Cervical cancer (CC) is the second most common gynecological malignancy worldwide with 569,847 new cases in 2018 and the leading cause of death among gynecologic cancers with 311,365 deaths expected in 2018.1 Human papillomavirus (HPV) is one of the major etiologies in the pathologic course from premalignant lesion to carcinoma, and it has been reported that 90% to 100% of patients with invasive CC were infected with HPV.2
More than 100 different papilloma family members have been reported to exist, and in humans, these are responsible for a distinct variety of benign and malignant conditions. Although genital HPV types have been subdivided into high-risk (HR-HPV) types (frequently associated with invasive CC) and low-risk (LR-HPV) types (found mainly in genital warts), there is still no consensus on categorization of many HPV subtypes with low prevalence according to CC risk. Current epidemiologic data identifies 15 HPV types as high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and 12 (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and CP6108) as low-risk types.3 Oncogenic potential of certain high-risk subtypes, such as 16, 18, 31, and 45, has been demonstrated in initiation or promotion of both cervical intraepithelial neoplasia (CIN) and invasive CCs. Virtually, all cases of CC are caused by HR-HPVs, with HPV 16/18 responsible for 70% to 80% of cases.4,5 Moreover, existing results on the relationship between HPV genotype and survival show that specific subtypes might be related to poor prognosis.6 Identifying women with LR-HPV apparently offers little benefit because these types rarely cause CC, whereas potentially adding the cost of repeat HPV/PAP tests, colposcopies, and biopsies, and unnecessary psychological stress to more women identified as HPV positive. However, LR-HPV 6 and 11 are identified in verrucous carcinoma of vulva, a tumor with local invasive potential that rarely metastasizes.7
Nevertheless, it is theoretically possible that the detection of these types could identify women at higher risk of subsequently acquiring HR-HPV genotypes that do cause CC because they are all identically transmitted through sexual contact.8 Despite the fact that LR-HPV types are not investigated in most laboratories around the world and because our laboratory does examine these types, we find it important to bring the information we collect about LR-HPV types to the public's knowledge.
The objective of the current study was to evaluate the incidence of LR-HPV types among women with CIN 2–3 lesions and CC.
MATERIALS AND METHODS
We conducted a nested cohort study of patients diagnosed with CIN 2–3 or CC from May 2008 to October 2017 at the Cervical Disease Clinic at the Carmel Medical Cancer. The investigation included a colposcopy that was done after application of 5% acetic acid solution. Human papillomavirus DNA testing that included both HR-HPV types and LR-HPV types. Cervical biopsy, endocervical curettage, endometrial sampling, or large loop excision of the transformation zone were performed as indicated. Initial HPV status was determined during the investigation process and was taken directly from the cervix. Cytological findings were classified according to the Bethesda Classification and cervical results were classified as normal, CIN 1, CIN 2, CIN 2–3, or cancer.9 The most severe pathological results from the cervical biopsy or from the large loop excision of the transformation zone operation were recorded. Inclusion criteria were as follows: diagnosis of CIN 2–3 or CC and available HPV-DNA testing. We also collected clinical and demographic data including age, marital status, ethnicity, presenting symptoms (complaints), results of cytology test, HPV type, and final histologic diagnosis. The study protocol was approved on the January 08, 2014, by the ethics review committee of the Carmel Medical Center (Protocol Number CMC 88-0069).
