High-risk Human Papillomavirus Testing in Young Japanese Women with Atypical Squamous Cells of Undetermined Significance.

INTRODUCTION: The mortality due to uterine cervical cancer has been gradually increasing in women under 40 years of age (U40) in Japan. We investigated the effect of high-risk human papillomavirus (HR-HPV) on U40 subjects without any overt cytological abnormalities.
MATERIALS AND METHODS: We retrospectively examined the clinical data, including the findings of a cobas 4800 HPV test that was approved in Japan in 2013 to triage women with atypical squamous cells of undetermined significance (ASC-US) and a histological examination in 589 Japanese women.
RESULTS: The overall prevalence rate of HR-HPV was 34.5%. Biopsy-confirmed cervical intraepithelial neoplasia (CIN) 2, or worse (CIN2+) was identified in 45.1% (23/51) of HR-HPV-positive women with ASC-US, who underwent colposcopy immediately. The mean period from the HPV test to the diagnosis of CIN2+ was 3.7 months. CIN2+ was more common (69.6%) in U40 patients. The rates of single or multiple infections of HPV-16, HPV-18, and 12 other HR-HPV (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) in CIN2+ U40 patients were 31.3%, 0%, and 81.3%, respectively. The relative risk for CIN 2+ among U40 women with HPV-16 was not significantly different from that of the patients with infection of any of the 12 other HR-HPVs.
CONCLUSION: The results of this study suggest that the 12 other HR-HPVs have a potential to generate high-grade cervical lesions among young women, and the examination rate of colposcopy should be increased.

INTRODUCTION

Cervical cancer is the fourth-most common cancer in women worldwide, and the most common cancer in women in many low- and middle-income countries.[12] In December 2003, the European Council adopted a recommendation to implement population-based screening for cancer of the uterine cervix for women in all member states of the European Union (EU).[3] A significant decline in the incidence and mortality has been seen in North America, parts of Europe, Australia, and New Zealand, where screening programs have been implemented.[4]

There is little doubt that well-organized cytology-based screening programs for cervical cancer have been effective in reducing the cancer incidence and preventing premature deaths. Potential reductions in disease of 60%–90% are possible within 3 years after implementing screening programs.[567] However, a previous study found that 47% of fully invasive cancers in women under the age of 70 occurred despite an apparently adequate screening history,[6] suggesting problems with the sensitivity of the test. Human papillomavirus (HPV) testing was subsequently found to be more sensitive than cytology, leading to a recommendation that HPV testing be introduced on a pilot basis for women with borderline and mild smears.[7] HPV testing is currently recommended to triage women with atypical squamous cells of undetermined significance (ASC-US) in the United States and as an adjunct to cytology (cotesting).[8] In Europe, the guidelines recommend the use of HPV testing to triage women with ASC-US, for surveillance after treatment of cervical intraepithelial neoplasia (CIN) and as a stand-alone primary screening test without cytology for cervical cancer screening (HPV primary screening).[9]

The mortality due to uterine cervical cancer has been gradually increasing in women under 40 years of age (40) in Japan in recent years. To assess the correlation between ASC-US and HPV type, the ATHENA study, in which the cobas 4800 HPV test (Roche Molecular Systems, Pleasanton, CA, USA) was performed, showed that the relative risk (RR) for CIN2 or worse (CIN2+) among women who were HPV-16- and HPV-18-positive, including multiple infections, were 42.0 (95% CI, 20.1-87.5) and 5.8 (95% CI, 1.3-26.5), respectively, compared with those who were HR-HPV-negative (14 types). The RR for CIN2+ among women who were HPV-16-positive was 3.7 (95% CI, 2.4-5.7) compared with those who were positive for the 12 other HR-HPVs, excluding HPV-16 and HPV-18.[10] The cobas test was approved in Japan in 2013 to triage women with ASC-US.

