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Cervical cytology of atypical squamous cells, cannot exclude high-grade squamous intra-epithelial lesion: significance of age, human papillomavirus DNA detection and previous abnormal cytology on follow-up outcomes
European Journal of Obstetrics & Gynecology and Reproductive Biology 159 (2011) 155–159
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Cervical cytology of atypical squamous cells cannot exclude high-grade squamous intra-epithelial lesion: significance of age, human papillomavirus DNA detection and previous abnormal cytology on follow-up outcomes Chang Ohk Sung, Young Lyun Oh *, Sang Yong Song * Department of Pathology, Samsung Medical Centre, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 June 2010 Received in revised form 23 October 2010 Accepted 19 May 2011
Objective: Despite the usefulness of Pap tests for cancer screening, outcomes can be difficult to predict when atypical squamous cells (ASCs) are identified. According to the 2001 Bethesda system, ASCs can be subdivided into two groups: ASCs of undetermined significance (ASC-US); and ASCs, cannot exclude high-grade squamous intra-epithelial lesion (ASC-H). ASC-H interpretations are uncommon, and studies involving this type of lesion are based on small numbers of cases. Study design: Cross-sectional, retrospective study of 392 ASC-H cases. The follow-up outcomes of ASC-H cases that were diagnosed during routine primary screening between 2002 and 2008 were investigated, and relationships between clinicopathological parameters were assessed, particularly positive test for high-risk HPV (HPV) DNA, patient age at diagnosis and previous abnormal cytology. Results: Of the 392 cases, high-grade squamous intra-epithelial lesion (HSIL) was detected in 111 (28.3%) cases, squamous cell carcinoma was detected in 15 (3.8%) cases, low-grade squamous intra-epithelial lesion was detected in 37 (9.4%) cases, reactive change was detected in 178 (45.4%) cases, atrophy was detected in 47 (12.0%) cases, and adenocarcinoma was detected in four (1.0%) cases. The prevalence of HSIL or greater was 27.8% for women aged 40 years, and 52.3% for women aged <40 years (p < 0.001). HPV positivity in ASC-H smears was significantly associated with HSIL or greater, irrespective of age (<40 years, p = 0.003; 40 years, p < 0.001). ASC-H with previous abnormal cytology greater than ASCUS showed a significantly higher detection rate for HSIL or greater at follow-up (p < 0.001). Conclusions: Patient age, positive HPV DNA test and previous abnormal cytology are useful predictors of underlying HSIL or greater in women with ASC-H. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cytology Human papillomavirus Cervix Atypical squamous cells Follow-up
1. Introduction Worldwide, uterine cervical cancer is the second most common cancer among women [1]. Fortunately, the incidence has decreased due to widespread use of the Papanicolaou (Pap) test as a screening method [2]. Consequently, the detection rates of early cervical lesions have increased. However, despite the usefulness of the Pap test for cancer screening, it still has limitations including high false-negative rates and frequent interobserver variability. The presence of atypical cells in smears is one of the major diagnostic problems for cervical cancer screening. Typically, it is difficult to predict outcomes for patients with atypical cells in their smears. According to the 2001 Bethesda system, atypical squamous cells (ASCs) can be subdivided into two groups: ASCs of undetermined significance (ASC-US); and ASCs, cannot
* Corresponding authors. Tel.: +82 2 3410 2803; fax: +82 2 3410 0025. E-mail addresses: [email protected] (Y.L. Oh), [email protected] (S.Y. Song). 0301-2115/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2011.05.022
exclude high-grade squamous intra-epithelial lesion (ASC-H). The ASC-H category includes ASCs that exhibit equivocal features which are suggestive of a high-grade squamous intra-epithelial lesion (HSIL) but which are not sufficient to diagnose HSIL. It was recently demonstrated that ASC-H is not a homogeneous category, and has a broad spectrum of cytomorphological patterns that are associated with different clinicopathological categories classified by biopsy and human papillomavirus (HPV) test results [3]. ASC-H interpretations are uncommon. ASC-H represents 5– 10% of ASC diagnoses and accounts for approximately 0.2% of cytological interpretations [4]. A woman with a cervical cytology result interpreted as ASC-US has a 5–17% chance of having cervical intra-epithelial neoplasia (CIN) 2 or 3 confirmed by biopsy, while ASC-H is associated with a significant incidence of HSIL on followup compared with ASC-US [4–7]. However, the rates of underlying HSIL in patients with ASC-H range widely from 24% to 94% [4–6,8– 10], and studies involving these ASCs have included small numbers of cases. The present study investigated follow-up outcomes of 392 ASC-H cases identified during routine primary
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Table 1 Summary of follow-up results for patients with smears interpreted as atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion, by preparation method. Type of preparation
All cases n (%)
HSIL n (%)
Squamous cell carcinoma n (%)
LSIL n (%)
Reactive change n (%)
Atrophy n (%)
Adenocarcinoma n (%)
CS LBP Total
263 (100) 129 (100) 392 (100)
80 (30.4) 31 (24.0) 111 (28.3)
11 (4.2) 4 (3.1) 15 (3.8)
19 (7.2) 18 (14.0) 37 (9.4)
125 (47.5) 53 (41.1) 178 (45.4)
26 (9.9) 21 (16.3) 47 (12.0)
2 (0.7) 2 (1.6) 4 (1.0)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; CS, conventional smear; LBP, liquid-based preparation.
screening, and analysed the relationships between outcomes and clinicopathological parameters, particularly positive test for highrisk HPV (HR-HPV) DNA, patient age at diagnosis and previous abnormal cytology.
