Prevalence of cervical precancers or cancers in women with ASC-H/HSIL cytology according to Aptima HPV (AHPV) assay-detected HPV genotypes and age

Objective: Aimed to potentially risk-stratify patients with different cervical cytology diagnoses, by HPV genotypes and/or age, we have conducted a series of studies to examine the prevalence of cervical precancers and cancers for women with different cytology diagnoses. This paper will be focusing on patients with ASC-H/HSIL cytology. Methods: In total, 1183 patients aged 20-78 years with atypical squamous cells, cannot rule out HSIL (ASC-H)/HSIL by cytology underwent AHPV assay and cervical biopsy in a developed region in southern China were included in this study. Results: Overall, 59.2% women with ASC-H/HSIL cytology had cervical intraepithelial neoplasia (CIN)2/3 lesions while 1.6% had adenocarcinoma in situ (AIS) lesions. Compared to other groups, HPV-16+ group (80.8%) showed a significantly higher prevalence of CIN2/3 than other genotype+ groups (p<0.0001). Further, HPV-16+ (9.3%) or HPV-18/45+ (6.3%) group showed a significantly higher prevalence of squamous cell carcinoma (SCC) than other genotype+ groups (p<0.0001). The prevalence of AIS glandular lesions in HPV-18/45+ group (13.8%) is significantly higher than other genotype groups (p<0.0001). When stratified by age, younger group showed a significantly higher prevalence of CIN2/3 (p=0.009) while older group presented an obvious higher prevalence of SCC (p<0.0001). Conclusions: In this patient population, among women with ASC-H/HSIL cytology, HPV positive groups are at significantly higher risk of CIN2/3 compared to HPV negative group. Specifically, prevalence of CIN2/3 and SCC is significantly higher in HPV-16+ group while AIS lesions are more prevalent among HPV-18/45+ patients. In addition, younger group showed a significantly higher prevalence of CIN2/3 while older group presented an obvious higher prevalence of SCC.


Introduction
Cervical cancer is a major gynecological malignancy that typically develops from the progression of cervical precancerous lesions.In most cases, chronic infection with high-risk types human papillomavirus (hrHPV) is necessary for the cervical precancerous lesion and cancer development [1,2].

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Although cervical cytology screening programs are widely used, they have shown relatively low sensitivity and inter-observer consistency [3][4][5].Consequently, co-testing, which combines cervical cytology and hrHPV testing, has been recommended as the screening regimen by American Society for Colposcopy and Cervical Pathology (ASCCP) [6].Current HPV detection methods detect HPV DNA and/or HPV mRNA [7][8][9][10].For example, the Aptima HPV (AHPV) assay magnifies E6/E7 mRNA transcripts that are linked to persistent HPV infections, and this method has a better specificity for detecting high-grade squamous intraepithelial lesions (HSIL+) than DNA-based assays [11].Therefore, AHPV testing is considered better suited as the primary cervical cancer screening method [12].
HSIL (High-grade squamous intraepithelial lesion) exhibits pronounced nuclear abnormalities, such as enlarged irregular nuclei, significant hyperchromasia with coarse and uneven chromatin, and increased nuclear-cytoplasmic ratio.ASC-H (Atypical squamous cells -cannot exclude high-grade squamous intraepithelial lesion), on the other hand, displays similar atypical cytological features but to a lesser degree qualitatively and/or quantitatively.From a molecular perspective, HSIL is strongly associated with human papillomavirus (HPV) infection.Common molecular alterations observed in HSIL cells encompass the overexpression of viral proteins E6/E7 and enhanced expression of p16INK4a protein [13].Techniques like immunohistochemistry can be employed to detect these molecular features.To summarize, ASC-H and HSIL present distinct morphological characteristics in cytological images, with HSIL demonstrating more prominent nuclear abnormalities.While most cervical cytology abnormalities are not severe and will not develop into cancer or its precursors, high-grade cervical cytology abnormalities, most of which were positive for hrHPV, have a higher risk of precancerous and cancerous cervical lesions [14], and these patients often undergo immediate treatment [6].However, different genotypes of hrHPV carry different risks of developing high-grade lesions [15].In our previous studies, we demonstrated the benefit of risk-stratifying patients with NILM and ASC-US cytology based on hrHPV genotype for optimizing patient management [16,17].However, there is limited research on the association between ASC-H/HSIL cytology and hrHPV infection using Aptima assays, particularly on a large scale in China.Therefore, the aim of this study was to investigate the prevalence and the distribution of hrHPV among women with ASC-H/HSIL cytology, to evaluate the immediate histological risk in women with ASC-H/HSIL cytology stratified by age and HPV genotypes based on Aptima assays, and to assess the applicability of the ASCCP guidelines in the population of women from developed southern China.

