Risk Profile of High-grade Cervical Lesions and Cervical Cancer Considering the Combination of Cytology, HPV Genotype, and Age among Women Undergoing Colposcopy

Objective  The present study aims to establish a risk profile for high-grade cervical lesions and cervical cancer (CIN2 + ) in women undergoing colposcopy at the Hospital do Câncer de Barretos, through the analysis of Human Papillomavirus (HPV) infection, cervical cytology, and patient's age. Methods  Retrospective cross-sectional study based on a computerized database of women aged ≥ 18 years old who underwent colposcopy at the Prevention Department of the Hospital do Câncer de Barretos from 2017 to 2019. Results  A total of 3,411 women were included, 58.0% were positive for high-risk-HPV test, with a higher prevalence of CIN2+ for HPV16 (30.3%) and other HPV (45.0%). Cytological findings that suggest invasive cervical cancer (squamous cells or adenocarcinoma), regardless of the status of HPV test, showed 100% diagnosis of CIN2 + , while atypias that suggest high-grade lesions, HSIL and ASC-H, positive for HPV test, showed in 86 and 55.2%, respectively, diagnosis of CIN2 + . ASC-H cytological results among women aged > 40 years old and negative HPV were mainly associated with benign findings. We observed that ≤ CIN1 has a higher prevalence among older women with negative HPV, while for high-grade lesions there is an increase among young women HPV16- and/or 18-positive. In cancer diagnosis, we observed a predominance of HPV 16/18 regardless of the age group. Conclusion  The highest risks of precursor lesions and cervical cancer were found among women with positive HPV 16/18 tests and severe cytological atypia in population screening tests. In addition, cytological findings of ASC-H HPV negative in women > 40 years old usually represent benign findings in histological investigation.


Introduction
Cervical cancer is one of the most frequent types of cancer among women worldwide, being the fourth in incidence and mortality, with an estimated 604,127 new cases in 2020. 1 Among the countries with the highest incidence of this disease, Brazil is fourth (17,743) in absolute numbers after India, China, and Indonesia. 1,2According to data from the National Cancer Institute (INCA, in the Portuguese acronym), cervical cancer is the third most common cancer among Brazilian women (7.0%). 3ersistent infection by human papillomavirus (HPV) is the leading cause of cervical cancer, with 14 types considered oncogenic (hrHPV).][6] The early detection of precursor lesions through population screening programs and the expansion of HPV vaccination coverage can minimize the impact of cervical cancer. 7,8n Brazil, cervical cytology is recommended for women aged between 25 and 64 years old.2][13][14][15] In addition, HPV16/18 coinfection is related to high rates of high-grade lesions (56.7%) and cancer (71.1%), 16 which demonstrates the importance of tests based on the polymerase chain reaction (PCR) technique with genotyping of the viral types.
The association of viral type with the age of the infected woman may also represent an increased risk factor for precursor lesions.Positivity for HPV16 in CIN2þ cases showed an increased risk among English women aged 24 to 44 years old, and for the group aged from 45 to 64 years old, there was a greater association with viral types other than 16/18. 17onsidering the cytological findings, the association of moderate to severe atypia and hrHPV infection among patients referred for colposcopy represents a high risk for histological diagnosis of high-grade and invasive lesions. 18he presence of severe atypia among women infected with types 16/18 indicates a high risk of CIN2 þ , with results varying between 60.2 and 83.6%. 19pulation screening tests.In addition, cytological findings of ASC-H HPV negative in women > 40 years old usually represent benign findings in histological investigation.

Palavras-chave
► lesão escamosa intraepitelial cervical ► neoplasias do colo do útero ► infecções por papillomavirus ► colposcopia ► risco Therefore, cytological results, HPV testing, and age have demonstrated their potential in the stratification of risk for precursor lesions and cervical cancer. 18,20At the Hospital do Câncer de Barretos (HCB, in the Portuguese acronym), all patients performed hr-HPV test in the colposcopy; therefore, the objective of the present study was to stratify the risk of precursor lesions and cancer among women undergoing colposcopy at the Hospital do Câncer de Barretos in São Paulo, state of São Paulo, Brazil.

