernatant was discarded and the tubes were dried at room temperature for 20 to 30 min. Finally, a
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nucleic acids pellet was suspended in 50 ml of TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA) and kept at 20˚C for further processing. The concentration of nucleic acids was quantified with a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE) according to the manufacturer’s instructions. HPV Detection and Genotyping Samples were genotyped by the 21 HPV GenoArray Diagnostic Kit (Hybribio Limited, Hong Kong, China) according to the manufacturer’s instructions. The Hybribio’s technique is based on E6 amplification and subsequent flow-through hybridization with specific probes within a membrane to identify 21 alpha HPV genotypes: 6, 11, 42, 43, 44, and 81 (low-risk) and 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 (high-risk). This technique has an internal amplification control and a biotin hybridization control for each sample. We used HPV-free human DNA and a no-DNA sample as negative controls. Molecular Analysis Variants of the most frequent HPV types (HPV16 and HPV58) were analyzed by amplifying genes E6 and L1 from mono-infected samples using a modification of the protocol described by Sotlar et al., 2004. In brief, E6 amplification was carried out in a final volume of 30 ml containing a 1X colorless reaction buffer, 1.6 mM MgCl2, 200 mM of each dNTP, 0.26 mM of primers GP-E6-3F and GP-E6-5B, 0.75U of GoTaq DNA Polymerase (PROMEGA Corporation, Madison) and 250 ng of DNA. The conditions for the amplification of a 630-bp product comprised an initial denaturation at 94˚C for 4 min, followed by 40 cycles of 94˚C for 1 min, 40˚C for 1 min, and 72˚C for 2 min. The last cycle was followed by an elongation step at 72˚C for 10 min. PCRs for L1 gene were performed in a final volume of 30 ml containing a 1X colorless reaction buffer, 2.5 mM MgCl2, 200 mM of each dNTP, 0.1 mM of primers MY09 and MY11, 2 U of GoTaq DNA Polymerase (PROMEGA Corporation, Madison) and 150 ng of DNA for the amplification of a 450-bp product. The amplification program was performed with an initial denaturation at 94˚C for 3 min followed by 40 cycles of 1-min denaturation at 94˚C, 1-min annealing at 55˚C, and 1-min elongation at 72˚C. The final extension step was done at 72˚C for 10 min. Genes E6 and L1 from samples infected with HPV16 (n ¼ 16) and HPV58 (n ¼ 12) were amplified and sequenced in Functional Biosciences (http://functionalbio. com/web/) and submitted to Genbank (Accession numbers for HPV16 E6 sequences are: KP794863-KP794878, HPV58 E6 sequences; KP794879- KP794890, HPV16 L1 sequences; KP794891- KP794906, HPV58 L1 sequences KP794907- KP794918). Each gene nucleotide sequence was aligned independently with HPV16 and HPV58 J. Med. Virol. DOI 10.1002/jmv
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reference variants from the GenBank (using MEGA software version 6.0 with ClustalW method with a gap opening penalty of 15). The E6 and L1 gene alignments were concatenated manually for each strain. Maximumlikelihood algorithm was performed using the same program. Node support was assessed by bootstrapping with 1000 replications. RESULTS Sample Screening and HPV Prevalence A total of 164 fresh cervical biopsies were included in the study. Patient age ranged from 19 to 77 years. The lesions were divided in three groups according to their histopathological results: 47 samples (28.7%) with CIN II (Cervical Intraepithelial Neoplasia type II), 62 samples (37.8%) with CIN III and 55 samples (33.5%) with cervical cancer (in situ or invasive) (Table I). Out the 164 samples, we found 141 (86.0%) to be positive for at least one HPV type distributed as follows: CIN II, 35 (74.5%); CIN III, 55 (88.7%); and cancer, 51 (92.7). From the total samples analyzed (n¼164), 96 (58.5%) were infected with only one HPV type, 45 (27.4%) had multiple infections with two or three types and 23 (14.0%) were negative for HPV DNA. HPV Genotyping We detected 19 out of 21 HPV types at least once; HPV16 and HPV58 were the most frequent in monoinfections (40.6%, and 25%, respectively) and coinfections (44.4%, and 42.2%, respectively). HPV16 was the most frequent genotype in both precancerous (CIN II and CIN III) and cancerous lesions, and
HPV58 was the second most frequent in all grade lesions, followed by HPV52, 6/11, 66, and 31 in decreasing frequency. HPV18 was only detected in four samples (2.8%). Other high risk types such as HPV51 and HPV81 were not found in any sample (Table I). All co-infections had at least one high-risk HPV type (HPV44, HPV53, HPV56, HPV59 were only found in co-infections). However, seven mono-infected samples had only low-risk types: HPV6 or HPV11 in five samples showing CIN II and CIN III lesions; HPV42 in one CIN II lesion, and HPV43 in a patient with a CIN III lesion. No low-risk genotypes were detected in cancer lesions. HPV16 and HPV58 Subtyping Nucleotide sequences from HPV16 amplicons grouped mostly into the European branch (Lineage A; Fig. 1), and only 1 (amplicon 88M) clustered with the Asian-American variants (sublineages D2/D3). Among sequences with high similarity to European variants (Table II), 14 sequences grouped into sublineage A2 (German sublineage), amplicon 20 M was identical to sublineage A1, and 69 M clustered within lineage A, although it showed one non-synonymous substitution which was also present in lineages B, C, and D (Table II). The majority (83.3%) of HPV58 amplicons grouped into sublineage A2 (Fig. 2) but the sequence from amplicon LM82 showed one synonymous substitution in common with sublineage A3; the amplicon LM51 had one synonymous substitution and four nonsynonymous substitutions which were also present in lineage C sequences. No samples aligned with sublineage A1, lineages B or D (Table III).