To investigate whether or not HR-HPV is correlated with subsequent CIN2+ in young Japanese women with ASC-US, we retrospectively examined the results of cytology, the cobas 4800 HPV test, and follow-up data, including the findings from a histological examination.

MATERIALS AND METHODS

Study population

This study retrospectively assessed a cohort of the women with both ASC-US and high-risk (HR) HPV at four centers in Hokkaido Prefecture, Japan. We enrolled 589 women ≥20 years of age undergoing cervical cancer screening from 2013 to 2016. The study inclusion criterion was no treatment for CIN in the past history. As a rule, we recommended women with HR-HPV-positive ASC-US undergo colposcopy. The vaccination history was not considered. The protocol was approved by the institutional review boards (Registration ID 2016-10).

Cytological testing, HPV testing, and colposcopy

Cytology was performed with either a Papanicolaou smear or liquid-based cervical cytology (ThinPrep, Hologic, Bedford, MA, USA). The cytology evaluation was conducted by full-boarded cytoscreeners and pathologists at each center. All HPV tests were performed within 6 weeks after cytology with a cobas 4800 HPV test. In brief, the cobas HPV test is according to two major processes: (1) automated specimen preparation to simultaneously extract HPV and cellular DNA; (2) PCR amplification of target DNA sequences using both HPV and beta-globin specific complementary primer pairs and real-time detection of cleaved fluorescent-labeled HPV and beta-globin specific oligonucleotide detection probes. A 2-ml aliquot removed from the liquid-based cervical cytology specimen was tested to detect HPV-16 individually, HPV-18 individually, and 12 pooled HR-HPV genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). For participants with only a Papanicolaou smear, a liquid-based specimen was obtained again after the results of cytology were revealed. The specimens for cytology were obtained by general gynecologists, and PCR was performed by laboratory medical technologists. The results of a cobas 4800 HPV were revealed within 3 weeks. We routinely recommend colposcopy with a simultaneous biopsy and/or endocervical curettage within 12 weeks after HPV testing in accordance with a standardized protocol that included a biopsy of all visible cervical lesions. For women with satisfactory colposcopy findings but no visible cervical lesions, a random biopsy at the squamocolumnar junction was performed. Biopsy specimens were evaluated by board qualified pathologists at each center and diagnosed using standard criteria and CIN terminology.

RESULTS

Baseline characteristics

We retrospectively investigated 589 women with ASC-US cytology in this study and 64.0% of them were U40. The average age was 45.4 years (range: 20–82 years). The average gravidity and parity were 1.8 (range: 0–8) and 1.4 (range: 0–5), respectively.

Human papillomavirus test and colposcopy/biopsy results

The overall prevalence of HR-HPV (14 genotypes), HPV-16, HPV-18, and any of the 12 other HR-HPVs, including multiple infection, were 34.3% (202/589), 6.6% (39/589), 1.5% (9/589), and 27.2% (160/589), respectively. The prevalence of HR-HPV (14 genotypes) was 63.6%, 46.5%, 30.4%, 20.7%, 22.6%, and 17.8% for subjects in their 20s, 30s, 40s, 50s, 60s, and 70s or older, respectively, declining with increasing age. The most common type was any of the 12 other HR-HPVs, and the prevalence was 50.6%, 33.3%, 23.4%, 19.8%, 18.9%, and 15.6% for subjects in their 20s, 30s, 40s, 50s, 60s, and 70s or older, respectively [Table 1].

