人乳头瘤病毒16/18分型在细胞学轻度异常的低度宫颈病变中的预测价值

Prognostic value of human papillomavirus 16/18 genotyping in low-grade cervical lesions preceded by mildly abnormal cytology 人乳头瘤病毒16/18分型在细胞学轻度异常的低度宫颈病变中的预测价值 first-author †E-mail:attwentyone@163.comchengxd@zju.edu.cn †E-mail:attwentyone@163.comchengxd@zju.edu.cn attwentyone@163.com corresp ‡Corresponding Author ‡Corresponding Author †E-mail:attwentyone@163.comchengxd@zju.edu.cn †E-mail:attwentyone@163.comchengxd@zju.edu.cn attwentyone@163.com Department of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China Histological low-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grade 1 (LSIL/CIN1) preceded by normal or mildly abnormal cytology is recommended for conservative follow-up, with no separated management. In this study, we assessed the triage value of human papillomavirus (HPV) 16/18 genotyping in 273 patients with LSIL/CIN1. HPV16/18 genotyping was performed at baseline and follow-up was at 6-monthly intervals for up to 2 years. At each follow-up, women positive for cytology or high-risk HPV (hrHPV) were referred for colposcopy. Enrollment cytology, HPV16/18 genotyping, and questionnaire-obtained factors were linked to the 2-year cumulative progression rate. Univariate and multivariate analyses were performed taking into account time-to-event with Cox proportional hazard regression. The results showed that 190 cases (69.6%) regressed, 37 (13.6%) persisted, and 46 (16.8%) progressed. HPV16/18 positivity (hazard ratio (HR), 2.708; 95% confidence interval (CI), 1.432–5.121; P =0.002) is significantly associated with higher 2-year cumulative progression rate. Sub-analysis by enrollment cytology and age restricted the positive association among patients preceded by mildly abnormal cytology and aged 30 years or older. Immediate treatment is a rational recommendation for the high-risk subgroup, when good compliance is not assured. 目的 2 评估人乳头瘤病毒（HPV）16/18分型在细胞学正常或轻度异常的低度宫颈病变中的预测价值，为临床分流决策提供依据。 创新点 2 目前对于细胞学正常或轻度异常的低度宫颈病变患者缺乏有效分流策略。本研究首次探索HPV16/18分型对这部分患者疾病转归的预测价值。 方法 2 新招募细胞学正常或轻度异常的低度宫颈病变患者，以6个月为间隔随访2年，采用Cox回归模型对入组HPV16/18分型结果与疾病转归进行关联分析。 结论 2 对于HPV16/18阳性，细胞学轻度异常，且30岁及以上的低度宫颈病变患者，可以考虑比保守随访更积极的诊疗方案。 Low-grade squamous intraepithelial lesion (LSIL) Cervical intraepithelial neoplasia grade 1 (CIN1) Human papillomavirus (HPV) HPV16/18 genotyping Prognostic value Prospective study 低度鳞状上皮内病变 宫颈上皮内瘤变1级 人乳头瘤病毒（HPV） HPV16/18分型 预测价值 前瞻性研究 Introduction 1 Histological low-grade squamous intraepithelial lesion (LSIL), also termed cervical intraepithelial neoplasia grade 1 (CIN1) in the prior edition of three-tier terminology, is limited to the basal one-third of the squamous epithelium. Up to 70%–80% of LSIL/CIN1 will regress spontaneously, while a subset is associated with residual risk for future precancerous lesion (Schiffman et al., 2007 2007 ; Martin and O'Leary, 2011 2011 ). No reliable biomarker, other than cytology, has been used to predict the evolution of LSIL/CIN1. Kaiser Permanente Northern California (KPNC) data showed a relatively low 5-year progression rate of LSIL/CIN1 preceded by normal cytology (negative for intraepithelial lesion or malignancy (NILM)) or mildly abnormal cytology (atypical squamous cells of undetermined significance (ASC-US) and LSIL), but a substantially higher progressive risk with more severe abnormalities (high-grade squamous intraepithelial lesion (HSIL), atypical squamous cells—cannot rule out HSIL (ASC-H), and atypical glandular cells (AGC)) (Katki et al., 2013 2013 ). Accordingly, LSIL/CIN1 preceded by normal or mildly abnormal cytology is considered low-risk and recommended for conservative follow-up only (Massad et al., 2013 2013 ), with no triage management available. Yet up to 10% of progression was reported for these patients (Katki et al., 2013 2013 ). Previous