GYNECOLOGIC ONCOLOGY

Cervical cytology with a diagnosis of atypical squamous cells,cannot exclude high-grade squamous intraepithelial lesion(ASC-H): a follow-up study with corresponding histologyand significance of predicting dysplasia by human papillomavirus(HPV) DNA testing

Syed M. Gilani • Randy Tashjian • Lamia Fathallah

Received: 2 February 2013 / Accepted: 23 August 2013 / Published online: 4 September 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract

Objectives To evaluate the clinical significance of

‘‘atypical squamous cells, cannot exclude high-grade

squamous intraepithelial lesion’’ ASC-H by comparing the

original cytologic findings with follow-up tissue biopsies,

and its association with high-risk HPV.

Methods A total of 235,518 ThinPrep Pap tests were

performed at our institution from January 2008 through

December 2010, but only 727 (0.3 %) of these cases were

diagnosed as ASC-H.

Results Of the 309 cases diagnosed as ASC-H on cytol-

ogy for which follow-up histologic material was available,

120 (38.8 %) were definitively diagnosed as high-grade

dysplasia (CIN 2/3) and 75 (24.2 %) showed features of

low-grade dysplasia (CIN 1). We observed that the inci-

dence of dysplasia in patients less than 30 years of age was

73.4 % (113/154) and 48.3 % (14/29) in patients greater

than 49 years of age (p = 0.001). There were 71 cases for

which high-risk HPV DNA testing was conducted. HPV

DNA was found to be positive in 41 of the dysplastic cases

(CIN 1 = 18 cases and CIN 2/3 = 23) and negative in six

of the dysplastic cases (CIN1 = 2 and CIN2/3 = 4).

Conclusion We conclude that cases diagnosed as ASC-H

should be followed-up with caution as they are strongly

associated with dysplasia of any grade (63.1 %), especially

high-grade dysplasia (38.8 %). Reflex HPV DNA testing is

an important predictor of dysplasia with a positive pre-

dictive value of 87.2 % in our study.

Keywords Atypical squamous cells � High grade �Cervical biopsy � HPV � Follow-up

Introduction

The Pap smear is simple, cost-effective, and accurate tool

for the initial screening of cervical pathology. Over the

course of its existence, the reporting nomenclature of cer-

vical pathology has been revised and standardized, with the

Bethesda System for Reporting Cervical Cytology cur-

rently in use. The majority of Pap smears are diagnosed

into specific categories with very little difficulty. Occa-

sionally, however, some borderline cases may be assigned

to the ‘‘atypical squamous cells’’ (ASC) category.

According to 2001 Bethesda System for Reporting Cervical

Cytology, two subcategories of ASC exist: the first is

‘‘atypical squamous intraepithelial lesion with undermined

significance’’ (ASC-US) and other is ‘‘atypical squamous

cells, cannot exclude high-grade squamous intraepithelial

lesion’’ (ASC-H). Patient management and follow-up is

different for each subcategory, in that patients with a

diagnosis of ASC-H require closer and more frequent fol-

low-up than those with a diagnosis of ASC-US. As such, it

is essential for the Pathologist to make a distinction

between the two subcategories to better guide patient

treatment.

A diagnosis of ASC-H is rendered infrequently, and the

result for human papilloma virus (HPV) DNA testing

varies depending on the patient’s age. Previous studies

have demonstrated that cases that are diagnosed as ASC-H

have a greater tendency to exhibit HPV DNA positivity as

compared to those cases that are diagnosed as ASC-US.