2. Materials and methods 2.1. Study design and case selection This was a cross-sectional study of retrospective data from patients who visited Samsung Seoul Hospital, Korea between July 2002 and August 2008. The hospital provides tertiary and secondary healthcare services, and also provides primary healthcare services through the healthcare centre. Most of the cervical smears in this study were obtained at the primary healthcare centre. Cases were identified by a electronic database search for diagnoses of ASC-H on cervicovaginal Pap smears in accordance with the 2001 Bethesda system. During the study period, most smears were read by the same group of 10 cytopathologists. Of 198,069 Pap smears read during this period, 448 (0.23%) cases were interpreted as ASC-H. Specifically, the rates were 0.22% and 0.23% for conventional and ThinPrep liquidbased preparations, respectively. All of the patients underwent colposcopy under supervision by experienced colposcopists. Of the 448 cases of ASC-H, 229 cases had follow-up tissue sampling within 6 months, which included colposcopic biopsy, loop electrosurgical excision procedure, conization and hysterectomy. Histological specimens (e.g., normal colposcopy or minor abnormalities) were not obtained for 163 of the 448 cases. These women underwent repeated follow-up cytological tests for 6–12 months after the initial abnormal smear. The remaining 56 cases, for whom colposcopy was unsatisfactory and there were no follow-up cytological tests, were considered to be lost to followup and were excluded from the study. As such, 392 Pap smears, including 263 conventional smears and 129 liquid-based cytological smears, were included in this study. The follow-up results were classified as HSIL, squamous cell carcinoma, low-grade squamous intra-epithelial lesion (LSIL), reactive change, atrophy or adenocarcinoma. For this study, histological diagnoses of CIN 1 were classified as LSIL, and diagnoses of CIN 2 and 3 were classified as HSIL. Cytological diagnoses and adequacy of specimens were classified using the 2001 Bethesda system for cervical cytology [11]. 2.2. Detection of HR-HPV DNA To determine the viral load, 137 smears were evaluated for HRHPV DNA using the Hybrid Capture II (Digene Corporation, Gaithersburg, MD, USA) or HPV DNA microchip (Mygene Co., Seoul, Korea) methods [12] in accordance with the manufacturers’ protocols. For the Hybrid Capture II test, the panel of HR-HPVcontaining specimens included 13 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). For HPV DNA microchip analysis, 15 HR-HPV types were included (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 54, 56, 58, 59, 68).
2.3. Statistical analysis Statistical significance was determined on the basis of Chisquared test or Fisher’s exact test, as appropriate. Independent two-sample t-tests were used for comparative analysis of continuous data. All tests were two-sided, and p-values <0.05 were considered to be statistically significant. Statistical analysis was performed using Statistical Package for the Social Sciences Version 12.0 (SPSS Inc., Chicago, IL, USA).
3. Results 3.1. Follow-up results The age of patients ranged from 23 to 85 years, with a mean of 51 years and a median of 51 years. One hundred and twenty-nine (32.9%) specimens were liquid-based preparations, and 263 (67.1%) were conventional smears. The age distribution of patients did not differ between the liquid-based and conventional methods (49.7 vs. 51.7 years, respectively; p = 0.152). Of all 392 cases, HSIL was detected in 111 cases (28.3%), squamous cell carcinoma was detected in 15 (3.8%) cases, LSIL was detected in 37 (9.4%) cases, reactive change was detected in 178 (45.4%) cases, atrophy was detected in 47 (12.0%) cases, and adenocarcinoma was detected in four (1.0%) cases. Follow-up results were stratified by cytological preparation method and are summarized in Table 1. There was no significant difference in the incidence of HSIL or greater between the conventional and liquid-based methods (35.4% and 28.7%, respectively; p = 0.187) (Table 2). 3.2. Significance of patient age for ASC-H smears In this study, 86 (21.9%) cases were aged <40 years. The followup results of these patients with ASC-H identified HSIL or greater in 45 (52.3%) cases. In the 306 (78.1%) cases who were aged 40
Table 2 Prevalence of high-grade squamous intraepithelial lesion (HSIL) or greater according to the clinicopathological parameters of women with atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion.
Method CS LBP Age (years) <40 40 HR-HPV DNA Positive Negative Previous Pap ASC-US >ASC-US
Total
HSIL or greater n (%)
LSIL or less n (%)
263 129
93 (35.4) 37 (28.7)
170 (64.6) 92 (71.3)
86 306
45 (52.3) 85 (27.8)
41(47.7) 221(72.2)
88 49
65 (73.9) 9 (18.4)
23 (26.1) 40 (81.6)
<0.001
160 35
30 (18.8) 17 (48.6)
130 (81.3) 18 (51.4)
<0.001
p-Value 0.187
<0.001
LSIL, low-grade squamous intraepithelial lesion; CS, conventional smear; LBP, liquid-based preparation; HR-HPV, high-risk human papillomavirus; ASC-US, atypical squamous cells of undetermined significance.
C.O. Sung et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 159 (2011) 155–159 Table 3 Summary of follow-up results for patients with atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion by age. Follow-up results
All ages n (%)
<40 years n (%)
HSIL Squamous cell carcinoma LSIL Reactive change Atrophy Adenocarcinoma Total
111 (28.3) 15 (3.8)
44 (51.2) 0 (0)
37 178 47 4 392
6 33 2 1 86
(9.4) (45.4) (12.0) (1.0) (100)
(7.0) (38.4) (2.3) (1.2) (100)
40 years n (%) 67 (21.9) 15 (4.9) 31 145 45 3 306
(10.1) (47.4) (14.7) (1.0) (100)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion.