TABLE I. Prevalence of HPV Types Found in Cervical Precancerous and Cancerous Lesions (n ¼ 164) in Ecuadorian Women Attending a Large Hospital in Quito. CIN: Cervical Intraepithelial Neoplasia; II and III: Lesion Grade II and Grade III, Respectively CIN II n (%) HPVþ (%) HPV16 (%) HPV58 (%) HPV52 (%) HPV6/11 (%) HPV66 (%) HPV31 (%) HPV43 (%) HPV39 (%) HPV68 (%) HPV35 (%) HPV33 (%) HPV18 (%) HPV59 (%) HPV45 (%) HPV56 (%) HPV53 (%) HPV42 (%) HPV44 (%) HPV51 (%) HPV81 (%)
47 35 13 7 5 4 3 3 3 2 1 1 1 0 1 2 1 0 1 0 0 0
(28.7) (74.5) (37.1) (20.0) (14.3) (11.4) (8.6) (8.6) (8.6) (5.7) (2.9) (2.9) (2.9) (0.0) (2.9) (5.7) (2.9) (0.0) (2.9) (0.0) (0.0) (0.0)
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CIN III
Cancer
62 55 24 20 5 7 4 1 4 1 2 1 2 4 0 0 0 0 0 1 0 0
58 54 22 16 7 2 4 6 3 2 2 2 1 0 2 1 0 1 0 0 0 0
(37.8) (88.7) (43.6) (36.4) (9.1) (12.7) (7.3) (1.8) (7.3) (1.8) (3.6) (1.8) (3.6) (7.3) (0.0) (0.0) (0.0) (0.0) (0.0) (1.8) (0.0) (0.0)
(33.5) (92.7) (43.1) (31.4) (13.0) (3.7) (7.4) (11.1) (5.6) (3.7) (3.7) (3.7) (1.9) (0.0) (3.7) (1.9) (0.0) (1.9) (0.0) (0.0) (0.0) (0.0)
Total 164 141 59 43 17 13 11 10 10 5 5 4 4 4 3 3 1 1 1 1 0 0
(100) (86.0) (41.8) (30.5) (12.1) (9.2) (7.8) (7.1) (7.1) (3.5) (3.5) (2.8) (2.8) (2.8) (2.1) (2.1) (0.7) (0.7) (0.7) (0.7) (0.0) (0.0)
Mono-infections 96 96 39 24 9 5 2 6 1 1 2 1 2 1 0 2 0 0 1 0 0 0
(58.5) (100) (40.6) (25.0) (9.09) (5.05) (2.02) (6.06) (1.01) (1.01) (2.02) (1.01) (2.02) (1.01) (0.0) (2.02) (0.0) (0.0) (1.01) (0.0) (0.0) (0.0)
Co-infections 45 45 20 19 8 8 9 4 9 4 3 3 2 3 3 1 1 1 0 1 0 0
(27.4) (100) (44.44) (42.22) (17.77) (17.77) (20.00) (8.88) (20.00) (8.88) (6.66) (6.66) (4.44) (6.66) (6.66) (2.22) (2.22) (2.22) (0.0) (2.22) (0.0) (0.0)
Total 141 141 59 43 17 13 11 10 10 5 5 4 4 4 3 3 1 1 1 1 0 0
(100) (100) (41.8) (30.5) (12.1) (9.2) (7.8) (7.1) (7.1) (3.5) (3.5) (2.8) (2.8) (2.8) (2.1) (2.1) (0.7) (0.7) (0.7) (0.7) (0.0) (0.0)
HPV Types on Cervical Cancer in Ecuadorian Women
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Fig. 1. Maximum-likelihood tree using nucleotide sequences of E6 and L1 genes from HPV16 variants obtained in this study (KP794863-KP794878 for E6 sequences and KP794891KP794906 for L1 sequences). Sequences reported here are denoted as CIN (cervical intraepithelial neoplasia) II, CIN III and cancer. The rest of the sequences were obtained from GenBank. Numbers indicate bootstrap values from 1,000
pseudoreplicates. Representative genomes for HPV16 lineages (termed A, B, C and D) and sub-lineages (termed A1-A4/B1-B2/ D1-D3) were used as variant references [Burk et al., 2013]. Regional variants are indicated as: E, European variant; As, East Asian; Afr, African; NA, North American; AA, AsianAmerican.
DISCUSSION
associated with precancerous and cancerous lesions; however, similar results have been described previously in non-cancerous lesions from Ecuador [Tornesello et al., 2008], and in lesions of different severity from Nicaragua and Mexico [Hindryckx 2006; Pi~ naS anchez et al., 2006]. The low prevalence of HPV18 found in the current study is in line with previous findings showing a lower prevalence in South America (only 5% of HPV infections) compared to North America (11%) and Europe (8%) [Clifford, 2005]. Our laboratory has detected HPV18 in 6.67% of 135 HPV positive samples (without cancerous or precancerous lesions) from Ecuadorian women (unpublished data). The reason for these differences in geographic distribution of HPV types is unknown, but some authors have proposed complex interactions between HPV types and HLA haplotypes as a possible explanation [Clifford, 2005]. In the current study we also found that 27.4% of total samples were infected with two or three HPV types. HPV6/11 were detected in cancer lesions but always in co-infections (Table I) with at least one high-risk HPV, which is in accordance with
Prevalence of HPV Genotypes This is the first report describing the distribution of HPV types among Ecuadorian women with cervical precancerous and cancerous lesions. The study contributes to the understanding of HPV epidemiology in Ecuador by showing that HPV16 (40.6% in monoinfections and 44.4% in co-infections) and HPV58 (25.0% and 42.2%, respectively) were more commonly associated with cervical precancer and cancer in Ecuadorian women attending a major hospital in Quito. We also found the same HPV types in 3 samples of vaginal cancer, HPV16 (n ¼ 2) and HPV58 (n ¼ 1), from patients attending the same hospital (data not shown). It was surprising to find a high percentage of precancerous and cancerous lesions associated with HPV58, although similar results have been described in Asia [Huang et al., 1997; Chen et al., 2006], Mexico [Gonz alez-Losa et al., 2004; Pi~ na-S anchez et al., 2006], Nicaragua [Hindryckx, 2006] and Brazil [Fernandes et al., 2013]. It was also surprising to find very little (2.8%) of HPV18
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TABLE II. Nucleotide Polymorphisms in E6 and L1 Genes From HPV16 Variants Obtained in This Study (Accession Numbers for HPV16 E6 Sequences are: KP794863-KP794878, HPV16 L1 Sequences; KP794891- KP794906). The Sequences Reported in This Manuscript Are Denoted as CIN (Cervical Intraepithelial Neoplasia) II, CIN III and Cancer; the Rest of the Sequences Were Obtained From GenBank. Representative Genomes for HPV16 Lineages (Termed A, B, C and D) and Sublineages (Termed A1-A4/B1-B2/D1-D3) Were Used as Variant References [Burk, Harari, and Chen 2013]. Regional Variants are Indicated as: E, European Variant; As, East Asian; Afr, African; NA, North American; AA, Asian-American.