Samples collected from the cervix by brush according to manufacturer's instructions and were transferred to the laboratory for HPV analysis.10 From 2008 to 2014, HPV DNA was extracted from the samples using an automated extractor (easyMag, Biomérieux, Belgium) according to manufacturer's instructions. Human papillomavirus genotype was determined by nested PCR; nucleotide sequencing was performed by Hylabs (Rehovot, Israel). Samples of undetermined genotype were analyzed by a reverse hybridization line probe assay (INNO-LiPA HPV Genotyping Extra, Innogenetics N.V., Belgium). This method enables the detection of 50 HPV types.11 From 2014 to 2017, HPV DNA was determined by HPV Direct Flow CHIP (Master Diagnóstica, Granada, Spain), which is intended for simultaneous screening and genotyping of 36 HPV types: HR-HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82, and low-risk HPV (LR-HPV) 6, 11, 40, 42, 44, 54, 55, 61, 62, 67, 69, 70, 71, 72, 81, 84, and 89 (=CP6108) by PCR, followed by reverse dot blot automatic hybridization, based on DNA-Flow Technology (e-BRID System). Fourteen HPV types were classified as HR-HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Twenty-seven types were classified as LR-HPV 6, 11, 26, 32, 40, 42, 43, 44, 54, 55, 61, 62, 64, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 87, 89, and 91, IS 39, and CP6108.12,13
Data were collected on Microsoft Excel, which was used to generate tables and descriptive statistics. Statistical analyses were performed using IBM Statistical Package for the Social Sciences statistics software, Version 21 (SPSS, IBM Corp, Armonk, NY). Mean, median, and relative proportions are presented in numbers and percentages. Sensitivity, specificity, and positive and negative predictive values were calculated. χ2 test and univariate analysis were used to test for correlation. p values <.05 were considered significant.
During the study period, we collected clinical data on 608 women, of whom 402 were with CIN 2–3 and 206 with diagnosis of CC. Patient demographic and clinical characteristics are presented in Table 1. The mean age of women with CIN 2–3 was 37.7 years (range = 21–85 years) and the mean age of women with CC was 50.6 years (range = 25–94 years). There were no differences noticed between patients with CIN 2–3 and CC regarding marital status or ethnicity. The initial presenting symptom was abnormal Pap among 347 women, of those 294 (73.1%) with CIN 2–3 compared with 53 (25.7%) with CC (p < .001). One hundred sixty patients were diagnosed because of clinical symptoms; of them, 108 (26.9%) with CIN 2–3 and 152 (74.1%) with CC (p < .001). The most common presenting symptom among CIN 2–3 patients was postcoital bleeding (33.3%) compared with postmenopausal bleeding (36.2%) among CC patients (which was present only among 5.6% of women with CIN 2–3, p < .001). The histology type of CC patients was squamous in 161 (78.2%) women, adenocarcinoma in 36 (17.5%), and other types in 9 (4.3%) women.
Among those diagnosed with CC due to abnormal screening Pap test, 77% were diagnosed at early stage compared with 55.3% of those diagnosed due to clinical symptoms (p = .002).
Table 2 describes the distribution of HPV types in women with CIN 2–3 and CC counting single- and multiple-type HPV infection. High-risk HPV types as a single HPV type was detected in 276 (68.7%) women with CIN 2–3 and in 164 (79.6%) women with CC. Multiple HPV types were detected in 87 (21.6%) and in 21 (10.2%) of women with CIN 2–3 and CC, respectively. In all the multiple types in CIN 2–3 women, there was at least one HR-HPV type, so we added those cases, resulting in HR-HPV detected in 363 (90.3%) women with CIN 2–3. In the multiple type CC patients, 21 women had at least one HR-HPV, so HR-HPV was detected in 185 (89.8%) of women with CC (see Table 3).
In CIN 2–3 patients, HPV 16 was detected in 167 (41.5%) women as a single type and in 38 (9.4%) women with multiple HPV types; altogether, HPV 16 was detected in 205 (51%) of women with CIN 2–3 lesions.
In CC women, HPV 16 as a single type was found in 97 (47.1%) women and in multiple types in 10 (4.85%) women; altogether, HPV 16 was detected in 107 (52%) of women with CC. In women with CIN 2–3, HPV 18 was detected in 15 women as a single infection and in 17 women as part of multiple infections; altogether, HPV 18 was found in 32 (8.0%) of CIN 2–3 cases. In CC patients, HPV 18 was detected as a single infection in 19 women (9.2%) and in 6 women (12.1%) with multiple infections. All together HPV 16 and 18 were detected in 237 (58.9%) of CIN 2–3 patients and in 132 (64%) of CC patients. Low-risk HPV types were detected in 18 (4.5%) CIN 2–3 patients and in 8 (3.9%) CC patients.