years, 85 (27.8%) patients were diagnosed with HSIL or greater at follow-up (Table 3). ASC-H smears of younger patients (<40 years) were significantly associated with HSIL or greater at follow-up (p < 0.001). Only two (2.3%) patients aged <40 years were diagnosed with atrophy at follow-up, compared with 45 (14.7%) patients aged 40 years. However, all of the 15 patients with invasive squamous cell carcinoma detected at follow-up were aged 40 years. Representative smears are shown in Fig. 1. 3.3. HR-HPV DNA positivity for ASC-H smears stratified by age Table 4 shows the HR-HPV status for the ASC-H smears according to follow-up findings. The HPV test was performed in 41 (47.7%) of 86 women aged <40 years and 96 (31.4%) of 306 women aged 40 years, with positive results observed in 73.2% (30/41) and 60.4% (58/96), respectively. Of the ASC-H smears with HSIL or greater diagnosed at follow-up, positive HR-HPV results were found in 86.7% (26/30) of women aged <40 years and 86.6% (39/ 44) of women aged 40 years. HR-HPV-positive cases were significantly more likely to be diagnosed with HSIL or greater at
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Table 4 Follow-up results of high-risk human papillomavirus (HPV) in women with atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion aged <40 years and 40 years.
Age <40 years HSIL Squamous cell carcinoma LSIL Reactive change Atrophy Adenocarcinoma Total (n = 86) Age 40 years HSIL Squamous cell carcinoma LSIL Reactive change Atrophy Adenocarcinoma Total (n = 306)
HPV testing
Positive results n (%)
30 0 2 8 1 0 41
26 0 1 2 1 0 30
(86.7) (0) (50) (25) (100) (0) (73.2)
36 7 12 34 6 1 96
31 7 6 11 2 1 58
(86.1) (100) (50) (32.3) (33.3) (100) (60.4)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion.
follow-up (p < 0.001). When stratified into two age groups (<40 years vs. 40 years), HR-HPV positivity in ASC-H smears was significantly associated with HSIL or greater, irrespective of age (p = 0.003 and p < 0.001, respectively) (Table 5). 3.4. Associations between previous abnormal cytology and follow-up results Nearly half (49.7%, 195/392) of the cases had had other cytology smears in the previous 2 years. Specifically, the median intervals were as follows: ASC-H, 89 days; ASC-US, 98 days; atrophy, 261 days; HSIL or greater, 76 days; LSIL, 131 days; negative, 188 days.
Fig. 1. Examples of atypical squamous cells when high-grade squamous intra-epithelial lesions cannot be excluded (ASC-H). (A and B) Smears from women aged <40 years and (C) smears from women aged 40 years that were confirmed as cervical intra-epithelial neoplasia 3 at histological follow-up. (D) Smears from women aged <40 years and (E and F) smears from women aged 40 years that were confirmed as reactive lesions at histological follow-up. Papanicolaou-stained ThinPrep preparation, original magnification 400.
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Table 5 Prevalence of high-grade squamous intraepithelial lesion (HSIL) or greater by age among women with atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion. HR-HPV DNA
Positive Negative
Age <40 years
Age 40 years
Total
HSIL or greater n (%)
LSIL or less n (%)
p-Value
Total
HSIL or greater n (%)
LSIL or less n (%)
p-Value
30 11
26 (86.7) 4 (36.4)
4 (13.3) 7 (63.6)
0.003
58 40
39(67.2) 5(12.5)
19(32.8) 35(87.5)
<0.001
LSIL, low-grade squamous intraepithelial lesion; HR-HPV, high-risk human papillomavirus.
Table 6 Relationship between previous abnormal cytology and follow-up results in women with atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H). Previous cytology results
n
HSIL or greater (%)
Normal Atrophy ASC-US ASC-H LSIL HSIL or greater Total
101 28 31 18 9 7 195
19 4 7 7 3 7 47
(18.8) (14.3) (22.6) (38.9) (33.3) (100) (24.1)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; ASC-US, atypical squamous cells of undetermined significance.
Associations between previous cytology findings and follow-up results for ASC-H smears are summarized in Table 6. Of 195 cases, 24.1% were diagnosed with HSIL or greater at follow-up. Previous ASC-US cytology showed a similar detection rate for HSIL or greater (22.6%, 7/31) at follow-up. However, ASC-H with previous abnormal cytology of more than ASC-US showed an increased detection rate for HSIL or greater at follow-up (ASC-H, 38.9%; LSIL, 33.3%; HSIL or greater, 100%); this increase was statistically significant (p < 0.001). 4. Discussion The prevalence of HSIL or greater in cases of ASC-H cytology was 33.2% [95% confidence interval (CI) 28.5–37.8%]. The follow-up results for cases diagnosed with ASC-H on Pap smear vary between studies. Mokhtar et al. [13] reported that 59.4% of 123 cases that were diagnosed cytologically as ASC-H had HSIL on subsequent biopsies. Cytryn et al. [5] showed that the prevalence of CIN 2/3 in 57 cases of ASC-H cytology was 19.3%. Kietpeerakool et al. [9] reported that the prevalence of underlying CIN 2 or greater in 85 ASC-H smears was 69.4%. The prevalence of underlying HSIL or greater in the present study was relatively low compared with previous studies, in which it ranged from 24% to 94% [4]. The relatively low incidence of underlying HSIL in this study may be related to the fact that a large number of older patients (40 years) were included in the sample. Actually, 52.3% patients aged <40 years with ASC-H on Pap smear showed underlying HSIL or greater at follow-up. In addition, cases with negative colposcopic findings who were not biopsied but who had repeat smears soon after were included. As expected, only 7.3% (12/163; 95% CI 3.3–11.4%) were diagnosed as HSIL or greater at follow-up. Cytryn et al. [5] reported that the prevalence of HSIL among ASC-H cases was 19.3%, which included cases with negative colposcopic findings, as in the present study. For direct comparison with previous studies that only included histological diagnoses, the prevalence of HSIL or greater was recalculated in this subgroup of patients, resulting in a prevalence rate of 51.5% (118/229; 95% CI 45.0–58.1%). In this study, women aged <40 years had a higher incidence of HSIL or greater compared with women aged 40 years. These findings are consistent with those of previous studies in which