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Fig. 2. Maximum-likelihood tree using nucleotide sequences of E6 and L1 genes from HPV58 variants obtained in this study (KP794879-KP794890 for E6 sequences and KP794907KP794918 for L1 sequences). Sequences reported here are denoted as CIN (cervical intraepithelial neoplasia) II, CIN III and cancer. The rest of the sequences were obtained from GenBank. Numbers indicate bootstrap values from 1,000 pseudoreplicates. Representative genomes for HPV58 lineages (termed A, B, C and D) and sub-lineages (termed A1-A3/B1-B2/ D1-D2) were used as variant references [Chen et al. 2011].
previously published results [zur Hausen, 2002]. As in previous reports [Thomas et al., 2000; zur Hausen 2002], no clear association between specific HPV combinations was observed. Seven precancerous lesions (but no cancerous lesions) were associated with infections with low-risk HPV types (data not shown), and this finding also concurs with other reports [zur Hausen, 2002]. Nevertheless, some authors have suggested that there are genetic defects in the host which may increase the susceptibility to develop cancer even when infected only with low-risk HPV types [Doorbar et al., 2012]. HPV16 and HPV58 Subtype Analysis The majority of HPV16 variants (93.75%) in precancer and cancer lesions were of European lineage and 6.25% belonged to the Asian-American sublineage, which coincides with reports from other LatinAmerican countries [Villa et al., 2000; Hildesheim
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et al., 2001; Picconi et al., 2003; Sichero et al., 2007; Schiffman et al., 2010]. However, one study from Mexico found that 23.8% of the HPV16 variants belonged to European lineages and 23.2% to the Asian-American sublineages [Berumen et al., 2001]. This is relevant because non-European HPV16 sublineages (especially the Asian-American) tend to be more persistent [Villa et al., 2000], more aggressive and more carcinogenic than European counterparts [Berumen et al., 2001; Hildesheim et al., 2001; Sichero et al., 2007; Chen et al., 2011; Burk et al., 2013]. Greater carcinogenicity may be associated with more efficient viral replication (higher copy number per cell), greater p53 degradation by E6 protein [Berumen et al., 2001], better binding and degradation of pRb by E7 protein, induction of genome instability and telomerase activation [Doorbar et al., 2012]. It is interesting that 14 (87.5%) out of 16 samples analyzed contained the polymorphism 350G which has been associated with higher risk of persistence and high grade lesions and cancer [Pande et al., 2008; Huertas-Salgado et al., 2011]; however, the alternative nucleotide found in this site (350T) has also been associated with an increased risk of persistence and cancer [Gheit et al., 2011; Cornet et al., 2013]. These contradictory results suggest that the oncogenic potential of HPV16 variants might be also associated to genetic differences in the host [Zehbe et al., 2001]. The phylogenetic analysis of Ecuadorian HPV58 sequences (12 samples) showed that most variants belonged to sublineage A2 which may be the oldest HPV58 sublineage and from which the other lineages emerged [Chan et al., 2011]. One amplicon sequence clustered with sublineage A3; two substitutions found in the latter sequence have been associated previously with higher risk of cancer: E7 T20I causes greater affinity to the RB protein and E7 G35S creates an additional phosphorylation site which may increase oncogenic activity [Chan et al., 2002]. Assessment of the oncogenic HPV types affecting Ecuadorian women is vital to analyzing the possible local impact of the vaccination program. Both bivalent and tetravalent vaccines have been available for more than 4 years in private settings in Ecuador [Ministerio de Salud P ublica 2014a]; however, our results may predict a lower effectiveness of these vaccines in Ecuador than in other countries with higher HPV18 and lower HPV58 prevalences. Additionally it would be important to monitor mutations in capsid and oncogenic proteins for they will provide information about predicted oncogenicity and vaccination success [Fleury et al., 2014]. The introduction of a 9-valent vaccine may reduce the combined incidence of high grade lesions in cervical/vulvar/ vaginal disease (caused by genotypes 16, 18, 31, 33, 45, 52, and 58) by 96.7% [Joura et al., 2015]. Our research had one limitation which was the small number of samples analyzed because we focused only in precancer and cancer samples. J. Med. Virol. DOI 10.1002/jmv
TABLE III. Nucleotide Polymorphisms in E6 and L1 Genes From HPV58 Variants Obtained in This Study (Accession Numbers for HPV58 E6 Sequences are KP794879- KP794890, and for HPV58 L1 Sequences are KP794907- KP794918). The Sequences Reported in This Manuscript Are Denoted as CIN (Cervical Intraepithelial Neoplasia) II, CIN III and Cancer. The Rest of the Sequences Were Obtained From GenBank. Representative Genomes for HPV58 Lineages (Termed A, B, C, and D) and Sub-Lineages (Termed A1-A3/B1-B2/D1-D2) Were Used as Variant References [Chen et al., 2011].
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HPV Types on Cervical Cancer in Ecuadorian Women
Nevertheless, the samples analyzed came from a nationwide oncologic referral center which may allow inference to the distribution of HPV genotypes and variants in different regions of Ecuador. It is also noteworthy that this study is the first report of HPV58 molecular variants done in Ecuador. Pathological characteristics of HPV58 variants and their associations with cancer needs further study. CONCLUSION In conclusion, the overall frequency of HPV types detected in precancerous and cancerous lesions was high for HPV16 and HPV58 and low for HPV18 in Ecuador. The introduction of the current 9-valent vaccine may be more suitable to reduce the combined incidence of high grade lesions and cancer associated with HPV in Ecuador. Additionally, the variants observed in this study were principally European for HPV16 and from sublineage A2 for HPV58. The HPV types and variants reported here were similar to other studies of malignant lesions in other Latin American countries. ACKNOWLEDGMENTS The authors would like to thank Kristin Tomey for her valuable comments and her editing support and Maria Elena Pe~ naranda for her manuscript suggestions. The authors also thank Hybribio Limited (Hong Kong, China) for their kind support in providing “21 HPV GenoArray Diagnostic Kits” for HPV genotyping. REFERENCES Berumen J, Ordonez R M, Lazcano E, Salmeron J, Galvan S C, Estrada R A, Yunes E, Garcia-Carranca A, Gonzalez-Lira G, Madrigal-de la Campa A. 2001. Asian-American variants of human papillomavirus 16 and risk for cervical cancer: A casecontrol study. JNCI J Natl Cancer Inst 93:1325–1330. Bruni L, Barrionuevo-Rosas L, Serrano B, Brotons M, Cosano R, Mu~ noz J, Bosch FX, de Sanjos e S, Castellsagu e X. 2014a. Human papillomavirus and related diseases in Ecuador. Summary Rep http://www.hpvcentre.net/statistics/reports/ECU.pdf. Bruni L, Barrionuevo-Rosas L, Serrano B, Brotons M, Cosano R, Mu~ noz J, Bosch FX, de Sanjos e S, Castellsagu e X. 2014b. Human papillomavirus and related diseases in world. Summary Rep http://www.hpvcentre.net/statistics/reports/XWX.pdf. Burk RD, Harari A, Chen Z. 2013. Human papillomavirus genome variants. Virology 445:232–243. Chan Pa‘ul KS, Ching-Wan Lam, Tak-Hong Cheung, William Li WH, Keith Lo WK, May Chan YM, Jo Cheung LK, Augustine Cheng. F. 2002. Association of Human Papillomavirus type 58 Variant with the risk of cervical cancer. J Natl Cancer Inst 94:1249–1253. Chan PKS, Luk ACS, Park J-S, Smith-McCune KK, Palefsky JM, Konno R, Giovannelli L, Coutl ee F, Hibbitts S, Chu TY, Settheetham-Ishida W, Picconi MA, Ferrera A, De Marco F, Woo YL, Raiol T, Pi~ na-S anchez P, Cheung JL, Bae JH, Chirenje MZ, Magure T, Moscicki AB, Fiander AN, Di Stefano R, Cheung TH, Yu MM, Tsui SK, Pim D, Banks L. 2011. Identification of Human Papillomavirus Type 58 Lineages and the Distribution Worldwide. J Infect Dis 203:1565–1573. Chen C-A, Liu C-Y, Chou H-H, Chou C-Y, Ho C-M, Twu N-F, Kan Y-Y, Chuang M-H, Chu T-Y, Hsieh C-Y. 2006. The distribution and differential risks of human papillomavirus genotypes in cervical preinvasive lesions: A Taiwan cooperative oncologic group study. Int J Gynecol Cancer 16:1801–1808.