Methods
Women !18 years old who underwent colposcopy at the Cancer Prevention Unit of the HCB from 2017 to 2019 were included.Patients undergoing colposcopy were previously screened by cervical cytology, and HPV tests were performed as a cotest on the same sample of screening cytology or collected during the colposcopy examination.Inconclusive results or findings related to other sites (vagina, vulva, and endometrium) were disregarded.
This was a retrospective cross-sectional study based on a computerized database of the Department of Cancer Prevention of the HCB.
The cervical cancer screening program includes conventional cervical smear and liquid based cytology (LBC, BD SurePath).Samples were sent to the Pathology laboratory for preparation of slides according to the manufacturer's instructions using BD PrepMate and BD PrepStain.
For the detection of HPV DNA, the Cobas HPV Test X480TM (Roche Molecular Systems, Pleasanton, CA, USA) was used.This test detects 14 types of HPV, with HPV 16 and 18 alone and HPV-others encompassing the oncogenic types in a grouping: HPV-31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. 21omen are referred for colposcopy due to abnormal cervical screening at the HCB.During colposcopy, biopsy is performed when the gynecologist identifies a lesion in the examination.Biopsy was not performed when squamocolumnar junction (SCJ) was visualized and no lesion was identified by the gynecologist.Finally, when SCJ was not visualized and no lesion was identified by the gynecologist, an endocervical curettage (ECC) was performed.
The gold standard considered was the combination of colposcopic examination and anatomopathological results for cases undergoing biopsies or excisional therapeutic procedures (excision of the transformation zone of the cervix [TZE]).The final histological diagnosis was established according to the most severe result obtained.
Analyses of comparisons of proportions were performed using the chi-squared test, considering that the differences between the groups were statistically significant when the pvalue was < 0.05.A confidence interval (CI) of 95% was considered.IBM SPSS Statistics for Windows version 27 (IBM Corp., Armonk, NY, USA) was used for data storage and statistical analysis.
The present study was conducted in accordance with the guidelines for good clinical practice and was approved by the Ethics Committee of the HCB under CAAE number 50357321.9.0000.5437.

Results
A total of 3,411 women aged from 18 to 93 years old (mean 42.5 years old) who underwent colposcopy between March 2017 and December 2019 were included; 27.4% of the participants were tested for HPV at the time of screening (associated with cytology), and 72.6% were tested during colposcopy.The positivity rate for HPV was 58.0% (1,979 women), with 802 positive for HPV16/18 (40.5%) and 1,177 positive for other HPV types (59.5%).HPV16 alone was detected in 19.6% of the cases.Among the women positive for types 16/18, there was a higher prevalence (38.4%) of CIN3.Among the women positive for other viral types than 16/18, 52.8% had no intraepithelial lesions or cancer.In the group of women who tested negative for HPV, 86% had benign findings on colposcopy associated with pathological findings.Our findings show the evidence of false positives in DNA-HPV results since we find $ 5% of CIN2þ findings in HPV-negative cases, which could be explained by the possibility of a few cases of cervical cancer not being caused by HPV (►Fig. 1).
Regarding cytology, the presence of atypical squamous cells of undetermined significance, which cannot rule out a high-grade lesion (ASC-H), was the most frequent finding (40.8%) and the cytological classification that most commonly pushes the patient to the complementary examination of colposcopy.The mean ages of the women diagnosed with high-grade intraepithelial lesions were 36.4 years old and, for cases of cervical cancer, 43.8 years old (►Table 1).
Considering the diagnoses of CIN2/3 and cervical cancer, the HPV positivity rates were 90.8% and 94.5%, respectively.Types 16 and 18 were detected in 50.8% of the women with CIN2/3 and in 81.7% of the patients diagnosed with cancer.HSIL and ASCH were the types of cytological atypia most frequently associated with the diagnosis of CIN2þ (►Table 2).
For the analysis of HPV positivity in the CIN2þ and CIN3þ populations, there was greater positivity in the group of other HPVs (45 and 41.5%, respectively) and HPV16 alone (30.3 and 34%, respectively).The most frequent combination was observed in the combined HPV16 and other HPV groups, namely, 14.4% for CIN2þ and 14.0% for CIN3þ (►Fig. 2).
Positivity for HPV 16 and 18 was associated with a higher prevalence of diagnosis of CIN2, CIN3 and cancer (8.4,39.3, and 12.5%, respectively), while other HPV types and negative HPV showed higher prevalence of diagnosis of CIN1 (67.4 and 94.6%, respectively).Regarding cytology, the diagnoses of benign and low-grade intraepithelial lesions were more frequent among women with cytology showing ASCUS, LSIL or NILM.CIN2/3 was more frequent among women with AIS, IA, SCC, HSIL (invasive) or HSIL cytology (►Table 3).
Patients classified as ASCH were the most frequent finding in cytology (►Table 1).In total, 54.0% of these patients are !40 years old.We observe in this group 64.6% negative HPV associated with 61.8% of diagnosis as benign/CIN1.Otherwise, women < 40 years old showed a positivity for HPV in 67,2%.In this group, 44,5% had the diagnosis of CIN2þ (►Fig.3).
►Fig. 4 shows the risk of CIN2þ according to the combination of cytological results and HPV tests.
There was a higher prevalence of infection by type 16/18 among younger women diagnosed with CIN2/3 and the involvement of the other viral types increased proportionally with increasing age.Among women diagnosed with cancer, a higher incidence of infection by type 16/18 was also found in all age groups, but there was a tendency for other viral types to increase beginning at 35 years old (►Fig. 5).