Human papillomavirus was not detected in 21 (5.2%) and 13 (6.3%) of women with CIN 2–3 and CC, respectively (see Table 3).
The histological type of HPV-negative women was squamous cell carcinoma in 8 women, adenocarcinoma in 4 women, and 1 woman was diagnosed with clear cell cancer.
Among the types included in the new nanovalent vaccine (6, 11, 16, 18, 31, 33, 45, 52, 58), 80.5% of women with CIN 2–3 and 82% of women with CC were found positive to one of those HPV types as single infection or part of multiple types. The distribution of HR-HPV among different age groups is delineated in Table 4. In women with CIN 2–3, most cases of HR-HPV were among women older than 30 years, whereas LR-HPV or negative cases were among women older than 35 years. In patients with CC, most of those positive for HR-HPV were older than 35 years, whereas most cases of LR-HPV or negative HPV were older than 30 years; however, those differences were not significant because of the low number of cases in each age group.
In this cohort of women from one tertiary center that concentrates most of the CIN 2–3 and CC patients from northern Israel, we demonstrated that 4.5% of those with CIN 2–3 and 3.9% of patients with CC were positive to only one LR-HPV and 5.2% of those with CIN 2–3 and 6.3% with CC and were negative to HPV types.
The incidence of LR-HPV among patients with CC has been evaluated. In a global assessment of HPV distribution among CC patients, de Sanjose et al.11 included 10,575 cases of invasive CC and found 3.5% cases with LR-HPV. In a report by Tjalma et al.,14 more than 6,000 women diagnosed with CIN 2–3 or CC from 17 European countries were enrolled in a cross-sectional study. A total of 3,103 women were diagnosed with CIN 2–3 and a total of 3,162 with CC, of whom 1.5% and 8.2% were HPV negative, respectively.14 In both studies, HPV was detected between 85% and 91.8% of cancer cases. In our study, 93.7% of CC patients were positive for any HPV type and 3.9% had only LR-HPV. In the CIN 2–3 women, HPV was detected in 94.8% of them with 4.5% positive for LR-HPV. In a previous study, we describe the prevalence of HPV types in 6,654 high-risk women, who were referred because of atypical squamous cells of undetermined significance (ASCUS) results on Pap tests or because of complaints suggestive of cervical neoplasia. Low-risk HPV was detected in 8.1% of women with ASCUS, 11.4% women with low squamous intraepithelial lesion (LSIL), 2.9% women with CIN 2–3, and 2.1% women with CC.15
Screening by HPV-DNA testing that includes only 14 HR-HPV types is gaining popularity and has been adopted by some as a reliable screening program instead of cytology, with increased sensitivity but decreased specificity.16,17
One important issue determining which HPV types should be screened for. Testing for only the HPV types that cause most of the cancers might increase specificity while preserving sensitivity. In screening programs based on testing for 12 to 14 HR-HPV types, a negative test usually indicates a low risk for CIN 2–3 or CC for up to 18 years.18–20 Moreover, previous prospective studies that estimated the absolute risk of CIN 3 or worse21,22 or invasive CC22 after LR-HPV infection also found that risks were low22,23 or zero.21,24
Sundström et al.25 found that women with LR-HPV had a statistically significantly increased risk for CIN 2–3 detection but not increased risk for CC compared with HPV-negative women.
The prevalence of CC and CIN 2–3 among HPV-negative women was also evaluated. In a large-scale community-based cohort of 10,123 women for 16 years to investigate the role of genotype-specific HPV persistence in predicting CC including invasive and in situ carcinoma, Chen et al.23 found the 16-year cumulative risks of subsequent CC for HPV-negative women was 0.26% compared with 4% to 13.5% for the HR-HPV–positive women.