studies have explored the values of immunohistochemical markers (such as p16 INK4A and cytokeratin 7) in predicting the behavior of LSIL/CIN1. However, microscopically interpretive variability confounded such studies, and results have been inconsistent (Liao et al., 2014 2014 ; Mills et al., 2015 2015 ; Huang et al., 2016 2016 ; Paquette et al., 2016 2016 ; Sagasta et al., 2016 2016 ). Human papillomavirus (HPV) genotypes have different carcinogenicities, leading us to wonder whether HPV genotyping could be of clinical value in refining the management of LSIL/CIN1. HPV16/18 genotyping is the most approved genotyping strategy. Progressive risk is elevated for women with positive HPV16/18, even when cytology is normal (Khan et al., 2005 2005 ). Thus, HPV16/18 positivity alone is an indication for colposcopy referral among women aged 30 years or older, according to the current American Society for Colposcopy and Cervical Pathology (ASCCP) management guidelines (Massad et al., 2013 2013 ). Yet no prospective study has addressed the prognostic value of HPV16/18 genotyping in LSIL/CIN1 preceded by non-severe cytology abnormalities. We conducted this hospital-based longitudinal study to evaluate the performance of HPV16/18 genotyping as a triage for LSIL/CIN1 preceded by normal or mildly abnormal cytology. Materials and methods 1 Patient recruitment 2 Women with LSIL/CIN1 diagnosed by colposcopy-guided biopsy (colposcopy is adequate and endocervical curettage is negative) were prospectively recruited in the Women’s Hospital, School of Medicine, Zhejiang University, China during June 2012 to December 2013. The diagnostic criterion was intraepithelial lesion limited to the basal one-third of the squamous epithelium, exclusive of flat condyloma, koilocytotic atypia, and koilocytosis. Women were excluded from this study according to the following criteria: (1) preceded by cytological HSIL, ASC-H, or AGC; (2) surgically or ablatively treated cervix; (3) previously confirmed cervical cancer or precursor, or other malignancies; (4) with immunosuppressive diseases; and, (5) pregnancy. All eligible patients underwent HPV16/18 genotyping at baseline. Information on socio-demographic characteristics, reproductive history, contraception, menstrual status, and sexual behavior was collected via an interviewer-administered structured questionnaire. Ethics approval and consent to participate 2 This study was approved by the Human Research Ethical Committee of the Women’s Hospital, School of Medicine, Zhejiang University, China with protocol No. 20110014. Informed consent was obtained from study participants according to institutional guidelines. Follow-up 2 Patients were followed up at 6-monthly intervals for up to 2 years. At each follow-up, liquid-based cytology and HPV genotyping were performed as first-level exams. Women with positive cytology or high-risk HPV (hrHPV) were referred for colposcopy. The endpoints were defined as follows: (1) progression: histology-confirmed HSIL or more; (2) regression: cytology normal and hrHPV negative, or cytology/hrHPV positive but histology negative; and, (3) persistence: histology-confirmed LSIL/CIN1. Women reaching an endpoint would then be handled according to the ASCCP management guidelines (Massad et al., 2013 2013 ). Cytology and HPV genotyping 2 Hospital cytologists performed cytological diagnosis according to the 2001 Bethesda System (Solomon et al., 2002 2002 ). HPV genotyping was carried out with HPV GenoArray test kit (Hybribio, Hong Kong, China), which was described in our previous studies (Ye et al., 2010a 2010a ; 2010b 2010b ). Briefly, this test kit can identify 14 hrHPVs (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) separately. Colposcopy and histological diagnosis 2 Hospital colposcopists performed