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152 variants in quechua aboriginals from Argentina. J Med Virol 69:546–552. Pi~ na-S anchez P, Hern andez-Hern andez DM, L opez-Romero R, V azquez-Ortız G, P erez-Plasencia C, Lizano-Sober on M, Gonz alez-S anchez JL, Cruz-Talonia F, Salcedo M. 2006. Human papillomavirus-specific viral types are common in Mexican women affected by cervical lesions.. Int J Gynecol Cancer 16:1041–1047. Saghai-Maroof, MA, KM Soliman, RA Jorgensen, RW Allard. 1984. Ribosomal DNA spacer-length polymorphisms in Barley: Mendelian inheritance, chromosomal location, and population dynamics 81 (December):8014–18. Schiffman M, Rodriguez AC, Chen Z, Wacholder S, Herrero R, Hildesheim A, Desalle R, et al. 2010. A population-based prospective study of carcinogenic human papillomavirus variant lineages, viral persistence, and cervical neoplasia. Cancer Res 70:3159–3169. Sichero L, Ferreira S, Trottier H, Duarte-Franco E, Ferenczy A, Franco EL, Villa LL. 2007. High grade cervical lesions are caused preferentially by non-european variants of HPVs 16 and 18. Int J Cancer 120:1763–1768. Sotlar K, Diemer D, Dethleffs A, Hack Y, Stubner A, Vollmer N, Menton S, et al. 2004. Detection and typing of human papillomavirus by E6 nested multiplex PCR. J Clin Microbiol 42:3176–3184. Thomas KK, Hughes JP, Kuypers JM, Kiviat NB, Lee SK, Adam DE, Koutsky LA. 2000. Concurrent and sequential acquisition of different genital human papillomavirus types. J Infect Dis 182:1097–1102. Tornesello ML, Buonaguro L, Izzo S, Lopez G, Vega X, Reyes CFM, Buonaguro FM. 2008. A pilot study on the distribution of human
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Mejıa et al. papillomavirus genotypes and HPV-16 variants in cervical neoplastic lesions from ecuadorian women. J Med Virol 80: 1959–1965. Villa LL, Sichero L, Rahal P, Caballero O, Ferenczy A, Rohan T, Franco EL. 2000. Molecular variants of human papillomavirus types 16 and 18 preferentially associated with cervical neoplasia. J Gen Virol 81:2959–2968. Walboomers, M.M. Jan, V. Marcel Jacobs, M. Michele Manos, F. Xavier Bosch, J. Alain Kummer, V. Keerti Shah, J.F. Peter Snijders, Julian Peto, J.L.M. Chris Meijer, Nubia Mu~ noz. Human Papillomavirus Is a Necessary Cause of Invasive Cervical Cancer Worldwide. J Pathol 189: 12–19. doi:10.1002/ (SICI) 1096-9896(199909) 189:13.0.CO; 2-F. WHO. 2013. Human papillomavirus (HPV) and cervical cancer. Fact sheet N˚380. World Health Organizaci on http://www.who. int/mediacentre/factsheets/fs380/en/. WHO IVB. 2010. Human papillomavirus laboratory manual. WHO Document Production Services http://whqlibdoc.who.int/hq/2010/ WHO_IVB_10.12_eng.pdf. Zehbe I, Tachezy R, Mytilineos J, Voglino G, Mikyskova I, Delius H, Marongiu A, Gissmann L, Wilander E, Tommasino M. 2001. Human papillomavirus 16 E6 polymorphisms in cervical lesions from Different european populations and their correlation with human leukocyte antigen class II haplotypes. Int J Cancer 94:711–716. Zheng Z-M, Baker CC. 2006. Papillomavirus genome structure, expression, and post-transcriptional regulation. Front Biosci: J Virt Libr 11:2286–2302. Zur Hausen H. 2002. Papillomaviruses and cancer: From basic studies to clinical application. Nat Rev Cancer 2:342–350.
Prevalence of Human Papillomavirus Types in Cervical Cancerous and Precancerous Lesions of Ecuadorian Women Lorena Mejıa,1 Diana Mu~ noz,1 Gabriel Trueba,1 Leopoldo Tinoco,2 and Sonia Zapata1* 1
Instituto de Microbiologıa, Universidad San Francisco de Quito, Quito, Ecuador Unidad de Ginecologıa y Colposcopıa, Hospital SOLCA, Quito, Ecuador
2
Human Papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of uterine cancer. In Ecuador there is limited information about HPV types (and variants) in cancerous lesions; however, identifying the type-specific HPV prevalence in cervical lesions of women living in Ecuador is important to better predict the impact of HPV prophylactic vaccination in this country. We studied the prevalence of HPV types in cervical cancerous or precancerous lesions from 164 Ecuadorian women and found that 86.0% were HPV positive. The most common types were HPV16 (41.8%) and HPV58 (30.5%). Interestingly, HPV18 was detected only in 2.8% of the HPV-positive samples. Fifteen DNA sequences (genes E6 and L1) from 16 samples positive for HPV16 belonged to the European lineage, considered one of the least carcinogenic lineages, and 1 (6.25%) to the Asian-American lineage. Similar analysis in 12 HPV58 positive samples showed that 10 (83.3%) sequences grouped in sublineage A2, which belongs to the oldest HPV58 lineage, 1 belonged to A3 and 1 to lineage C. This study suggests that the currently used HPV vaccines (bivalent and tetravalent) may have lower effectiveness in Ecuador than in other geographic locations where HPV18 is more prevalent. J. Med. Virol. 88:144–152, 2016. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
HPV16 variants; HPV58 variants; cervical cancer; Ecuador
INTRODUCTION Human Papillomavirus (HPV) is responsible for 100% of cervical cancer cases [Walboomers et al., 1999; Khan et al., 2005] which is the fourth most important cause of cancer associated deaths in women worldwide [Bruni et al., 2014b]; in Ecuador, it C 2015 WILEY PERIODICALS, INC.