Population study
In total, 293920 women with cytology results were recorded in our pathology database between September 2016 and May 2020 at the Zhejiang University School of Medicine Women's Hospital.As demonstrated in Figure 1, a total of 1183 women aged between 20 and 78 years who were previously diagnosed as ASC-H/HSIL cytology with concurrent hrHPV results and underwent follow-up biopsy according to the American Society of Colposcopy & Cervical Pathology (ASCCP) guidelines [18,19].Inclusion Criteria: Females with a history of sexual activity who are non-pregnant; all subjects with a clear diagnosis of cervical biopsy pathology; all subjects underwent AHPV testing and TCT testing, with results indicating ASC-H/HSIL; subjects with no autoimmune disorders.Exclusion Criteria: subjects with incomplete clinical data or unreliable follow-up; subjects with history of hysterectomy or malignant tumors; subjects with history of other malignancies or pelvic radiotherapy/chemotherapy. The colposcopists were informed of the cytology and AHPV results before the colposcopy visit.The permission was obtained from the ethics committee of the Zhejiang University School of Medicine Women's Hospital.

Colposcopy, cervical biopsy and follow-up histopathological diagnoses in patients
The cervical tissue samples obtained during colposcopy were paraffin embedded and sectioned, and the slides were dewaxed and stained with hematoxylin and eosin.Finally, the slides were read by two senior pathologists in the pathology department.The histopathological results included three general groups: (1) Benign; (2) CIN1; (3) CIN2/3; (4) dysplastic cervical glandular lesion including AIS and ADC.CIN2/3 and AIS lesions were regarded as cervical precancers.p16/Ki-67 immunohistochemical staining assays were used to verify CIN2/3.If different sites or multiple biopsies, the highest pathological grade is recorded as the final diagnosis.

Statistical analysis
The database was established by Excel and the data were statistically analyzed by SPSS 23.0 software (IBM).Pearson χ2 or Fisher exact test was used to compare the distribution of cervical precancers or cancers in different hrHPV types and age groups, with the histopathological results used as the gold standard.A p<0.05 was considered statistically significant.

Comparison of precancer and cancer in different age groups among women with ASC-H/HSIL cytology
Among different age groups, prevalence of the hrHPV types and histopathological results were further compared.Among the 1183 ASC-H/HSIL cases with following histopathological results, 1.6% (19/1183) patients were younger than 25  ).When we chose 40 years or 50 years as the age cutoffs for further analysis, the prevalence of CIN2/3 was significantly higher in younger group than that in older group (p<0.0001),while SCC prevalence is significantly higher in older age groups than in younger (p<0.0001).However, there were no significant differences for glandular lesions among different age groups.Detailed analysis is included in tables 4 and 5.