patients with a histological diagnosis of HSIL were younger than patients with a benign histological diagnosis after a Pap smear interpretation of ASC-H [6,14–16]. ASC-H is primarily considered when cells show small aggregates of metaplastic-appearing cells that demonstrate increased nucleus-to-cytoplasm ratios with finely granular chromatin and nuclear irregularities. ACS-H may show nuclear atypia associated with metaplasia, repair or atropy [17]. ASC-H can be difficult to distinguish from secondary changes, such as metaplasia, inflammation-associated change or atrophyrelated change. These secondary changes occur more frequently in older women, which may explain why ASC-H smears from younger patients have a higher incidence of HSIL or greater. At follow-up, no significant difference was found between conventional and liquid-based preparations for the incidence of HSIL or greater. Most published studies have shown no significant difference between the two preparation methods [6,8,10]. However, Sherman et al. [15] described a higher incidence of HSIL (CIN 2 or greater) when ThinPrep liquid-based preparations were used compared with conventional preparations (40.5% vs. 27.2%, respectively). However, in their study, women with ASC-H who had conventional smears tended to be older than those who had liquid-based smears. In contrast, in the present study and the study by Louro et al. [10], the age distributions for the two preparation methods were similar. In the present study, the HPV test was performed in 41 of 86 (47.7%) women aged <40 years and 96 of 306 (31.4%) women aged 40 years; positive results were observed in 73.2% (30/41) and 60.4% (58/96) of cases, respectively. HPV positivity was significantly related to underlying HSIL or greater at follow-up. HR-HPV DNA was detected more frequently in ASC-H smears than ASC-US smears [15], and it has been reported that HPV positivity is significantly related to secondary genetic alterations, such as p16 overexpression, in ASC-H smears compared with ASC-US smears [18]. For all histological outcomes, the false-positive rate in the HPV DNA screening test decreased significantly with increasing age; that is, the specificity of the HPV screening test decreased with decreasing age [19]. In this study, HR-HPV was detected more frequently in the younger age group than the older age group with ASC-H (73.2% vs. 60.4%, respectively). However, HR-HPV positivity in ASC-H smears was significantly related to underlying HSIL or greater, irrespective of age. This study explored the relationship between previous abnormal cytology and follow-up outcomes among women with ASC-H. Many studies have reported that approximately 10–15% of LSIL Pap tests are associated with HSIL or greater at histological follow-up [20–23]. However, the present study demonstrated that LSIL in ASC-H patients was more frequently associated with underlying HISL or greater at follow-up. This finding suggests that knowledge of previous abnormal cytology can help to predict underlying HSIL or greater in ASC-H smears. ASC-H diagnoses are poorly reproducible due to interobserver variability. Quddus et al. [6] reported poor interobserver agreement (kappa = 0.11) in 20 cases with ThinPrep liquid-based preparation. Although ASC-H is of clinical use because of its association with underlying HSIL or greater compared with that for ASC-US, it is still difficult to predict ASC-H outcomes and to
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understand it as a biological entity. This study has demonstrated that ASC-H is closely related to underlying HSIL or greater, especially in younger women (aged <40 years), women with smears testing positive for HPV DNA, and women with previous abnormal cytology. In conclusion, patient age, positive HPV DNA test and previous abnormal cytology can help to predict underlying HSIL or greater in women with ASC-H. References [1] Waggoner SE. Cervical cancer. Lancet 2003;361:2217–25. [2] Mathew A, George PS. Trends in incidence and mortality rates of squamous cell carcinoma and adenocarcinoma of cervix – worldwide. Asian Pac J Cancer Prev 2009;10:645–50. [3] Chivukula M, Shidham VB. ASC-H in Pap test – definitive categorization of cytomorphological spectrum. Cytojournal 2006;10:14. [4] Wright Jr TC, Cox JT, Massad LS, Twiggs LB, Wilkinson EJ. 2001 Consensus guidelines for the management of women with cervical cytological abnormalities. JAMA 2002;287:2120–9. [5] Cytryn A, Russomano FB, Camargo MJ, et al. Prevalence of cervical intraepithelial neoplasia grades II/III and cervical cancer in patients with cytological diagnosis of atypical squamous cells when high-grade intraepithelial lesions (ASC-H) cannot be ruled out. Sao Paulo Med J 2009;127:283–7. [6] Quddus MR, Sung CJ, Steinhoff MM, Lauchlan SC, Singer DB, Hutchinson ML. Atypical squamous metaplastic cells: reproducibility, outcome, and diagnostic features on ThinPrep Pap test. Cancer 2001;93:16–22. [7] Selvaggi SM. Reporting of atypical squamous cells, cannot exclude a highgrade squamous intraepithelial lesion (ASC-H) on cervical samples: is it significant? Diagn Cytopathol 2003;29:38–41. [8] Duncan LD, Jacob SV. Atypical squamous cells, cannot exclude a high-grade squamous intraepithelial lesion: the practice experience of a hospital-based reference laboratory with this new Bethesda system diagnostic category. Diagn Cytopathol 2005;32:243–6. [9] Kietpeerakool C, Srisomboon J, Tantipalakorn C, et al. Underlying pathology of women with ‘‘atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion’’ smears, in a region with a high incidence of cervical cancer. J Obstet Gynaecol Res 2008;34:204–9. [10] Louro AP, Roberson J, Eltoum I, Chhieng DC. Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion. A follow-up study of conventional and liquid-based preparations in a high-risk population. Am J Clin Pathol 2003;120:392–7.