In this study, our primary objective was to compare Pap

smears findings with the results obtained from detailed

S. M. Gilani (&) � R. Tashjian � L. Fathallah

Department of Pathology, St. John Hospital and Medical Center,

22101 Moross Road, CCB-SB, Detroit, MI 48236, USA

e-mail: magilani@hotmail.com

123

Arch Gynecol Obstet (2014) 289:645–648

DOI 10.1007/s00404-013-3015-5

microscopic examination of the subsequent cervical tissue

material for each patient who received a diagnosis of

ASC-H. Our aim was to determine the degree of correlation

between a diagnosis of ASC-H rendered on Pap smear and

biopsy-proven high-grade cervical dysplasia. The second

objective of this study was to determine the incidence of a

positive HPV DNA test in patients diagnosed with ASC-H

on Pap smear and high-grade dysplasia on follow-up.

Material and methods

Prior to its initiation, this study was reviewed and approved

by the Institutional Review Board Committee (IRB) at the

parent institution, Saint John Hospital and Medical Center

in Detroit, Michigan. We retrospectively reviewed our

laboratory information system database for all cases of

adult female patients diagnosed with ‘‘atypical squamous

intraepithelial lesion, cannot rule out high grade’’ (ASC-H)

on cytology from January 2008 through December 2010.

The cytology (Pap smear) and corresponding histology

(biopsy and excision) reports and slides were reviewed by a

Pathologist for specific inclusion criteria. The Pap smears

were prepared using a liquid-based method technique fol-

lowing the manufacturer’s guidelines (ThinPrep 3000;

Hologic, Inc., Marlborough, MA) and stained utilizing the

standard Papanicolaou technique. These cytology slides

were screened by cytotechnologists, and cases requiring

review by a Cytopathologist were assessed for accuracy.

The final reports for HPV DNA testing performed on all

patients with a diagnosis of ASC-H diagnosed during the

above-mentioned time period were also reviewed. HPV

DNA testing was performed with the Digene Hybrid

Capture II method (Qiagen, Gaithersburg, MD). Follow-up

biopsy material, when available, was used for comparison

purposes and to establish a definitive diagnosis.

The inclusion criteria included all ASC-H cases diag-

nosed on Pap smear with subsequent histology with an age

range of 18–90 years. Histologic follow-up that was

included in this study consisted of cervical biopsies,

endocervical curettages, cervical conizations, and vaginal

biopsies. The cases that did not meet the inclusion criteria,

as well as those cases with previous history of dysplasia of

any grade, were excluded from the study. A total of

235,518 ThinPrep Pap tests were performed at our insti-

tution during the time period mentioned above, but only

727 (0.3 %) of these cases were diagnosed as ASC-H. Of

these 727 cases, histologic follow-up was available for 309

cases; the remaining 418 cases were excluded from the

study. The average age of the patients in this population

was 32.53 years with a standard deviation of ±11.52 years,

and the mean follow-up period was 4.09 months with a

standard deviation of ±5.32 months.

Results

Of the 309 cases diagnosed as ASC-H on cytology for

which follow-up histologic material was available, 120

(38.8 %) were definitively diagnosed as high-grade dys-

plasia [cervical intraepithelial neoplasia (CIN) 2/3].

Another 75 (24.2 %) cases showed features of low-grade

dysplasia (CIN 1) on subsequent evaluation of the corre-

sponding biopsy. Inflammation and/or reactive atypia were

detected in 55 (17.8 %) cases. Interestingly, no transfor-

mation zone was present for evaluation in 20 (6.5 %) of the

cases (Table 1). We observed that the incidence of dys-

plasia in patients below the age of 30 years was 73.4 %

(113/154) and it was 48.3 % (14/29) in patients over

49 years of age (p = 0.001). Of the patients in our study

who were diagnosed with ASC-H on cytology, CIN 1 was

observed in 48 (31.1 %) individuals under the age of

30 years, 24 (19 %) individuals between ages 30 and

49 years, and 3 (10.3 %) individuals over the age of

49 years. Sixty-five patients (42.2 %) under 30 years of

age, 44 (35 %) between the ages of 30 and 49 years, and 11

(38.1 %) over 49 years of age who were diagnosed as

having ASC-H on Pap smear were found to have CIN 2/3

on assessment of the follow-up biopsy samples (Table 2).

