It is well recognized that the persistent infection of the uterine cervix with a high-risk human papillomavirus (HR-HPV) is a major cause of invasive cervical cancer and its precursor [1]. Infections with HPV-16 and HPV-18 are associated with the highest risk for cervical intraepithelial neoplasia (CIN) 2 or worse (≥CIN 2). HPV-16 and HPV-18 are responsible for 70% of all invasive cervical carcinomas, followed by HPV-45/31/33 [234].

The cytology test, which is used as a cervical cancer screening tool, has been successfully contributing to a decrease in cervical cancer incidence and its associated mortality. However, recent guidelines favor performing HPV DNA testing in addition to the cytology test (co-testing) [4]. Because the cytology test has low sensitivity and reproducibility, adding a HPV DNA test to cytology increases the sensitivity for detecting high grade squamous intraepithelial lesions (HSILs) or invasive cancers. Moreover, the screening interval is extended to every 5 years compared to every 3 years when the cytology test is used alone. In 2015, new interim guidance on HPV primary screening for cervical cancer was established [4]. It recommends primary HPV testing for women who are age 25 or older. For women with HR-HPV (–), follow-up after 3 years is recommended. For women with HPV-16/18 (+), direct colposcopy is advised, and for women with other non-16/18 HR-HPV (+), the cytology test is advised. If the cytology test shows a negative intraepithelial lesion or malignancy (NILM) result, follow-up with both cytology and HR-HPV testing at 12 months is indicated. If the cytology result is atypical squamous cells of undetermined significance (ASCUS) or worse (≥ASCUS), the appropriate treatment guideline is followed [4].

Among the HR-HPV genotypes, some genotypes that are closely related evolutionarily (e.g., HPV-16 and HPV-31) exhibit different degrees of cancer risk, which are thought to be related to different protein functions and patterns of gene expression [5]. Numerous studies have revealed information about the biology and life-cycle of HPV, but these processes are not fully understood. Although the prevalence of HPV and its detected genotypes is variable across the world, HPV-16, -18, -31, -52, and -58 are consistently found worldwide [67].

The U.S. Food and Drug Administration (FDA) has recently approved a recombinant 9-valent HPV vaccine (Gardasil 9, Merck, Kenilworth, NJ, USA) for the prevention of anogenital cancer caused by HPV-16, -18, -31, -33, -45, -52, and -58 and for the prevention of genital warts caused by HPV-6 and -11. Four genotypes are from the licensed Gardasil vaccine (HPV-6, -11, -16, -18), and five are new additions (HPV-31, -33, -45, -52, -58) [89]. The five additional HPV types covered by Gardasil 9 account for approximately 20% of cervical cancers.

Current screening algorithms of both co-testing or HPV primary screening guidelines do not reflect the difference in risk for ≥CIN 2 among women with various non-16/18 HR-HPV genotypes. However, a number of studies revealed that, in Asia (unlike America, Australia, Europe, and Africa), a higher percentage of HPV-52 and HPV-58 is associated with cervical cancer and HPV-52 and HPV-58 are comparable to HPV-16 and HPV-18 in prevalence for invasive cervical cancer [67]. This is consistent with several investigations that studied Korean women [101112]. Previous studies showed that HPV-16, -18, -52, -45, -58, -35, -33, and -31 were strongly associated with cervical cancer and precancer lesions in Asian women, especially Korean women [13]. In this study, we examined the HPV genotype distribution and odds ratios for histologically confirmed ≥CIN 2 for each HR-HPV genotype using a commercially available HPV DNA chip test (HPV 9G DNA chip, Biometrix Technology Inc., Chuncheon, Korea) to test the hypothesis that HPV genotyping can classify non-16/18 HR-HPV infection by risk stratification.

Because HR-HPV infection is a necessary cause of cervical cancer, tests for HR-HPV have been proposed as an adjunctive tool to cervical cytology [16]. In our study, we evaluated whether there are differences in risk among 12 non-16/18 HR-HPV genotypes. There are a number of commercial HPV tests, including nucleic acids hybridization assays, such as in situ hybridization; signal amplification assays, such as Hybrid Capture 2 (Qiagen, Gaithersburg, MD); and nucleic acids amplification, such as microarray and PCR (Cervista [Hologic, Bedford, MA, USA], Cobas 4800 HPV test [Roche Molecular Systems Inc., Pleasanton, CA, USA], and APTIMA [Gen-Probe, San Diego, CA, USA]) [17]. Among them, 9G DNA chip technology is based on the phenomenon of molecular recognition to immobilize oligonucleotides for the production of DNA chips. The 9G probes are immobilized by multiple interactions of the nine consecutive guanines on the aminomethylcoumarin acetate (AMCA) monolayer [14]. The HPV 9G DNA chip test, which has been approved by the Korea Food and Drug Administration (KFDA), displays a promising diagnostic characteristic of 100% clinical sensitivity and specificity, making it a promising diagnostic tool for HPV genotyping [1518]. Considering concordance rate between HPV 9G DNA test and Cobas 4800 HPV test which was approved by FDA, both tests were agreed in 97.2% for detecting HPV-16 and -18. HPV 9G DNA test seemed effective in detecting high risk HPV [19].