Table 1

The correlation between the HR-HPV* type and age in women with ASC-US‡

Age	70 - 79	45	17.8% (8)	0.0% (0)	2.2% (1)	0.0% (0)	0.0% (0)	0.0% (0)	15.6% (7)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	82.2% (37)	0.0% (0)
	60 - 69	53	22.6% (12)	0.0% (0)	1.9% (1)	0.0% (0)	1.9% (1)	0.0% (0)	18.9% (10)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	0.0% (0)	77.4% (41)	0.0% (0)
	50 - 59	116	20.7% (24)	1.7% (2)	0.9% (1)	0.0% (0)	0.0% (0)	0.0% (0)	17.2% (20)	0.9% (1)	0.0% (0)	0.0% (0)	1.7% (2)	0.9% (1)	0.9% (1)	0.0% (0)	0.0% (0)	0.0% (0)	79.3% (92)	0.0% (0)
	40 - 49	184	30.4% (56)	2.7% (5)	4.3% (8)	1.1% (2)	0.5% (1)	0.0% (0)	21.2% (39)	1.1% (2)	0.0% (0)	0.0% (0)	1.6% (3)	0.5% (1)	0.5% (1)	0.0% (0)	2.2% (4)	0.0% (0)	69.6% (128)	0.0% (0)
	30 - 39	114	46.5% (53)	8.8% (10)	8.8% (10)	2.6% (3)	1.8% (2)	0.0% (0)	29.8% (34)	5.3% (6)	0.0% (0)	0.0% (0)	2.6% (3)	0.9% (1)	0.9% (1)	0.0% (0)	2.6% (3)	0.0% (0)	53.5% (61)	0.0% (0)
	20 - 29	77	63.6% (49)	7.8% (6)	6.5% (5)	0.0% (0)	1.3% (1)	0.0% (0)	42.9% (33)	6.5% (5)	0.0% (0)	0.0% (0)	6.5% (5)	1.3% (1)	1.3% (1)	0.0% (0)	5.2% (4)	0.0% (0)	36.5% (28)	0.0% (0)
	Overall	589	34.3% (202)	3.9% (23)	4.4% (26)	0.8% (5)	0.8% (5)	0.0% (0)	24.3% (143)	2.4% (14)	0.0% (0)	0.0% (0)	2.2% (13)	0.7% (4)	0.7% (4)	0.0% (0)	1.9% (11)	0.0% (0)	65.7% (387)	0.0% (0)
			Overall	CIN2+†	Overall	CIN2+	Overall	CIN2+	Overall	CIN2+	Overall	CIN2+	Overall	CIN2+	Overall	CIN2+	Overall	CIN2+	Overall	CIN2+
	% (n)		HR-HPV positive		HPV-16		HPV-18		12 other		HPV-16 and HPV-18		HPV-16 and 12 other		HPV-18 and 12 other		HR-HPV positive subtype NA§		HR-HPV negative

HR-HPV* high-risk human papillomavirus; ASC-US‡ atypical squamous cells of undetermined significance; CIN2+† cervical intraepithelial neoplasia 2 or worse; NA§ not applicable, HPV-16 includes positivity for HPV-16 without HPV-18 or any of the 12 other HR-HPVs, HPV-18 includes positivity for HPV-18 without HPV-16 or any of the 12 other HR-HPVs, “12 other” includes positivity for any of the 12 other HR-HPVs without HPV-16 or HPV-18

Fifty-one of the 202 subjects with positive HPV test results immediately underwent colposcopy. Biopsy-confirmed CIN2+ was identified in 45.1% (23/51) of HR-HPV positive women with ASC-US. CIN2+ was more common in the subjects U40 than in older ones (69.6% vs. 30.4%). The correlations between HPV type and CIN2 + by age in women with ASC-US are shown in [Table 1]. The most common type was any of the 12 other HR-HPVs without HPV-16 or HPV-18, especially in the subjects U40. The rates of single infection of HPV-16, HPV-18, and any of the 12 other HR-HPVs or multiple infections of HPV-16, HPV-18, and 12 other HR-HPV in CIN2+ patients were 21.7% (5/23), 0% (0/23), 60.9% (14/23), and 17.4% (4/23), respectively, in all women with biopsy -confirmed CIN2+. For women with biopsy-confirmed CIN2+ U40, the rates of single infection of HPV-16, HPV-18, and any of the 12 other HR-HPVs or multiple infections of HPV-16, HPV-18, and 12 other HR-HPV in CIN2+ patients were 18.8% (3/16), 0% (0/16), 68.8% (11/16), and 12.5% (2/16), respectively. The odds ratio for CIN2+ with single or multiple infections of HPV-16 versus single or multiple infections of any of the 12 other HR-HPVs was 2.9 (95% CI 0.5-17.5) in women U40 and 1.9 (95% CI 0.3-12.3) in women over 40 years of age, respectively.