There were 71 cases for which high-risk HPV DNA

testing was conducted. HPV DNA was found to be positive

in 41 of the dysplastic cases (18 cases of CIN 1 and 23

cases of CIN 2/3) and negative in six of the dysplastic cases

(two cases of CIN 1 and four cases of CIN 2/3). As for the

24 cases that lacked dysplasia (i.e., those cases diagnosed

as benign, inflammation, and reactive atypia), 14 were

HPV DNA positive and 10 were HPV DNA negative

[sensitivity 74.5 %, specificity 62.5 %, positive predictive

value (PPV) 87.2 %, negative predictive value (NPV)

41.7 %, p = 0.006] (Table 3).

Discussion

Atypical squamous cells, cannot exclude high-grade squa-

mous intraepithelial lesion (ASC-H) is a term that was

introduced in the 2001 Bethesda Reporting System for

Cervical Cytology, which provides an elaboration of the

morphologic criteria of the squamous cells that fall into this

category [1]. These cells usually exhibit focal features

suggestive of, but not diagnostic of, a high-grade cervical

intraepithelial lesion. Significant interobserver variability is

inherent in the diagnosis of ASC-H. Several studies have

reported a wide spectrum of concordance between cases

diagnosed as ASC-H on cytology specimens and follow-up

biopsy specimens with a diagnosis of high-grade cervical

intraepithelial neoplasia (CIN 2/3), ranging from 10 to 85 %

[2–7]. Galliano et al. [2] reported a 68.3 % risk of

646 Arch Gynecol Obstet (2014) 289:645–648

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high-grade intraepithelial lesion irrespective of the patient’s

HPV status. Mokhtar et al. [6] studied 123 cases of ASC-H

and found that high-grade dysplasia was present in 59.4 %

of these cases, while McHale et al. [7] reported a cumulative

risk of high-grade dysplasia of 12.2 %. Due to an increased

risk of developing dysplasia, especially high-grade dys-

plasia, in cases diagnosed as ASC-H on cytology, an initial

triage colposcopic examination is the recommended man-

agement approach [8]. According to guidelines established

by the American Society for Colposcopy and Cervical

Pathology (ASCCP), an initial colposcopy is required for

patients diagnosed with ASC-H. Many premalignant lesions

and malignant lesions may be visualized directly by an

experienced clinician during colposcopic examination,

allowing for targeted retrieval of biopsy specimens. Accu-

rate histologic evaluation of these samples may then be

possible. If no high-grade dysplasia is identified on the

colposcopic biopsy sample, then the recommended follow-

up algorithm consists of either reflex HPV DNA testing at

12 months or repeat cervical cytologic evaluation at 6 or

12 months [9]. It is clear that the importance of a colpo-

scopic examination cannot be overstated. Indeed,

patient management differs significantly based on the col-

poscopy and biopsy results. While the use of HPV DNA

testing may aid in the triage of patients diagnosed with

ASC-H, it certainly is not the only tool in accomplishing

this goal. Ultimately, accurate diagnosis is mandatory, as

patients diagnosed with ASC-H are usually managed more

aggressively than are patients with a diagnosis of ASC-US.

In our study, the observed risk for high-grade dysplasia in

patients diagnosed with ASC-H was 38.8 %. The overall

incidence of both low-grade dysplasia and high-grade dys-

plasia was 63.1 %, which is within the range reported in other

studies [2–7]. Patton et al. [10] emphasized the significance of

age in the predicting the risk of dysplasia in ASC-H cases,

while Kietpeerakool et al. [11] found no statistical correlation

in predicting high-grade dysplasia in ASC-H cases when

comparing female patients under the age of 40 years with

those over the age of 40 years. Similarly, we found statistical

significance in the incidence of both low- and high-grade

dysplasia between women under the age of 30 years and

women over the age of 49 years (p value = 0.001).