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[11] Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda system: terminology for reporting results of cervical cytology. JAMA 2002;287:2114–9. [12] Jeong JH, Cho HY, Kim NR, Chung DH, Park S, Ha SY. The analysis and clinical usefulness of HPV DNA Chip test in the uterine cervix. Korean J Pathol 2010;44:77–82. [13] Mokhtar GA, Delatour NL, Assiri AH, Gilliatt MA, Senterman M, Islam S. Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion: cytohistologic correlation study with diagnostic pitfalls. Acta Cytol 2008;52:169–77. [14] Saad RS, Dabbs DJ, Kordunsky L, et al. Clinical significance of cytologic diagnosis of atypical squamous cells, cannot exclude high grade, in perimenopausal and postmenopausal women. Am J Clin Pathol 2006;126:381– 8. [15] Sherman ME, Solomon D, Schiffman M. Qualification of ASCUS. A comparison of equivocal LSIL and equivocal HSIL cervical cytology in the ASCUS LSIL Triage Study. Am J Clin Pathol 2001;116:386–94. [16] Srodon M, Parry Dilworth H, Ronnett BM. Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion: diagnostic performance, human papillomavirus testing, and follow-up results. Cancer 2006;108:32–8. [17] Sherman ME, Tabbara SO, Scott DR, et al. ‘‘ASCUS, rule out HSIL’’: cytologic features, histologic correlates, and human papillomavirus detection. Mod Pathol 1999;12:335–42. [18] Sung CO, Kim SR, Oh YL, Song SY. The use of p16(INK4A) immunocytochemistry in ‘‘atypical squamous cells which cannot exclude HSIL’’ compared with ‘‘atypical squamous cells of undetermined significance’’ in liquid-based cervical smears. Diagn Cytopathol 2010;38:168–71. [19] Leinonen M, Nieminen P, Kotaniemi-Talonen L, et al. Age-specific evaluation of primary human papillomavirus screening vs conventional cytology in a randomized setting. J Natl Cancer Inst 2009;101:1612–23. [20] Elsheikh TM, Kirkpatrick JL, Wu HH. The significance of ‘‘low-grade squamous intraepithelial lesion, cannot exclude high-grade squamous intraepithelial lesion’’ as a distinct squamous abnormality category in Papanicolaou tests. Cancer 2006;108:277–81. [21] Kir G, Cetiner H, Gurbuz A, Karateke A. Reporting of ‘‘LSIL with ASC-H’’ on cervicovaginal smears: is it a valid category to predict cases with HSIL followup? Eur J Gynaecol Oncol 2004;25:462–4. [22] Nasser SM, Cibas ES, Crum CP, Faquin WC. The significance of the Papanicolaou smear diagnosis of low-grade squamous intraepithelial lesion cannot exclude high-grade squamous intraepithelial lesion. Cancer 2003;99:272–6. [23] Shidham VB, Kumar N, Narayan R, Brotzman GL. Should LSIL with ASC-H (LSILH) in cervical smears be an independent category? A study on SurePath specimens with review of literature. Cytojournal 2007;4:7.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Cervical cytology of atypical squamous cells cannot exclude high-grade squamous intra-epithelial lesion: significance of age, human papillomavirus DNA detection and previous abnormal cytology on follow-up outcomes Chang Ohk Sung, Young Lyun Oh *, Sang Yong Song * Department of Pathology, Samsung Medical Centre, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 June 2010 Received in revised form 23 October 2010 Accepted 19 May 2011
Objective: Despite the usefulness of Pap tests for cancer screening, outcomes can be difficult to predict when atypical squamous cells (ASCs) are identified. According to the 2001 Bethesda system, ASCs can be subdivided into two groups: ASCs of undetermined significance (ASC-US); and ASCs, cannot exclude high-grade squamous intra-epithelial lesion (ASC-H). ASC-H interpretations are uncommon, and studies involving this type of lesion are based on small numbers of cases. Study design: Cross-sectional, retrospective study of 392 ASC-H cases. The follow-up outcomes of ASC-H cases that were diagnosed during routine primary screening between 2002 and 2008 were investigated, and relationships between clinicopathological parameters were assessed, particularly positive test for high-risk HPV (HPV) DNA, patient age at diagnosis and previous abnormal cytology. Results: Of the 392 cases, high-grade squamous intra-epithelial lesion (HSIL) was detected in 111 (28.3%) cases, squamous cell carcinoma was detected in 15 (3.8%) cases, low-grade squamous intra-epithelial lesion was detected in 37 (9.4%) cases, reactive change was detected in 178 (45.4%) cases, atrophy was detected in 47 (12.0%) cases, and adenocarcinoma was detected in four (1.0%) cases. The prevalence of HSIL or greater was 27.8% for women aged 40 years, and 52.3% for women aged <40 years (p < 0.001). HPV positivity in ASC-H smears was significantly associated with HSIL or greater, irrespective of age (<40 years, p = 0.003; 40 years, p < 0.001). ASC-H with previous abnormal cytology greater than ASCUS showed a significantly higher detection rate for HSIL or greater at follow-up (p < 0.001). Conclusions: Patient age, positive HPV DNA test and previous abnormal cytology are useful predictors of underlying HSIL or greater in women with ASC-H. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cytology Human papillomavirus Cervix Atypical squamous cells Follow-up
1. Introduction Worldwide, uterine cervical cancer is the second most common cancer among women [1]. Fortunately, the incidence has decreased due to widespread use of the Papanicolaou (Pap) test as a screening method [2]. Consequently, the detection rates of early cervical lesions have increased. However, despite the usefulness of the Pap test for cancer screening, it still has limitations including high false-negative rates and frequent interobserver variability. The presence of atypical cells in smears is one of the major diagnostic problems for cervical cancer screening. Typically, it is difficult to predict outcomes for patients with atypical cells in their smears. According to the 2001 Bethesda system, atypical squamous cells (ASCs) can be subdivided into two groups: ASCs of undetermined significance (ASC-US); and ASCs, cannot