DISCUSSION

The accuracy of cytology depends on multiple factors, including sampling errors,[7] as well as the skill of individual screeners.[11] On the basis of the evidence concerning the role of oncogenic HPV infections in the development of cervical cancer and CIN,[12] HPV testing has been proposed as a triage method for identifying women at increased risk of cervical cancer requiring a referral for colposcopy among those with ASC-US.[13] Several HPV screening tests are available for ASC-US triage. The HC2 (Qiagen) and Aptima HPV assay (HOLOGIC) detect 13 (16/18/31/33/35/39/45/51/52/56/58/59/68) or 14 (16/18/31/33/35/39/45/51/52/56/58/59/66/68) HR-HPV, respectively and do not discriminate among high-risk types. In contrast, the cobas 4800 HPV test and Abbott RealTime HPV (Abbott) detect HPV-16 individually, HPV-18 individually, and 12 pooled HR-HPV genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). In Japan, all of these tests cost about $30 USD, resulting in a final patient burden of about $10 USD with national health insurance coverage. Previous reports have shown that the HR-HPV prevalence as determined by the cobas 4800 test ranged from approximately 30% to 45%[1014] and declined with age[151617] in women with ASC-US; we obtained similar results to those previous reports.

In general, when HR-HPV is divided into three categories of HPV-16, HPV-18, and other HR types, the positivity is highest for other HR types, followed by HPV-16 and HPV-18, among women with ASC-US.[101418] A previous meta-analysis showed that the average risk for a high-grade cervical lesion is higher in HPV-16- and HPV-18-positive women with ASC-US than in HR-HPV-positive but HPV-16- and HPV-18-negative women with ASC-US.[19] However, more recent reports have shown that infection with non-16/18-HR-HPVs is more common in women with biopsy-confirmed, high-grade squamous intraepithelial lesions after a cytological diagnosis of lower-grade cervical lesions, including low-grade squamous intraepithelial lesion and ASC-US.[20] Our results are similar to those of these later reports. However, because there were no age-specific data in the meta-analysis, young women with CIN 2+ lesions in that report may have had a higher infection rate of non-16/18 HR-HPV than HPV-16 and HPV-18.

This study showed that a diagnosis with any of the 12 other HR-HPVs using the cobas test are more likely than that of HPV-16 or HPV-18 in women diagnosed with ASC-US, especially in those U40. This study also showed that the 12 other HR-HPVs were most frequently found in CIN2+ lesions, regardless of single or multiple infections with HPV-16.

Previous reports have shown that CIN2+ declines with age,[2122] including young women of reproductive age.[23] However, the mortality rate due to uterine cervical cancer has been gradually increasing in women U40 in Japan in recent years,[24] suggesting both insufficient screening and the fact that some cervical lesions progress in young women without demonstrating any overt cytological abnormalities. Although HPV16 is strongly correlated with carcinogenesis of the female genital tract,[25] the results of this study suggest that the 12 other HR-HPVs also have a great potential to generate high-grade cervical lesions in young women with low rates of cytological abnormalities. Further studies in larger cohorts are needed to clarify the mechanisms that induce the development of CIN in young women with HR-HPV infection and a history of being vaccinated for HPV.

CONCLUSION

Infections of HR-HPVs without HPV-16 or HPV-18 have a great potential to generate high-grade cervical lesions in young Japanese women with low rates of overt cytological abnormalities. The examination rate of colposcopy should be increased in Japanese women with HR-HPV-positive ASC-US.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.