colposcopy according to the standardized protocol and hospital pathologists made histological diagnoses according to the Lower Anogenital Squamous Terminology (LAST) recommendations (Darragh et al., 2012 2012 ). Consensus on histodiagnosis was reached by an expert panel in the event of disagreement. Statistical analysis 2 Enrollment cytology, HPV16/18 genotyping, and potential risk factors identified from the questionnaire were assessed as independent prognostic markers for 2-year cumulative progression. In both univariate and multivariate analyses, Cox proportional hazards model was used to estimate hazard ratio (HR) and 95% confidence interval (CI). Factors associated with progression at P < 0.1 in univariate analysis would be included in multivariate analysis and mutually adjusted. Further, analysis was stratified by enrollment cytology (“normal cytology” (NILM) versus “mildly abnormal cytology” (ASC-US or LSIL)) and age ( < 30 years versus ≥30 years) to optimize the application scope of triage markers. SPSS software version 19.0 (SPSS Inc., Chicago, IL, USA) was used. All statistical tests were two-sided. A level of 0.05 was chosen to indicate statistical significance. Results 1 Two hundred and seventy-three patients, with a mean age of 36.5 years (range 22–62 years) at registration, completed follow-up. Fifty patients (18.3%) were HPV16/18-positive, while 191 cases (70.0%) were preceded by mildly abnormal cytology (ASC-US/LSIL) at enrollment. During the 2-year follow-up, 190 cases (69.6%) regressed, 37 cases (13.6%) persisted, and 46 cases (16.8%) progressed. Among the progression, 37.0% (17/46) occurred at the first follow-up, 30.4% (14/46) at the second, 17.4% (8/46) at the third, and 15.2% (7/46) at the end. In univariate analysis, age, marital status, reproductive history, contraception, menopausal status, sexual debut, and antecedent cytology were excluded at P > 0.1 (Table 1 1 ). With mutual adjustment, HPV16/18 positivity was confirmed as the independent prognostic marker for progression in the multivariate analysis (Table 1 1 ). The 2-year cumulative progression rate was 34.0% (95% CI, 20.9%–47.1%) among HPV16/18 positive patients, while 13.0% (95% CI, 8.6%–17.4%) for HPV16/18 negative patients (Fig. 1a 1a ). (a) All patients ( n =273); (b) Patients preceded by ASC-US/LSIL cytology and aged 30 years or older ( n =150) NILM: negative for intraepithelial lesion or malignancy; ASC-US: atypical squamous cells of undetermined significance; LSIL: low-grade squamous intraepithelial lesion; HPV: human papillomavirus; HR: hazard ratio; CI: confidence interval. Factors associated with 2-year cumulative progression rate at P < 0.1 in the univariate analysis were included in the multivariate analysis and mutually adjusted. Refer to the lifetime number of sexual partners 2 Characteristics 3 Univariate analysis 2 Multivariate analysis a a HR (95% CI) P HR (95% CI) P Age  <30 years Reference  ≥30 years 1.231 (0.574–2.642) 0.594 Marital status  Married Reference  Single 0.488 (0.118–2.020) 0.322 Childbearing  0 Reference  ≥1 2.968 (0.717–12.290) 0.133 Contraception  Oral contraceptive 0.474 (0.065–3.473) 0.462  Condom 0.576 (0.282–1.175) 0.129  Other Reference Menopausal status  Premenopause Reference  Menopause 0.608 (0.147–2.512) 0.492 Sexual debut  <20 years old 1.262 (0.495–3.216) 0.626  ≥20 years old Reference Sexual partner b b  1 Reference  ≥2 0.452 (0.190–1.074) 0.072 0.436 (0.183–1.039) 0.061 Enrollment cytology  NILM Reference  ASC-US/LSIL 1.498 (0.743–3.020) 0.259 HPV genotyping  HPV16/18 positive 2.777 (1.524–5.059) 0.001 2.708 (1.432–5.121) 0.002  HPV16/18 negative Reference 2 To improve the triage strategy, we stratified our analysis by cytology and age at enrollment to examine the risk in subgroups who might be managed with