* Corresponding authors. Tel.: +82 2 3410 2803; fax: +82 2 3410 0025. E-mail addresses: [email protected] (Y.L. Oh), [email protected] (S.Y. Song). 0301-2115/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2011.05.022
exclude high-grade squamous intra-epithelial lesion (ASC-H). The ASC-H category includes ASCs that exhibit equivocal features which are suggestive of a high-grade squamous intra-epithelial lesion (HSIL) but which are not sufficient to diagnose HSIL. It was recently demonstrated that ASC-H is not a homogeneous category, and has a broad spectrum of cytomorphological patterns that are associated with different clinicopathological categories classified by biopsy and human papillomavirus (HPV) test results [3]. ASC-H interpretations are uncommon. ASC-H represents 5– 10% of ASC diagnoses and accounts for approximately 0.2% of cytological interpretations [4]. A woman with a cervical cytology result interpreted as ASC-US has a 5–17% chance of having cervical intra-epithelial neoplasia (CIN) 2 or 3 confirmed by biopsy, while ASC-H is associated with a significant incidence of HSIL on followup compared with ASC-US [4–7]. However, the rates of underlying HSIL in patients with ASC-H range widely from 24% to 94% [4–6,8– 10], and studies involving these ASCs have included small numbers of cases. The present study investigated follow-up outcomes of 392 ASC-H cases identified during routine primary
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Table 1 Summary of follow-up results for patients with smears interpreted as atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion, by preparation method. Type of preparation
All cases n (%)
HSIL n (%)
Squamous cell carcinoma n (%)
LSIL n (%)
Reactive change n (%)
Atrophy n (%)
Adenocarcinoma n (%)
CS LBP Total
263 (100) 129 (100) 392 (100)
80 (30.4) 31 (24.0) 111 (28.3)
11 (4.2) 4 (3.1) 15 (3.8)
19 (7.2) 18 (14.0) 37 (9.4)
125 (47.5) 53 (41.1) 178 (45.4)
26 (9.9) 21 (16.3) 47 (12.0)
2 (0.7) 2 (1.6) 4 (1.0)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; CS, conventional smear; LBP, liquid-based preparation.
screening, and analysed the relationships between outcomes and clinicopathological parameters, particularly positive test for highrisk HPV (HR-HPV) DNA, patient age at diagnosis and previous abnormal cytology.
2. Materials and methods 2.1. Study design and case selection This was a cross-sectional study of retrospective data from patients who visited Samsung Seoul Hospital, Korea between July 2002 and August 2008. The hospital provides tertiary and secondary healthcare services, and also provides primary healthcare services through the healthcare centre. Most of the cervical smears in this study were obtained at the primary healthcare centre. Cases were identified by a electronic database search for diagnoses of ASC-H on cervicovaginal Pap smears in accordance with the 2001 Bethesda system. During the study period, most smears were read by the same group of 10 cytopathologists. Of 198,069 Pap smears read during this period, 448 (0.23%) cases were interpreted as ASC-H. Specifically, the rates were 0.22% and 0.23% for conventional and ThinPrep liquidbased preparations, respectively. All of the patients underwent colposcopy under supervision by experienced colposcopists. Of the 448 cases of ASC-H, 229 cases had follow-up tissue sampling within 6 months, which included colposcopic biopsy, loop electrosurgical excision procedure, conization and hysterectomy. Histological specimens (e.g., normal colposcopy or minor abnormalities) were not obtained for 163 of the 448 cases. These women underwent repeated follow-up cytological tests for 6–12 months after the initial abnormal smear. The remaining 56 cases, for whom colposcopy was unsatisfactory and there were no follow-up cytological tests, were considered to be lost to followup and were excluded from the study. As such, 392 Pap smears, including 263 conventional smears and 129 liquid-based cytological smears, were included in this study. The follow-up results were classified as HSIL, squamous cell carcinoma, low-grade squamous intra-epithelial lesion (LSIL), reactive change, atrophy or adenocarcinoma. For this study, histological diagnoses of CIN 1 were classified as LSIL, and diagnoses of CIN 2 and 3 were classified as HSIL. Cytological diagnoses and adequacy of specimens were classified using the 2001 Bethesda system for cervical cytology [11]. 2.2. Detection of HR-HPV DNA To determine the viral load, 137 smears were evaluated for HRHPV DNA using the Hybrid Capture II (Digene Corporation, Gaithersburg, MD, USA) or HPV DNA microchip (Mygene Co., Seoul, Korea) methods [12] in accordance with the manufacturers’ protocols. For the Hybrid Capture II test, the panel of HR-HPVcontaining specimens included 13 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). For HPV DNA microchip analysis, 15 HR-HPV types were included (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 54, 56, 58, 59, 68).
2.3. Statistical analysis Statistical significance was determined on the basis of Chisquared test or Fisher’s exact test, as appropriate. Independent two-sample t-tests were used for comparative analysis of continuous data. All tests were two-sided, and p-values <0.05 were considered to be statistically significant. Statistical analysis was performed using Statistical Package for the Social Sciences Version 12.0 (SPSS Inc., Chicago, IL, USA).