In our study, the cytology test showed a higher sensitivity of 95.9% (95% CI, 91.7 to 97.0) compared to that of the HPV 9G DNA chip test, which was 86.3% (95% CI, 82.2 to 89.8) and the HPV 9G DNA chip test showed a higher specificity of 60.0% (95% CI, 58.5 to 61.2) compared to that of the cytology test, which was 41.3% (95% CI, 40.1 to 42.0) (Table 3). These results are opposite of previous findings, which show generally higher sensitivity of HPV test than that of cytology test and lower specificity of HPV test than that of cytology test [20]. It is probably due to different study population such as age and regional distribution. Furthermore, we suspect that there is possibility that a clinician had scraped the cervix less extensively, because he might have been aware of abnormal cytology result from referred clinics. Too small specimen might give more negative influence to the result of cytology than HPV DNA test.

Previous studies reported that some genotypes of high risk HPV are strongly associated with high grade cervical lesions (≥CIN 2). According to Cervical Lesions Observed by Papillomavirus types-A Research in Europe II (CLEOPARE II) study, 12 HPV types were associated with high grade cervical lesion, of which especially HPV-16, -18, -31, -33, -35, -45, -51, -52, and -58 were important in Portugal women [21]. Similarly, HPV-16 was the most strongly associated with ≥CIN 3 cervical lesion and 60.6% of high grade cervical lesions were associated with HPV-31, -33, -45, -52, or -58 in French women [22]. A multicenter, prospective study to evaluate the prevalence of HPV types in Asian women with invasive cervical cancer showed that HPV-16, -18, -52, -45, -58, -33, or -31 were strongly associated with cervical cancer. Among them, HPV-33 was prevalent in Korean cervical cancer women than other Southeast Asian women [13]. These results were similar to our study. In our study, we obtained a statistically significant, age-adjusted odds ratio for ≥CIN 2 for each HR-HPV genotype, except the HPV-39, -59, and -66 genotypes (Table 4). Among the groups of HPV-39, -59, and -66, only five patients were ≥CIN 2. The age-adjusted odds ratio of patients with HPV-16 (+) was 21.7 (95% CI, 13.5 to 34.8) and that of patients with HPV-18 (+) was 8.0 (95% CI, 3.7 to 17.5). We used the odds ratio of a group of HPV-18 (+) patients, which was 8.0, as a cut-off, to categorize the 12 non-16/18 HR-HPV genotypes into the HR group and the LR group. Non-16/18 HR-HPV genotypes were divided accordingly as follows: HPV-31/33/35/45/52/58 (+) and HPV-39/51/56/59/66/68 (+). Non-16/18 HR HPV (+), which initially showed an odds ratio for ≥CIN 2 of 6.6 (95% CI, 4.4 to 0.9) as a whole, showed stratified odds ratios when divided into these two groups: 11.9 (95% CI, 7.6 to 18.8) for HPV-31/33/35/45/52/58 (+) and 2.4 (95% CI, 1.4 to 4.3) for HPV-39/51/56/59/66/68 (+). Although the current guidelines do not reflect this difference in risk, the higher risk HPV-31/33/35/45/52/58 genotypes might need more aggressive actions, such as direct colposcopy, rather than follow-up after 12 months. Large scale clinical trials or cohort studies are necessary especially in Asia, where cervical cancer prevalence is high and where HPV genotyping has been widely used in routine screening. This study has some limitations, (1) the study has a retrospective design based on the pathology archive database records. Selection bias may have occurred because we excluded some subjects with missing data. (2) It is single institution study of referral hospital and has small sample size; therefore, this study may not represent general population. Future prospective studies with larger sample size are needed to confirm our study.

In conclusion, the HPV 9G DNA chip test is a useful tool to detect patient with ≥CIN 2, in addition to cytology test. Furthermore, the 12 non-16/18 HR-HPV genotypes obtained from HPV 9G DNA chip test can be further categorized (HPV-31/33/35/45/52/58 vs. HPV-39/51/56/59/66/68) by risk stratification. The HPV-31/33/35/45/52/58 genotypes might need more aggressive action. Large scale clinical trials or cohort studies are necessary to confirm our suggestion.