different clinic strategies. When separately stratified, the progressive risk associated with HPV16/18 was not significantly elevated in patients preceded by normal cytology or aged below 30 years (Table 2 2 ). Further cross stratification showed that the elevated progressive risk associated with HPV16/18 was only remarkable among cases preceded by mildly abnormal cytology (ASC-US or LSIL) and aged 30 years or older (Table 2 2 ). In this subset, the 2-year cumulative progression rate was 50.0% (95% CI, 29.1%–70.9%) among HPV16/18-positive patients, while 14.8% (95% CI, 8.6%–21.0%) for HPV16/18 negative cases (Fig. 1b 1b ). HPV: human papillomavirus; NILM: negative for intraepithelial lesion or malignancy; ASC-US: atypical squamous cells of undetermined significance; LSIL: low-grade squamous intraepithelial lesion; HR: hazard ratio; CI: confidence interval Stratification HR (95% CI) P Cytology  NILM 2.757 (0.797–9.534) 0.109  ASC-US/LSIL 3.055 (1.527–6.116) 0.002 Age  <30 years 1.265 (0.255–6.276) 0.774  ≥30 years 3.327 (1.733–6.386) <0.001 Cytology & age  NILM & <30 years 1.612 (0.099–26.197) 0.737  NILM & ≥30 years 3.650 (0.911–14.627) 0.068  ASC-US/LSIL & <30 years 1.888 (0.220–16.241) 0.563  ASC-US/LSIL & ≥30 years 3.314 (1.575–6.972) 0.002 Discussion 1 To our knowledge, this study is the first to assess the triage value of HPV16/18 genotyping in LSIL/CIN1 preceded by normal or mildly abnormal cytology. Among the four HPV tests approved by the US Food and Drug Administration (FDA), only the Cobas HPV test could genotype HPV16/18 separately. However, when we started our research in June 2012, the Cobas HPV test had not yet been approved by the FDA. In view of the good performance of the HPV GenoArray test in our previous studies (Ye et al., 2010a 2010a ; 2010b 2010b ), as well as the excellent concordance with other HPV tests such as Amplicor HPV test and Roche Linear Array (Grisaru et al., 2008 2008 ; Liu et al., 2010 2010 ), we adopted the HPV GenoArray test in the current research. It is well known that HPV infection is the causal agent underlying cervical carcinogenesis (Walboomers et al., 1999 1999 ). The strong etiological link between HPV infection and cervical cancer has led to the routine application of HPV testing in improving existing cytology-based cervical cancer screening, managing women with equivocal cytology following surgically or ablatively treated cervical lesions, and so on. Compared with HPV testing, HPV genotyping has not been widely applied in clinical practice. Among HPV genotypes, HPV16 and 18 were considered to have the most potent carcinogenic potential (Dahlström et al., 2010 2010 ; de Sanjose et al., 2010 2010 ; Rijkaart et al., 2012 2012 ; Bzhalava et al., 2013 2013 ; Tjalma et al., 2013 2013 ; Wheeler et al., 2014 2014 ), being associated with about 70% of cervical cancers worldwide (de Sanjose et al., 2010 2010 ; Li et al., 2011 2011 ). The powerful carcinogenicity of HPV16/18 made them the most meaningful genotyping targets in clinical practice. In cross-sectional studies, HPV16/18 genotyping stratified HPV-positive women by their risk of prevalent high-grade cervical lesions (Castle et al., 2011 2011 ; Wright et al., 2011 2011 ; Lagos et al., 2015 2015 ; McKenna et al., 2016 2016 ). It also served as a reliable predictor of residual disease following conization for cervical precancerous lesion (Kang et al., 2016 2016 ). In prospective studies, HPV16/18 positivity is linked with a higher risk of future high-grade cervical lesions (Khan et al., 2005 2005 ; Bulk et al., 2007 2007 ; Persson et al., 2015 2015 ). Based on evidence in the research literature, ASCCP guidelines recommend that women aged 30 years or older with normal cytology but positive HPV16/18 should be referred for immediate colposcopy, because