3. Results 3.1. Follow-up results The age of patients ranged from 23 to 85 years, with a mean of 51 years and a median of 51 years. One hundred and twenty-nine (32.9%) specimens were liquid-based preparations, and 263 (67.1%) were conventional smears. The age distribution of patients did not differ between the liquid-based and conventional methods (49.7 vs. 51.7 years, respectively; p = 0.152). Of all 392 cases, HSIL was detected in 111 cases (28.3%), squamous cell carcinoma was detected in 15 (3.8%) cases, LSIL was detected in 37 (9.4%) cases, reactive change was detected in 178 (45.4%) cases, atrophy was detected in 47 (12.0%) cases, and adenocarcinoma was detected in four (1.0%) cases. Follow-up results were stratified by cytological preparation method and are summarized in Table 1. There was no significant difference in the incidence of HSIL or greater between the conventional and liquid-based methods (35.4% and 28.7%, respectively; p = 0.187) (Table 2). 3.2. Significance of patient age for ASC-H smears In this study, 86 (21.9%) cases were aged <40 years. The followup results of these patients with ASC-H identified HSIL or greater in 45 (52.3%) cases. In the 306 (78.1%) cases who were aged 40
Table 2 Prevalence of high-grade squamous intraepithelial lesion (HSIL) or greater according to the clinicopathological parameters of women with atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion.
Method CS LBP Age (years) <40 40 HR-HPV DNA Positive Negative Previous Pap ASC-US >ASC-US
Total
HSIL or greater n (%)
LSIL or less n (%)
263 129
93 (35.4) 37 (28.7)
170 (64.6) 92 (71.3)
86 306
45 (52.3) 85 (27.8)
41(47.7) 221(72.2)
88 49
65 (73.9) 9 (18.4)
23 (26.1) 40 (81.6)
<0.001
160 35
30 (18.8) 17 (48.6)
130 (81.3) 18 (51.4)
<0.001
p-Value 0.187
<0.001
LSIL, low-grade squamous intraepithelial lesion; CS, conventional smear; LBP, liquid-based preparation; HR-HPV, high-risk human papillomavirus; ASC-US, atypical squamous cells of undetermined significance.
C.O. Sung et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 159 (2011) 155–159 Table 3 Summary of follow-up results for patients with atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion by age. Follow-up results
All ages n (%)
<40 years n (%)
HSIL Squamous cell carcinoma LSIL Reactive change Atrophy Adenocarcinoma Total
111 (28.3) 15 (3.8)
44 (51.2) 0 (0)
37 178 47 4 392
6 33 2 1 86
(9.4) (45.4) (12.0) (1.0) (100)
(7.0) (38.4) (2.3) (1.2) (100)
40 years n (%) 67 (21.9) 15 (4.9) 31 145 45 3 306
(10.1) (47.4) (14.7) (1.0) (100)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion.
years, 85 (27.8%) patients were diagnosed with HSIL or greater at follow-up (Table 3). ASC-H smears of younger patients (<40 years) were significantly associated with HSIL or greater at follow-up (p < 0.001). Only two (2.3%) patients aged <40 years were diagnosed with atrophy at follow-up, compared with 45 (14.7%) patients aged 40 years. However, all of the 15 patients with invasive squamous cell carcinoma detected at follow-up were aged 40 years. Representative smears are shown in Fig. 1. 3.3. HR-HPV DNA positivity for ASC-H smears stratified by age Table 4 shows the HR-HPV status for the ASC-H smears according to follow-up findings. The HPV test was performed in 41 (47.7%) of 86 women aged <40 years and 96 (31.4%) of 306 women aged 40 years, with positive results observed in 73.2% (30/41) and 60.4% (58/96), respectively. Of the ASC-H smears with HSIL or greater diagnosed at follow-up, positive HR-HPV results were found in 86.7% (26/30) of women aged <40 years and 86.6% (39/ 44) of women aged 40 years. HR-HPV-positive cases were significantly more likely to be diagnosed with HSIL or greater at
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Table 4 Follow-up results of high-risk human papillomavirus (HPV) in women with atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion aged <40 years and 40 years.
Age <40 years HSIL Squamous cell carcinoma LSIL Reactive change Atrophy Adenocarcinoma Total (n = 86) Age 40 years HSIL Squamous cell carcinoma LSIL Reactive change Atrophy Adenocarcinoma Total (n = 306)
HPV testing
Positive results n (%)
30 0 2 8 1 0 41
26 0 1 2 1 0 30
(86.7) (0) (50) (25) (100) (0) (73.2)
36 7 12 34 6 1 96
31 7 6 11 2 1 58
(86.1) (100) (50) (32.3) (33.3) (100) (60.4)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion.
follow-up (p < 0.001). When stratified into two age groups (<40 years vs. 40 years), HR-HPV positivity in ASC-H smears was significantly associated with HSIL or greater, irrespective of age (p = 0.003 and p < 0.001, respectively) (Table 5). 3.4. Associations between previous abnormal cytology and follow-up results Nearly half (49.7%, 195/392) of the cases had had other cytology smears in the previous 2 years. Specifically, the median intervals were as follows: ASC-H, 89 days; ASC-US, 98 days; atrophy, 261 days; HSIL or greater, 76 days; LSIL, 131 days; negative, 188 days.
Fig. 1. Examples of atypical squamous cells when high-grade squamous intra-epithelial lesions cannot be excluded (ASC-H). (A and B) Smears from women aged <40 years and (C) smears from women aged 40 years that were confirmed as cervical intra-epithelial neoplasia 3 at histological follow-up. (D) Smears from women aged <40 years and (E and F) smears from women aged 40 years that were confirmed as reactive lesions at histological follow-up. Papanicolaou-stained ThinPrep preparation, original magnification 400.
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Table 5 Prevalence of high-grade squamous intraepithelial lesion (HSIL) or greater by age among women with atypical squamous cell, cannot exclude high-grade squamous intraepithelial lesion. HR-HPV DNA
Positive Negative
Age <40 years
Age 40 years
Total
HSIL or greater n (%)
LSIL or less n (%)
p-Value
Total
HSIL or greater n (%)
LSIL or less n (%)
p-Value
30 11
26 (86.7) 4 (36.4)
4 (13.3) 7 (63.6)
0.003
58 40
39(67.2) 5(12.5)
19(32.8) 35(87.5)
<0.001
LSIL, low-grade squamous intraepithelial lesion; HR-HPV, high-risk human papillomavirus.