is the leading cause of cancer deaths in women of all ages [Bruni et al., 2014a]. Human Papillomavirus is a non-enveloped, double stranded circular DNA virus, protected by an icosahedral protein capsid [Zheng and Baker, 2006], which belongs to the Papillomaviridae family. Approximately 40 types (classified in the Alphapapillomavirus genus) can infect mucosal tissue in the anogenital area [Dunne and Markowitz, 2006] and each has different associations with cancer. Viral types known as “low-risk” can cause condyloma acuminata, respiratory papillomatosis, and low-grade cervical intraepithelial lesions; those classified as “high-risk” can cause squamous and glandular highgrade intraepithelial lesions and cancer (cervical, anal, vulvar, vaginal, penile and oropharyngeal) [Jong 2004; Dunne and Markowitz 2006; Doorbar et al., 2012]. However, 90% of high-risk HPV infections are cleared within two years by host immune responses [Dunne and Markowitz 2006; WHO, 2010]. Types HPV16 and HPV18 are the two most commonly associated with cancerous lesions in the cervix around the world (70% of cases) [Dunne and Markowitz, 2006] and these types are covered by the vaccines used in many countries including Ecuador [WHO 2013; Ministerio de Salud P ublica, 2014b]. Identification of genome variants could provide important information about the virulence, distribution and spread of the virus in specific geographic regions. High-risk HPV types have diverged into lineages and sublineages (1.5–10% and 0.5–1.4% of nucleotide divergence, respectively) with varying
Grant sponsor: Universidad San Francisco de Quito Disclaimer: The authors declare no conflicts of interest. Correspondence to: Sonia Zapata, Instituto de Microbiologıa, Universidad San Francisco de Quito, Diego de Robles s/n y Pampite, 170157 Quito - Ecuador. E-mail: [email protected] Accepted 22 June 2015 DOI 10.1002/jmv.24310 Published online 16 July 2015 in Wiley Online Library (wileyonlinelibrary.com).
HPV Types on Cervical Cancer in Ecuadorian Women
carcinogenicity [Schiffman et al., 2010; Chen et al., 2011]. Furthermore, variants of HPV16 and HPV18, are associated with specific geographic locations and even to ethnic populations, whereas in others, such as HPV58, the association is less clear. To better forecast the impact of HPV prophylactic immunization in Ecuador, we analyzed the prevalence of HPV types in precancerous and cancerous lesions from the genital tract of 164 Ecuadorian women. HPV lineages and sublineages were identified in samples infected with either HPV16 or HPV58, the most frequent types in this study. MATERIALS AND METHODS Clinical Samples Each cervical precancerous or cancer biopsy was obtained by a gynecologist from patients that underwent colposcopy in the SOLCA (Sociedad de Lucha contra el C ancer) Hospital in Quito, from October to December 2011 and from May 2012 to June 2013. This hospital is a national referral hospital for cancer patients. Informed consent was obtained from all the participants, and the study was approved by Universidad San Francisco de Quito (USFQ) bio-ethics committee. Pathological tissue was collected by biopsy and split in two parts; one part was sent to SOLCA’s histopathology laboratory and the other was preserved in 70% ethanol at 20˚C for further processing. All of the samples with high-grade intraepithelial neoplasia (CIN II and CIN III) and cancer were subjected to DNA extraction and amplification of HPV sequences. Nucleic Acid Extraction Total nucleic acids were isolated by a modified version of the CTAB method described elsewhere [Saghai-Maroof et al., 1984]. Biopsies (in 75% ethanol) were washed twice in 1X PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4.7H2O, 1.4 mM KH2PO4, pH 7). Each sample was split in half, one part was kept in a freezer at 20˚C and the other was cut into small pieces with a sterile scalpel and digested with 20 ml proteinase K (20 mg/ml) in 700 ml CTAB solution (2% CTAB weight/vol, 1.4 M NaCl, 20 mM EDTA pH 8, 100 mM TrisHCl pH 8) for 2 hr at 65˚C with occasional mixing (by inverting the 1.5 ml tubes). Chloroform/isoamyl alcohol (24:1 vol/vol, 700 ml) was added and the solution was vigorously mixed to form an emulsion that was centrifuged at 13,300g for 5 min at room temperature. The aqueous phase (500 ml) was removed and placed into a new tube with 1 ml of cold ethanol 100%, mixed by threeto-five inversions and kept at 20˚C overnight. After centrifugation at 16,110g for 10 min, the supernatant was discarded followed by washing it with 1 ml of 70% ethanol and centrifugation at 16,110g for 10 min. The supernatant was discarded and the tubes were dried at room temperature for 20 to 30 min. Finally, a
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nucleic acids pellet was suspended in 50 ml of TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA) and kept at 20˚C for further processing. The concentration of nucleic acids was quantified with a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE) according to the manufacturer’s instructions. HPV Detection and Genotyping Samples were genotyped by the 21 HPV GenoArray Diagnostic Kit (Hybribio Limited, Hong Kong, China) according to the manufacturer’s instructions. The Hybribio’s technique is based on E6 amplification and subsequent flow-through hybridization with specific probes within a membrane to identify 21 alpha HPV genotypes: 6, 11, 42, 43, 44, and 81 (low-risk) and 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 (high-risk). This technique has an internal amplification control and a biotin hybridization control for each sample. We used HPV-free human DNA and a no-DNA sample as negative controls. Molecular Analysis Variants of the most frequent HPV types (HPV16 and HPV58) were analyzed by amplifying genes E6 and L1 from mono-infected samples using a modification of the protocol described by Sotlar et al., 2004. In brief, E6 amplification was carried out in a final volume of 30 ml containing a 1X colorless reaction buffer, 1.6 mM MgCl2, 200 mM of each dNTP, 0.26 mM of primers GP-E6-3F and GP-E6-5B, 0.75U of GoTaq DNA Polymerase (PROMEGA Corporation, Madison) and 250 ng of DNA. The conditions for the amplification of a 630-bp product comprised an initial denaturation at 94˚C for 4 min, followed by 40 cycles of 94˚C for 1 min, 40˚C for 1 min, and 72˚C for 2 min. The last cycle was followed by an elongation step at 72˚C for 10 min. PCRs for L1 gene were performed in a final volume of 30 ml containing a 1X colorless reaction buffer, 2.5 mM MgCl2, 200 mM of each dNTP, 0.1 mM of primers MY09 and MY11, 2 U of GoTaq DNA Polymerase (PROMEGA Corporation, Madison) and 150 ng of DNA for the amplification of a 450-bp product. The amplification program was performed with an initial denaturation at 94˚C for 3 min followed by 40 cycles of 1-min denaturation at 94˚C, 1-min annealing at 55˚C, and 1-min elongation at 72˚C. The final extension step was done at 72˚C for 10 min. Genes E6 and L1 from samples infected with HPV16 (n ¼ 16) and HPV58 (n ¼ 12) were amplified and sequenced in Functional Biosciences (http://functionalbio. com/web/) and submitted to Genbank (Accession numbers for HPV16 E6 sequences are: KP794863-KP794878, HPV58 E6 sequences; KP794879- KP794890, HPV16 L1 sequences; KP794891- KP794906, HPV58 L1 sequences KP794907- KP794918). Each gene nucleotide sequence was aligned independently with HPV16 and HPV58 J. Med. Virol. DOI 10.1002/jmv
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Mejıa et al.