Discussion
In this study, our objective was to investigate the prevalence of cervical precancers or cancers (HSIL, AIS, SCC and adenocarcinoma) for women with ASC-H/HSIL cytology stratified by AHPV assay-detected HPV genotypes or age in a developed region in southern China.In total, 59.2% women with ASC-H/HSIL cytology had cervical intraepithelial neoplasia (CIN)2/3 lesions while 1.6% had adenocarcinoma in situ (AIS) lesions.One 2015 large sample study from China on HSIL and hrHPV infection showed that the CIN2+ rate in women with HSIL cytology within 6 months was 82.6% (1994/2414) [20].Another recent study reported that the following CIN2+ rates in cases with abnormal cytology: ASC-US (18.58%),ASC-H (53.76%),LSIL (16.62%),HSIL (82.07%),SCC/AC (100.00%),AGC (63.77%) [21].A study conducted in a South Korean population demonstrated an 83.5% CIN2+ rate in HSIL cytology [22].In our study, the prevalence of CIN2/3 in cases with ASC-H and HSIL cytology were 49.8% and 79.2%, respectively, which was lower than the rates reported in the aforementioned studies.Factors contributing to this difference may include variations in examination methods, screening populations (economically developed region vs. developing region) and the increasing prevalence of cervical cancer screening in recent years.A study conducted in Shanghai, a region with a similar economic status to our study population, showed that 71.5% of patients with HSIL cytology were diagnosed with CIN2+ lesions, including 3.2% with invasive squamous cell carcinoma (SCC) [23].Additionally, they conducted another study revealing a CIN2+ rate of 76.8% in hrHPV mRNA-positive cases [15].Similarly, our study observed a CIN2+ rate of 66.1% in hrHPV mRNA-positive cases.
Our study represents the first analysis of HSIL cytology and AHPV results based on a large sample in China.Among the 1183 cases with ASC-H/HSIL cytology, hrHPV was detected in 91.8% of cases (1086/1183).The HPV-negative rate was 8.2% (97/1183) for women with HSIL cytology, which coincides with that previously reported, which showed an ~5% to 8% hrHPV-negative HSIL cytology rate [24,25].The results from immediate histopathological examinations demonstrated a significantly higher prevalence of CIN2/3 in the hrHPV positive group compared to the hrHPV negative group (p<0.0001).However, we observed a CIN2+ rate of 8.2% (8/97) among the 97 cases with HSIL/HPV negative results, which is significantly lower than another study conducted from Shanghai (53.3%) in 2020 [23].A blinded second cytology test for HSIL/HPV-negative cases exhibited the probability that some HSIL interpretations in hrHPV-negative cases might be actually false positives [25].The findings from this study provide additional evidence to support the notion that combining cytology and hrHPV testing offers enhanced safety advantages, reducing the risk of undetected significant lesions.Furthermore, individuals positive for HPV-16 or HPV-16, 18/45 dual positive had a significantly elevated risk of developing CIN2/3 compared to other groups (p<0.0001).
Given the rising incidence of ADC among young females in recent times, there has been increased focus on the assessment of glandular cells in cervical smears [26].This study encompassed 19 cases of AIS.Several studies have reported that LBC smears showed a high sensitivity for the detection of cervical ADC [27], possibly due to the morphologic characteristics of the samples [28].Nevertheless, other studies have demonstrated that a co-testing strategy may be advantageous in the detection of cervical carcinoma, particularly ADC or its precursor lesions [29][30][31].It is worth noting that among the 97 patients with ASC-H/HSIL cytology but negative hrHPV results in our study, 5 cases (5.2%) were diagnosed as CIN2+ squamous lesions, and 3 cases (3.1%) were diagnosed as AIS+ glandular lesions upon histological follow-up.This further demonstrates the suitability of co-testing as an effective screening method for cervical cancer, given its low rate of missed diagnoses in both cancer and precancerous lesions.Different from the previous studies, our studies distinguished between squamous and glandular lesions.As is known, the distribution of hrHPV genotypes varies among distinct histological subcategories of cervical malignancies [32].HPV16 is the dominant genotype in squamous cell carcinomas (SCCs),32 whereas HPV18 frequently, though not always, emerges as the predominant genotype in AIS [33,34].In our study, we compared the prevalence of AIS among different hrHPV genotype groups.The prevalence of AIS lesions in HPV-18/45 positive group was 13.8%, while it was 1.6% and 1.0% in HPV-16 positive and hrHPV-negative group respectively (p<0.0001).These findings support the notion that women with HPV 18/45+ are at higher risk of cervical ADC.
In a study conducted by Wang et [28], it was found that the incidence of high-grade squamous intraepithelial lesion (HSIL+) in younger women (≤30 years) was significantly higher compared to older women (51-60 years) [35].This finding was supported by another independent study which also reported a comparable risk of HSIL+ in older women relative to their younger counterparts [36].In our study, the rate of CIN2/3 decreased significantly with increasing age, while the rate of SCC increased.This finding is consistent with a large sample study conducted by Kaiser Permanente Northern California (KPNC) [24].One possible explanation for this phenomenon is that as cervical cancer screening becomes more prevalent in developed areas of China, the number of screening tests disproportionately increases among older patients, leading to a decrease in the CIN2/3 rate.However, as age increases, the risk of developing cancer accumulates, resulting in a higher SCC reporting rate among older people.Hence, the data suggest that age is a significant risk factor for the progression from cervical precancerous lesions to cancer, with perimenopausal and postmenopausal women with HSIL cytology showing a higher risk of developing cancer.
To summarize, the study concluded that the incidence of CIN2/3 was significantly higher among the hrHPV-positive group in comparison to the hrHPV-negative group of women in developed southern China with ASC-H/HSIL cytology.Specifically, the prevalence of CIN2/3 and SCC is significantly higher in HPV-16+ group, while AIS lesions are more prevalent among HPV-18/45+ patients.Furthermore, the younger age group showed a significantly higher prevalence of CIN2/3, while the older age group presented an obvious higher prevalence of SCC.Our data demonstrate that AHPV testing is an effective method for HPV phenotyping.These results are compatible with international findings and support the applicability of the ASCCP guideline in developed southern China.requirement for informed written consent, as the study was a retrospective study and there was no additional risk to patients.All data were anonymized to maintain patient privacy.

Figure 2 .
Figure 2. Pathological image of ASC-H/HSIL by liquid-based cytology.(A) ASC-H displays non-canonical cytological features, including nuclear enlargement, hyperchromasia, and increased nuclear-cytoplasmic ratio; (B) HSIL exhibits more pronounced nuclear abnormalities, such as enlarged nuclei, hyperchromasia with coarse and uneven chromatin, and increased nuclear-cytoplasmic ratio.

Figure 3 .
Figure 3.The stacked column chart showing the proportions of different HPV subtypes in the histological outcomes of women with ASC-H/HSIL cytology.The distribution of HPV genotypes in ASC-H/HSIL women (a), ASC-H women (b), and HSIL women (c).ASC-H, atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; CIN, cervical intraepithelial neoplasia grade; ADC, adenocarcinoma; AIS, adenocarcinoma in situ; ASC, adenosquamous carcinoma; SCC, squamous cell carcinoma; G-EAC, gastric-type endocervical adenocarcinoma.

Table 1 .
Demographic characteristics of patients with ASC-H and HSIL cytology.

Table 2 .
Distribution of HPV infection patterns among the women with ASC-H/HSIL cytology.

Table 3 .
The prevalence of precancer and cancer among women with ASC-H/HSIL.

Table 5 .
Immediate risk of high-grade squamous lesions/glandular lesions between older and younger group with ASC-H/HSIL.