they are at particularly high risk of current or future high-grade cervical lesions (Massad et al., 2013 2013 ). In this study, we have confirmed the triage value of HPV16/18 genotyping in LSIL/CIN1, which is comparable and complementary to earlier published data. In cytology sub-analysis, HPV16/18 genotyping provided little predictive value to LSIL/CIN1 preceded by normal cytology. It is inconsistent with early data, which showed an elevated 10-year risk of high-grade cervical lesions in women with normal cytology but positive HPV16/18 (Khan et al., 2005 2005 ). However, it is worth noting that this previous research had no baseline data of histodiagnosis (Khan et al., 2005 2005 ), so could not exclude patients with already existing high-grade cervical lesions at enrollment. Given the high current prevalence of high-grade cervical lesions among HPV16/18-positive women with normal cytology (Wright et al., 2011 2011 ), the positive follow-up association obtained without baseline histodiagnosis needs further verification. Another possible explanation for our negative relationship between HPV16/18 genotyping and LSIL/CIN1 preceded by normal cytology is low statistical power. Further study with a larger sample size and longer follow-up period is required to test this possibility. To balance risk versus benefit, we also tailored our strategy by age. Because of maximal exposure but minimal acquired immunity, HPV prevalence is high in young women (Bosch et al., 2008 2008 ; McKenna and McMenamin, 2014 2014 ). However, most young women have an effective immune response to clear the acute infection within a short time (Rodriguez et al., 2008 2008 ). Considering the high spontaneous regression rate among this age group, HPV testing is not recommended (Davey et al., 2014 2014 ). Similarly, colposcopy associated with HPV16/18 is restricted to women aged 30 years or older (Massad et al., 2013 2013 ). HPV16/18 genotyping showed no triage value under age of 30 years. Our observation supported the notion that HPV strategy should be age-related. Young women should be managed conservatively, especially for minor abnormalities (Massad et al., 2013 2013 ). Conclusions 1 HPV16/18 positivity is predictive of progression for women with LSIL/CIN1 preceded by mildly abnormal cytology, who are aged 30 years or older. Closer follow-up is required for this high-risk subset. 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We thank Dr. Hong-yun WANG (Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, China) for assistance with data entry. Contributors Jing YE, Xing XIE, Wei-guo LU, and Xiao-dong CHENG conceived and designed the project. Bei CHENG, Yi-fan CHENG, and Ye-li YAO participated in the recruitment and follow-up of patients. Jing YE analyzed the data and wrote the manuscript. Xing XIE, Wei-guo LU, and Xiao-dong CHENG revised the manuscript. All authors agreed to be accountable for all aspects of the work. All authors read and approved the final manuscript. Compliance with ethics guidelines Jing YE, Bei CHENG, Yi-fan CHENG, Ye-li YAO, Xing XIE, Wei-guo LU, and Xiao-dong CHENG declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. 10.1631/jzus.B1600473 Project supported by the Zhejiang Provincial Natural Science Foundation of China (No. LQ14H160007), the National Natural Science Foundation of China (No. 81402364), the Zhejiang Provincial Medical & Hygienic Science and Technology Project of China (No. 2013KYA104), the Special Fund for Scientific Research in the Public Interest from the National Health and Family Planning Commission of the People’s Republic of China (No. 2015SQ00243), and the National Key Research and Development Program of China (No. 2016YFC1302900) 2016-10-17 2017-02-13 2016-12-19 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 18