reference variants from the GenBank (using MEGA software version 6.0 with ClustalW method with a gap opening penalty of 15). The E6 and L1 gene alignments were concatenated manually for each strain. Maximumlikelihood algorithm was performed using the same program. Node support was assessed by bootstrapping with 1000 replications. RESULTS Sample Screening and HPV Prevalence A total of 164 fresh cervical biopsies were included in the study. Patient age ranged from 19 to 77 years. The lesions were divided in three groups according to their histopathological results: 47 samples (28.7%) with CIN II (Cervical Intraepithelial Neoplasia type II), 62 samples (37.8%) with CIN III and 55 samples (33.5%) with cervical cancer (in situ or invasive) (Table I). Out the 164 samples, we found 141 (86.0%) to be positive for at least one HPV type distributed as follows: CIN II, 35 (74.5%); CIN III, 55 (88.7%); and cancer, 51 (92.7). From the total samples analyzed (n¼164), 96 (58.5%) were infected with only one HPV type, 45 (27.4%) had multiple infections with two or three types and 23 (14.0%) were negative for HPV DNA. HPV Genotyping We detected 19 out of 21 HPV types at least once; HPV16 and HPV58 were the most frequent in monoinfections (40.6%, and 25%, respectively) and coinfections (44.4%, and 42.2%, respectively). HPV16 was the most frequent genotype in both precancerous (CIN II and CIN III) and cancerous lesions, and
HPV58 was the second most frequent in all grade lesions, followed by HPV52, 6/11, 66, and 31 in decreasing frequency. HPV18 was only detected in four samples (2.8%). Other high risk types such as HPV51 and HPV81 were not found in any sample (Table I). All co-infections had at least one high-risk HPV type (HPV44, HPV53, HPV56, HPV59 were only found in co-infections). However, seven mono-infected samples had only low-risk types: HPV6 or HPV11 in five samples showing CIN II and CIN III lesions; HPV42 in one CIN II lesion, and HPV43 in a patient with a CIN III lesion. No low-risk genotypes were detected in cancer lesions. HPV16 and HPV58 Subtyping Nucleotide sequences from HPV16 amplicons grouped mostly into the European branch (Lineage A; Fig. 1), and only 1 (amplicon 88M) clustered with the Asian-American variants (sublineages D2/D3). Among sequences with high similarity to European variants (Table II), 14 sequences grouped into sublineage A2 (German sublineage), amplicon 20 M was identical to sublineage A1, and 69 M clustered within lineage A, although it showed one non-synonymous substitution which was also present in lineages B, C, and D (Table II). The majority (83.3%) of HPV58 amplicons grouped into sublineage A2 (Fig. 2) but the sequence from amplicon LM82 showed one synonymous substitution in common with sublineage A3; the amplicon LM51 had one synonymous substitution and four nonsynonymous substitutions which were also present in lineage C sequences. No samples aligned with sublineage A1, lineages B or D (Table III).
TABLE I. Prevalence of HPV Types Found in Cervical Precancerous and Cancerous Lesions (n ¼ 164) in Ecuadorian Women Attending a Large Hospital in Quito. CIN: Cervical Intraepithelial Neoplasia; II and III: Lesion Grade II and Grade III, Respectively CIN II n (%) HPVþ (%) HPV16 (%) HPV58 (%) HPV52 (%) HPV6/11 (%) HPV66 (%) HPV31 (%) HPV43 (%) HPV39 (%) HPV68 (%) HPV35 (%) HPV33 (%) HPV18 (%) HPV59 (%) HPV45 (%) HPV56 (%) HPV53 (%) HPV42 (%) HPV44 (%) HPV51 (%) HPV81 (%)
47 35 13 7 5 4 3 3 3 2 1 1 1 0 1 2 1 0 1 0 0 0
(28.7) (74.5) (37.1) (20.0) (14.3) (11.4) (8.6) (8.6) (8.6) (5.7) (2.9) (2.9) (2.9) (0.0) (2.9) (5.7) (2.9) (0.0) (2.9) (0.0) (0.0) (0.0)
J. Med. Virol. DOI 10.1002/jmv
CIN III
Cancer
62 55 24 20 5 7 4 1 4 1 2 1 2 4 0 0 0 0 0 1 0 0
58 54 22 16 7 2 4 6 3 2 2 2 1 0 2 1 0 1 0 0 0 0
(37.8) (88.7) (43.6) (36.4) (9.1) (12.7) (7.3) (1.8) (7.3) (1.8) (3.6) (1.8) (3.6) (7.3) (0.0) (0.0) (0.0) (0.0) (0.0) (1.8) (0.0) (0.0)
(33.5) (92.7) (43.1) (31.4) (13.0) (3.7) (7.4) (11.1) (5.6) (3.7) (3.7) (3.7) (1.9) (0.0) (3.7) (1.9) (0.0) (1.9) (0.0) (0.0) (0.0) (0.0)
Total 164 141 59 43 17 13 11 10 10 5 5 4 4 4 3 3 1 1 1 1 0 0
(100) (86.0) (41.8) (30.5) (12.1) (9.2) (7.8) (7.1) (7.1) (3.5) (3.5) (2.8) (2.8) (2.8) (2.1) (2.1) (0.7) (0.7) (0.7) (0.7) (0.0) (0.0)
Mono-infections 96 96 39 24 9 5 2 6 1 1 2 1 2 1 0 2 0 0 1 0 0 0
(58.5) (100) (40.6) (25.0) (9.09) (5.05) (2.02) (6.06) (1.01) (1.01) (2.02) (1.01) (2.02) (1.01) (0.0) (2.02) (0.0) (0.0) (1.01) (0.0) (0.0) (0.0)
Co-infections 45 45 20 19 8 8 9 4 9 4 3 3 2 3 3 1 1 1 0 1 0 0