Table 6 Relationship between previous abnormal cytology and follow-up results in women with atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H). Previous cytology results
n
HSIL or greater (%)
Normal Atrophy ASC-US ASC-H LSIL HSIL or greater Total
101 28 31 18 9 7 195
19 4 7 7 3 7 47
(18.8) (14.3) (22.6) (38.9) (33.3) (100) (24.1)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; ASC-US, atypical squamous cells of undetermined significance.
Associations between previous cytology findings and follow-up results for ASC-H smears are summarized in Table 6. Of 195 cases, 24.1% were diagnosed with HSIL or greater at follow-up. Previous ASC-US cytology showed a similar detection rate for HSIL or greater (22.6%, 7/31) at follow-up. However, ASC-H with previous abnormal cytology of more than ASC-US showed an increased detection rate for HSIL or greater at follow-up (ASC-H, 38.9%; LSIL, 33.3%; HSIL or greater, 100%); this increase was statistically significant (p < 0.001). 4. Discussion The prevalence of HSIL or greater in cases of ASC-H cytology was 33.2% [95% confidence interval (CI) 28.5–37.8%]. The follow-up results for cases diagnosed with ASC-H on Pap smear vary between studies. Mokhtar et al. [13] reported that 59.4% of 123 cases that were diagnosed cytologically as ASC-H had HSIL on subsequent biopsies. Cytryn et al. [5] showed that the prevalence of CIN 2/3 in 57 cases of ASC-H cytology was 19.3%. Kietpeerakool et al. [9] reported that the prevalence of underlying CIN 2 or greater in 85 ASC-H smears was 69.4%. The prevalence of underlying HSIL or greater in the present study was relatively low compared with previous studies, in which it ranged from 24% to 94% [4]. The relatively low incidence of underlying HSIL in this study may be related to the fact that a large number of older patients (40 years) were included in the sample. Actually, 52.3% patients aged <40 years with ASC-H on Pap smear showed underlying HSIL or greater at follow-up. In addition, cases with negative colposcopic findings who were not biopsied but who had repeat smears soon after were included. As expected, only 7.3% (12/163; 95% CI 3.3–11.4%) were diagnosed as HSIL or greater at follow-up. Cytryn et al. [5] reported that the prevalence of HSIL among ASC-H cases was 19.3%, which included cases with negative colposcopic findings, as in the present study. For direct comparison with previous studies that only included histological diagnoses, the prevalence of HSIL or greater was recalculated in this subgroup of patients, resulting in a prevalence rate of 51.5% (118/229; 95% CI 45.0–58.1%). In this study, women aged <40 years had a higher incidence of HSIL or greater compared with women aged 40 years. These findings are consistent with those of previous studies in which
patients with a histological diagnosis of HSIL were younger than patients with a benign histological diagnosis after a Pap smear interpretation of ASC-H [6,14–16]. ASC-H is primarily considered when cells show small aggregates of metaplastic-appearing cells that demonstrate increased nucleus-to-cytoplasm ratios with finely granular chromatin and nuclear irregularities. ACS-H may show nuclear atypia associated with metaplasia, repair or atropy [17]. ASC-H can be difficult to distinguish from secondary changes, such as metaplasia, inflammation-associated change or atrophyrelated change. These secondary changes occur more frequently in older women, which may explain why ASC-H smears from younger patients have a higher incidence of HSIL or greater. At follow-up, no significant difference was found between conventional and liquid-based preparations for the incidence of HSIL or greater. Most published studies have shown no significant difference between the two preparation methods [6,8,10]. However, Sherman et al. [15] described a higher incidence of HSIL (CIN 2 or greater) when ThinPrep liquid-based preparations were used compared with conventional preparations (40.5% vs. 27.2%, respectively). However, in their study, women with ASC-H who had conventional smears tended to be older than those who had liquid-based smears. In contrast, in the present study and the study by Louro et al. [10], the age distributions for the two preparation methods were similar. In the present study, the HPV test was performed in 41 of 86 (47.7%) women aged <40 years and 96 of 306 (31.4%) women aged 40 years; positive results were observed in 73.2% (30/41) and 60.4% (58/96) of cases, respectively. HPV positivity was significantly related to underlying HSIL or greater at follow-up. HR-HPV DNA was detected more frequently in ASC-H smears than ASC-US smears [15], and it has been reported that HPV positivity is significantly related to secondary genetic alterations, such as p16 overexpression, in ASC-H smears compared with ASC-US smears [18]. For all histological outcomes, the false-positive rate in the HPV DNA screening test decreased significantly with increasing age; that is, the specificity of the HPV screening test decreased with decreasing age [19]. In this study, HR-HPV was detected more frequently in the younger age group than the older age group with ASC-H (73.2% vs. 60.4%, respectively). However, HR-HPV positivity in ASC-H smears was significantly related to underlying HSIL or greater, irrespective of age. This study explored the relationship between previous abnormal cytology and follow-up outcomes among women with ASC-H. Many studies have reported that approximately 10–15% of LSIL Pap tests are associated with HSIL or greater at histological follow-up [20–23]. However, the present study demonstrated that LSIL in ASC-H patients was more frequently associated with underlying HISL or greater at follow-up. This finding suggests that knowledge of previous abnormal cytology can help to predict underlying HSIL or greater in ASC-H smears. ASC-H diagnoses are poorly reproducible due to interobserver variability. Quddus et al. [6] reported poor interobserver agreement (kappa = 0.11) in 20 cases with ThinPrep liquid-based preparation. Although ASC-H is of clinical use because of its association with underlying HSIL or greater compared with that for ASC-US, it is still difficult to predict ASC-H outcomes and to
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