Discussion
The present study aimed to stratify the risk of precursor lesions and cervical cancer among women who were attended at a reference center for colposcopy, considering age, the cytological test result, and HPV genotype.
The possibility of molecular tests associated with colposcopic and cytological evaluation increases the accuracy of diagnosis, and the high negative predictive value of HPV tests confers a low risk of relevant tissue damage among women who test negative.In our study, 41.19% of the women were negative for hrHPV, of which 86% were negative for intraepithelial lesions and cervical cancer.Other studies have demonstrated the association between colposcopic and histological findings and HPV infection status. 18,20,24Among women referred for colposcopy, a large group showed cytological results of mild atypia or undetermined findings; for these cases, some groups of low-risk HPV (not identified by the method used in the study) and some results related to tissue repair, inflammation, and atrophy are expected.
In the qualitative evaluation of the composition of viral types among women who tested positive, it was observed   that the majority (59.5%) was infected with non-16/18 types.However, there is a clear trend toward benign findings and low-grade intraepithelial lesions in this group of women, whereas type 16/18 increases the risk of CIN3 þ .The ESTAMPA study conducted in Latin American countries also found an association between HPV16/18 positivity and the severity of tissue lesions (30.6% CIN2, 56.7% CIN3, and 71.1% cancer). 16][27][28][29][30] Cytological atypia classified as ASCH was the most frequent, and in 40.8% of the cases, the need for colposcopic  examination was justified.Among the women who received this cytological report, 50% tested positive for HPV, and a trend of CIN2/3 diagnoses was observed for the group of younger women.][33] There is a clear association of ASCH results with factors of atrophy of the cervical epithelium among postmenopausal women, and when associated with negative HPV > 40 years old, they usually represent benign findings in histological investigation.
Women with SCC and AI cytology, regardless of the HPV test status, had a 100% diagnosis of CIN2 þ/CIN3 þ , which reflects the high specificity of cytology directly associated with the degree of atypia.When associated with positive HPV, the cytology HSIL and ASCH presented a diagnosis of CIN2þ in 86 and 55.2% of patients, respectively.In the absence of HPV infection, CIN2þ detection decreases to < 50% for these same cytological results.These results agree with those of another study that reported a specificity of 86.6% in the diagnosis of CIN2þ among women with HSIL and positive HPV16/18. 184][35] Several studies have implemented the use of HPV tests in population screening of precursor lesions and cervical cancer in place of cytology, which makes it possible to extend the interval of repetition of the exams to periods of 5 years. 36,37In Brazil, HPV testing is not available for population screening in the public health system, which is still based on cytological tests. 10Some isolated and regional initiatives have proposed the use of HPV tests with results that endorse its applicability, including positive results in cost-effectiveness evaluations among Brazilian women. 11,38ur study did not aim to evaluate the primary screening of cervical lesions by molecular tests but focused on the importance of stratifying the risk of women previously screened by cytological tests, considering age and the results of the investigation for HPV by genotyping.The results demonstrate the importance of additional molecular tests, especially among women with cytological tests classified as inconclusive changes and atypia of undetermined significance.
In addition, the possibility of partial HPV genotyping may indicate the need for clinical procedures and personalized follow-up protocols for each patient with individual risk stratification.Although it was not the focus of the present study, the HPV vaccinated populations may also be an additional benefit with HPV genotyping for those viral types that are not protected by the vaccine.The results described here reinforce the importance of types 16/18 in the carcinogenesis of cervical cancer and add evidence regarding the participation of other viral types in a large proportion of women diagnosed with precursor lesions.

Conclusion
The highest risks of precursor lesions and cervical cancer were found among women with positive HPV 16/18 tests and severe cytological atypia in population screening tests.Patients with cytology classified as ASCH may have a risk of CIN2þ stratified by the HPV test, and the number of positive tests will be higher among younger women < 40 years old.

Contributors
All authors were involved in substantial contributions to conception and design, data collection, data analysis and interpretation; article writing, relevant critical review of the intellectual content; and final approval of the version to be published.

Fig. 1
Fig. 1 Women undergoing colposcopy in relation to the HPV test and final diagnosis.

Fig. 4
Fig. 4 Prevalence of CIN2þ according to cytology and HPV test results.

Fig. 5
Fig. 5 Association between HPV genotype and age at final diagnosis.
a Endometrial cells (in a woman !45 years old.b CIN2/3: cervical intraepithelial neoplasia grade 2 or 3, not possible to differentiate.

Table 2
Characterization of the study population according to the final diagnosis 22breviations: AIS, adenocarcinoma in situ; ASC-H, atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion; HPV, human papillomavirus; HSIL (inv), high grade squamous intraepithelial lesion cannot exclude invasion22; IA, Invasive adenocarcinoma; SCC, squamous cell carcinoma.

Table 3
Prevalence of HPV genotyping and cytology according to final diagnosis