(27.4) (100) (44.44) (42.22) (17.77) (17.77) (20.00) (8.88) (20.00) (8.88) (6.66) (6.66) (4.44) (6.66) (6.66) (2.22) (2.22) (2.22) (0.0) (2.22) (0.0) (0.0)
Total 141 141 59 43 17 13 11 10 10 5 5 4 4 4 3 3 1 1 1 1 0 0
(100) (100) (41.8) (30.5) (12.1) (9.2) (7.8) (7.1) (7.1) (3.5) (3.5) (2.8) (2.8) (2.8) (2.1) (2.1) (0.7) (0.7) (0.7) (0.7) (0.0) (0.0)
HPV Types on Cervical Cancer in Ecuadorian Women
147
Fig. 1. Maximum-likelihood tree using nucleotide sequences of E6 and L1 genes from HPV16 variants obtained in this study (KP794863-KP794878 for E6 sequences and KP794891KP794906 for L1 sequences). Sequences reported here are denoted as CIN (cervical intraepithelial neoplasia) II, CIN III and cancer. The rest of the sequences were obtained from GenBank. Numbers indicate bootstrap values from 1,000
pseudoreplicates. Representative genomes for HPV16 lineages (termed A, B, C and D) and sub-lineages (termed A1-A4/B1-B2/ D1-D3) were used as variant references [Burk et al., 2013]. Regional variants are indicated as: E, European variant; As, East Asian; Afr, African; NA, North American; AA, AsianAmerican.
DISCUSSION
associated with precancerous and cancerous lesions; however, similar results have been described previously in non-cancerous lesions from Ecuador [Tornesello et al., 2008], and in lesions of different severity from Nicaragua and Mexico [Hindryckx 2006; Pi~ naS anchez et al., 2006]. The low prevalence of HPV18 found in the current study is in line with previous findings showing a lower prevalence in South America (only 5% of HPV infections) compared to North America (11%) and Europe (8%) [Clifford, 2005]. Our laboratory has detected HPV18 in 6.67% of 135 HPV positive samples (without cancerous or precancerous lesions) from Ecuadorian women (unpublished data). The reason for these differences in geographic distribution of HPV types is unknown, but some authors have proposed complex interactions between HPV types and HLA haplotypes as a possible explanation [Clifford, 2005]. In the current study we also found that 27.4% of total samples were infected with two or three HPV types. HPV6/11 were detected in cancer lesions but always in co-infections (Table I) with at least one high-risk HPV, which is in accordance with
Prevalence of HPV Genotypes This is the first report describing the distribution of HPV types among Ecuadorian women with cervical precancerous and cancerous lesions. The study contributes to the understanding of HPV epidemiology in Ecuador by showing that HPV16 (40.6% in monoinfections and 44.4% in co-infections) and HPV58 (25.0% and 42.2%, respectively) were more commonly associated with cervical precancer and cancer in Ecuadorian women attending a major hospital in Quito. We also found the same HPV types in 3 samples of vaginal cancer, HPV16 (n ¼ 2) and HPV58 (n ¼ 1), from patients attending the same hospital (data not shown). It was surprising to find a high percentage of precancerous and cancerous lesions associated with HPV58, although similar results have been described in Asia [Huang et al., 1997; Chen et al., 2006], Mexico [Gonz alez-Losa et al., 2004; Pi~ na-S anchez et al., 2006], Nicaragua [Hindryckx, 2006] and Brazil [Fernandes et al., 2013]. It was also surprising to find very little (2.8%) of HPV18
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TABLE II. Nucleotide Polymorphisms in E6 and L1 Genes From HPV16 Variants Obtained in This Study (Accession Numbers for HPV16 E6 Sequences are: KP794863-KP794878, HPV16 L1 Sequences; KP794891- KP794906). The Sequences Reported in This Manuscript Are Denoted as CIN (Cervical Intraepithelial Neoplasia) II, CIN III and Cancer; the Rest of the Sequences Were Obtained From GenBank. Representative Genomes for HPV16 Lineages (Termed A, B, C and D) and Sublineages (Termed A1-A4/B1-B2/D1-D3) Were Used as Variant References [Burk, Harari, and Chen 2013]. Regional Variants are Indicated as: E, European Variant; As, East Asian; Afr, African; NA, North American; AA, Asian-American.
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Fig. 2. Maximum-likelihood tree using nucleotide sequences of E6 and L1 genes from HPV58 variants obtained in this study (KP794879-KP794890 for E6 sequences and KP794907KP794918 for L1 sequences). Sequences reported here are denoted as CIN (cervical intraepithelial neoplasia) II, CIN III and cancer. The rest of the sequences were obtained from GenBank. Numbers indicate bootstrap values from 1,000 pseudoreplicates. Representative genomes for HPV58 lineages (termed A, B, C and D) and sub-lineages (termed A1-A3/B1-B2/ D1-D2) were used as variant references [Chen et al. 2011].