Despite the above, and despite the popularity and high sensitivity of DNA-based screening, it should be taken with caution, because some women might have LR-HPV and some might be negative. Some types of adenocarcinoma of the cervix are considered to be HPV-negative cancers. In a retrospective study from China that included 630 patients with squamous CC and 718 patients with adenocarcinoma, HPV was negative among 2.4% and 25.5% of the cases, respectively.26 Others showed that of 371 CC patients, 31 (8.3%) were negative for HR-HPV. After performing surgical staging, the diagnosis of CC was found to be wrong in 21 (68%) of 31 cases. Non–HR-HPV subtypes were detected in 5 cases. They concluded that the main explanation for HPV-negative CC was a false diagnosis, followed by cancers associated with non–HR-HPV types.27 In our study, we also addressed age and noticed that most cases of CIN 2–3 were older than 30 years, preceding the cases of CC by 5 to 10 years, indicating the length of time from persistent HR-HPV infection to CC as previously studied.28 In our study, we look at the distribution of LR-HPV among CIN 2–3 and CC patients to see that most cases of both are older than 35 years.
The HPV nanovalent vaccine is aimed at the 9 strains of HR-HPV types related to CIN 2–3 and cancer and includes types 16, 18, 11, 6, 31, 33, 45, 52, and 58.29 This vaccine has the potential to prevent 80.5% of cases of CIN 2–3 and 82% of cases of CC in our population. Moreover, 6.3% of CC patients were HPV-negative, and 3.9% were positive to LR-HPV but negative to HR-HPV, which raises the question if we should increase the spectrum of HPV types being analyzed to include LR-HPV to improve the sensitivity of HPV screening. Another unanswered question is if we should treat CIN 2–3 women with LR-HPV (4.5% in our study), who rarely progress to invasive cancer, similarly to CIN 2–3 patients with HR-HPV.
The importance of LR-HPV in CIN 2–3 and CC will higher in future after the bi-, quadri-, and nanovalent vaccines decrease neoplasia due to HR-HPV types and accordingly lead to an increase of LR-HPV incidence. Although HR-HPV negative in high-grade squamous intraepithelial lesion or CC cannot be ignored, it does not mean that screening has failed but that it should be taken with the clinical context. More cost-effectiveness studies are needed to decide whether LR-HPV types should be added to the HPV tested in the screening programs.
Our study is not clear of limitations. First, its retrospective nature makes it susceptible to selection bias; second, we had no data regarding other risk factors of premalignant and malignant lesions for CC, including smoking, socioeconomic status, sexual history, and sexually transmitted diseases.
The prevalence of the LR-HPV in high-grade squamous intraepithelial lesion cervical lesions is low but is expected to increase in the future because of the expected decrease in CC caused by HPV types that are included in the bi-, quadri-, and nanovalent vaccine. The CIN 2–3 and CC patients with LR-HPV types and with negative HPV challenge HPV screening sensitivity, which is based on a limited number of HR-HPV types.
1. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin
2. Bosch FX, Lorincz A, Munoz N, et al. The causal relation between human papillomavirus and cervical cancer
. J Clin Pathol
3. Abreu AL, Souza RP, Gimenes F, et al. A review of methods for detect human papillomavirus infection. Virol J
4. Das P, Thomas A, Kannan S, et al. Human papillomavirus (HPV) genome status & cervical cancer
outcome—a retrospective study. Indian J Med Res
5. Saranath D, Khan Z, Tandle AT, et al. HPV16/18 prevalence in cervical lesions/cancers and p53 genotypes in cervical cancer
patients from India. Gynecol Oncol
6. Hang D, Jia M, Ma H, et al. Independent prognostic role of human papillomavirus genotype in cervical cancer
. BMC Infect Dis
7. Prat J, Mutch DG. Pathology of cancers of the female genital tract including molecular pathology. Int J Gynaecol Obstet
8. Schiffman M, Castle PE, Jeronimo J, et al. Human papillomavirus and cervical cancer
9. Davey DD. Cervical cytology classification and the Bethesda System. Cancer J
10. Fuessel Haws AL, He Q, Rady PL, et al. Nested PCR with the PGMY09/11 and GP5(+)/6(+) primer sets improves detection of HPV DNA in cervical samples. J Virol Methods
11. de Sanjose S, Quint WG, Alemany L, et al. Human papillomavirus genotype attribution in invasive cervical cancer
: a retrospective cross-sectional worldwide study. Lancet Oncol
12. Poynor EA, Barakat RR, Hoskins WJ. Management and follow-up of patients with adenocarcinoma in situ of the uterine cervix. Gynecol Oncol
13. Clifford GM, Smith JS, Plummer M, et al. Human papillomavirus types in invasive cervical cancer
worldwide: a meta-analysis. Br J Cancer
14. Tjalma WA, Fiander A, Reich O, et al. Differences in human papillomavirus type distribution in high-grade cervical intraepithelial neoplasia and invasive cervical cancer
in Europe. Int J Cancer
15. Siegler E, Shiner M, Segev Y, et al. Prevalence and genotype distribution of HPV types in women at risk for cervical neoplasia in Israel. Isr Med Assoc J
16. Castle PE, de Sanjosé S, Qiao YL, et al. Introduction of human papillomavirus DNA screening in the world: 15 years of experience. Vaccine
17. Ronco G, Dillner J, Elfstrom KM, et al. Efficacy of HPV-based screening for prevention of invasive cervical cancer
: follow-up of four European randomised controlled trials. Lancet
18. Mesher D, Szarewski A, Cadman L, et al. Long-term follow-up of cervical disease in women screened by cytology and HPV testing: results from the HART study. Br J Cancer
19. Dillner J, Rebolj M, Birembaut P, et al. Long term predictive values of cytology and human papillomavirus testing in cervical cancer
screening: joint European cohort study. BMJ
20. Katki HA, Kinney WK, Fetterman B, et al. Cervical cancer
risk for women undergoing concurrent testing for human papillomavirus and cervical cytology: a population-based study in routine clinical practice. Lancet Oncol
21. Schiffman M, Herrero R, Desalle R, et al. The carcinogenicity of human papillomavirus types reflects viral evolution. Virology
22. Castle PE, Solomon D, Schiffman M, et al. Human papillomavirus type 16 infections and 2-year absolute risk of cervical precancer in women with equivocal or mild cytologic abnormalities. J Natl Cancer Inst
23. Chen HC, Schiffman M, Lin CY, et al. Persistence of type-specific human papillomavirus infection and increased long-term risk of cervical cancer
. J Natl Cancer Inst
24. Thomsen LT, Frederiksen K, Munk C, et al. High-risk and low-risk human papillomavirus and the absolute risk of cervical intraepithelial neoplasia or cancer. Obstet Gynecol
25. Sundström K, Ploner A, Arnheim-Dahlström L, et al. Interactions between high- and low-risk hpv
types reduce the risk of squamous cervical cancer
. J Natl Cancer Inst
2015;107. pii: djv185.
26. Chen W, Sun H, Molijn A, et al. The variable characteristics of human papillomavirus in squamous cell carcinoma and adenocarcinoma of cervix in China. J Low Genit Tract Dis
27. Petry KU, Liebrich C, Luyten A, et al. Surgical staging identified false HPV-negative cases in a large series of invasive cervical cancers. Papillomavirus Res
28. Kjaer SK, Munk C, Junge J, et al. Carcinogenic HPV prevalence and age-specific type distribution in 40,382 women with normal cervical cytology, ASCUS/LSIL, HSIL, or cervical cancer
: what is the potential for prevention? Cancer Causes Control
29. Van Damme P, Bonanni P, Bosch FX, et al. Use of the nonavalent HPV vaccine in individuals previously fully or partially vaccinated with bivalent or quadrivalent HPV vaccines. Vaccine