previously published results [zur Hausen, 2002]. As in previous reports [Thomas et al., 2000; zur Hausen 2002], no clear association between specific HPV combinations was observed. Seven precancerous lesions (but no cancerous lesions) were associated with infections with low-risk HPV types (data not shown), and this finding also concurs with other reports [zur Hausen, 2002]. Nevertheless, some authors have suggested that there are genetic defects in the host which may increase the susceptibility to develop cancer even when infected only with low-risk HPV types [Doorbar et al., 2012]. HPV16 and HPV58 Subtype Analysis The majority of HPV16 variants (93.75%) in precancer and cancer lesions were of European lineage and 6.25% belonged to the Asian-American sublineage, which coincides with reports from other LatinAmerican countries [Villa et al., 2000; Hildesheim
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et al., 2001; Picconi et al., 2003; Sichero et al., 2007; Schiffman et al., 2010]. However, one study from Mexico found that 23.8% of the HPV16 variants belonged to European lineages and 23.2% to the Asian-American sublineages [Berumen et al., 2001]. This is relevant because non-European HPV16 sublineages (especially the Asian-American) tend to be more persistent [Villa et al., 2000], more aggressive and more carcinogenic than European counterparts [Berumen et al., 2001; Hildesheim et al., 2001; Sichero et al., 2007; Chen et al., 2011; Burk et al., 2013]. Greater carcinogenicity may be associated with more efficient viral replication (higher copy number per cell), greater p53 degradation by E6 protein [Berumen et al., 2001], better binding and degradation of pRb by E7 protein, induction of genome instability and telomerase activation [Doorbar et al., 2012]. It is interesting that 14 (87.5%) out of 16 samples analyzed contained the polymorphism 350G which has been associated with higher risk of persistence and high grade lesions and cancer [Pande et al., 2008; Huertas-Salgado et al., 2011]; however, the alternative nucleotide found in this site (350T) has also been associated with an increased risk of persistence and cancer [Gheit et al., 2011; Cornet et al., 2013]. These contradictory results suggest that the oncogenic potential of HPV16 variants might be also associated to genetic differences in the host [Zehbe et al., 2001]. The phylogenetic analysis of Ecuadorian HPV58 sequences (12 samples) showed that most variants belonged to sublineage A2 which may be the oldest HPV58 sublineage and from which the other lineages emerged [Chan et al., 2011]. One amplicon sequence clustered with sublineage A3; two substitutions found in the latter sequence have been associated previously with higher risk of cancer: E7 T20I causes greater affinity to the RB protein and E7 G35S creates an additional phosphorylation site which may increase oncogenic activity [Chan et al., 2002]. Assessment of the oncogenic HPV types affecting Ecuadorian women is vital to analyzing the possible local impact of the vaccination program. Both bivalent and tetravalent vaccines have been available for more than 4 years in private settings in Ecuador [Ministerio de Salud P ublica 2014a]; however, our results may predict a lower effectiveness of these vaccines in Ecuador than in other countries with higher HPV18 and lower HPV58 prevalences. Additionally it would be important to monitor mutations in capsid and oncogenic proteins for they will provide information about predicted oncogenicity and vaccination success [Fleury et al., 2014]. The introduction of a 9-valent vaccine may reduce the combined incidence of high grade lesions in cervical/vulvar/ vaginal disease (caused by genotypes 16, 18, 31, 33, 45, 52, and 58) by 96.7% [Joura et al., 2015]. Our research had one limitation which was the small number of samples analyzed because we focused only in precancer and cancer samples. J. Med. Virol. DOI 10.1002/jmv
TABLE III. Nucleotide Polymorphisms in E6 and L1 Genes From HPV58 Variants Obtained in This Study (Accession Numbers for HPV58 E6 Sequences are KP794879- KP794890, and for HPV58 L1 Sequences are KP794907- KP794918). The Sequences Reported in This Manuscript Are Denoted as CIN (Cervical Intraepithelial Neoplasia) II, CIN III and Cancer. The Rest of the Sequences Were Obtained From GenBank. Representative Genomes for HPV58 Lineages (Termed A, B, C, and D) and Sub-Lineages (Termed A1-A3/B1-B2/D1-D2) Were Used as Variant References [Chen et al., 2011].
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Nevertheless, the samples analyzed came from a nationwide oncologic referral center which may allow inference to the distribution of HPV genotypes and variants in different regions of Ecuador. It is also noteworthy that this study is the first report of HPV58 molecular variants done in Ecuador. Pathological characteristics of HPV58 variants and their associations with cancer needs further study. CONCLUSION In conclusion, the overall frequency of HPV types detected in precancerous and cancerous lesions was high for HPV16 and HPV58 and low for HPV18 in Ecuador. The introduction of the current 9-valent vaccine may be more suitable to reduce the combined incidence of high grade lesions and cancer associated with HPV in Ecuador. Additionally, the variants observed in this study were principally European for HPV16 and from sublineage A2 for HPV58. The HPV types and variants reported here were similar to other studies of malignant lesions in other Latin American countries. ACKNOWLEDGMENTS The authors would like to thank Kristin Tomey for her valuable comments and her editing support and Maria Elena Pe~ naranda for her manuscript suggestions. The authors also thank Hybribio Limited (Hong Kong, China) for their kind support in providing “21 HPV GenoArray Diagnostic Kits” for HPV genotyping. REFERENCES Berumen J, Ordonez R M, Lazcano E, Salmeron J, Galvan S C, Estrada R A, Yunes E, Garcia-Carranca A, Gonzalez-Lira G, Madrigal-de la Campa A. 2001. Asian-American variants of human papillomavirus 16 and risk for cervical cancer: A casecontrol study. JNCI J Natl Cancer Inst 93:1325–1330. Bruni L, Barrionuevo-Rosas L, Serrano B, Brotons M, Cosano R, Mu~ noz J, Bosch FX, de Sanjos e S, Castellsagu e X. 2014a. Human papillomavirus and related diseases in Ecuador. Summary Rep http://www.hpvcentre.net/statistics/reports/ECU.pdf. Bruni L, Barrionuevo-Rosas L, Serrano B, Brotons M, Cosano R, Mu~ noz J, Bosch FX, de Sanjos e S, Castellsagu e X. 2014b. Human papillomavirus and related diseases in world. Summary Rep http://www.hpvcentre.net/statistics/reports/XWX.pdf. Burk RD, Harari A, Chen Z. 2013. Human papillomavirus genome variants. Virology 445:232–243. Chan Pa‘ul KS, Ching-Wan Lam, Tak-Hong Cheung, William Li WH, Keith Lo WK, May Chan YM, Jo Cheung LK, Augustine Cheng. F. 2002. Association of Human Papillomavirus type 58 Variant with the risk of cervical cancer. J Natl Cancer Inst 94:1249–1253. Chan PKS, Luk ACS, Park J-S, Smith-McCune KK, Palefsky JM, Konno R, Giovannelli L, Coutl ee F, Hibbitts S, Chu TY, Settheetham-Ishida W, Picconi MA, Ferrera A, De Marco F, Woo YL, Raiol T, Pi~ na-S anchez P, Cheung JL, Bae JH, Chirenje MZ, Magure T, Moscicki AB, Fiander AN, Di Stefano R, Cheung TH, Yu MM, Tsui SK, Pim D, Banks L. 2011. Identification of Human Papillomavirus Type 58 Lineages and the Distribution Worldwide. J Infect Dis 203:1565–1573. Chen C-A, Liu C-Y, Chou H-H, Chou C-Y, Ho C-M, Twu N-F, Kan Y-Y, Chuang M-H, Chu T-Y, Hsieh C-Y. 2006. The distribution and differential risks of human papillomavirus genotypes in cervical preinvasive lesions: A Taiwan cooperative oncologic group study. Int J Gynecol Cancer 16:1801–1808.
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