Over the last few years, a growing debate has developed

over the utility of HPV DNA testing as the initial approach to

patients with a diagnosis of ASC-H on cytology. Several

studies have highlighted the important role that reflex HPV

DNA testing plays in the management of ASC-H patients

because cases that are negative for HPV DNA may be fol-

lowed-up with repeat cytology instead of an extensive and

invasive workup for a high-grade lesion [12–14]. However,

many studies have reported a wide range of HPV DNA

positivity in ASC-H cases, ranging between 33.3 and 85.6 %

[12–15]. This variability may possibly be due to the patient

population being studied. Sherman et al. [13] found a 40 %

HPV DNA positivity rate in women over the age of 35 years,

whereas Bandyopadhyay et al. [12] stated a rate of 36.5 %

HPV DNA positivity in the women over the age of 40 years

and 54.7 % HPV DNA positivity in women under the age of

Table 1 Detailed breakdown of the number of cases in each category

Years of age CIN 1 (%) CIN 2/3 (%) No transformation zone (%) Reactive/normal (%) Total

\30 48 (31.1) 65 (42.2) 7 (4.5) 34 (22.1) 154

30–49 24 (19.0) 44 (35) 9 (7.1) 49 (38.9) 126

[49 3 (10.3) 11 (38) 4 (13.7) 11 (38.0) 29

Total 75 (24.3) 120 (38.8) 20 (6.5) 94 (30.4) 309

Table 2 Comparison of the number of cases in patients \30 years of age and [49 years of age

Years of age CIN 1 (%) CIN 2/3 (%) No transformation zone (%) Inflammation (%) Normal (%) Total

\30 48 (31.1) 65 (42.2) 7 (4.5) 16 (10.4) 18 (11.7) 154

[49 3 (10.3) 11 (37.9) 4 (13.8) 6 (20.7) 5 (17.2) 29

Total 51 (27.8) 76 (41.5) 11 (6.0) 22 (12.0) 23 (12.6) 183

Table 3 Overall breakdown of HPV status in each category

HPV status CIN1 CIN2/3 No transformation zone Reactive/normal Total

Positive 18 23 2 12 55

Negative 2 4 2 8 16

Total 20 27 4 20 71

Arch Gynecol Obstet (2014) 289:645–648 647

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40 years. In our study, we found an overall PPV of 88.6 %

for HPV DNA testing, such that a positive test for HPV DNA

correlated to dysplasia of any grade in the majority of cases.

This value is near the upper limit of the range reported in the

literature. Close to two-thirds (62.4 %) of our cases with a

positive HPV DNA test were found to have high-grade

dysplasia (CIN 2/3) on microscopic evaluation of follow-up

tissue samples, translating into a relatively low specificity.

Four out of the 28 cases diagnosed as CIN 2/3 were negative

for HPV DNA testing, and one out of 15 cases diagnosed as

CIN 1 was negative for HPV DNA testing.

Based on our findings, it is advisable to perform HPV

DNA testing in combination with other diagnostic modal-

ities when assessing high-grade squamous intraepithelial

lesions (HSIL), because the majority of CIN 2/3 cases are

positive for HPV DNA [16]. In such instances, a colpo-

scopic examination with histologic assessment of biopsy

material is necessary to properly triage and treat these

patients, as management is dependent upon the histopath-

ologic findings. A negative HPV DNA test result, however,

does not definitively exclude the presence of high-grade

dysplasia, as 4 out of 27 (14.8 %) of our cases with high-

grade dysplasia were negative for the presence of HPV

DNA. ASC-H patients with a negative result still require

regular and close follow-up, and colposcopy in such

patients is also crucial for accurate triage and management.

Conclusion

We conclude that cases diagnosed as ASC-H should be fol-

lowed-up with caution, as they are strongly associated with

dysplasia of any grade (63.1 %), and especially high-grade

dysplasia (38.8 %). Reflex HPV DNA testing is an important

predictor of dysplasia with a positive predictive value of 87.2 %

in our study. Reactive and atrophic changes may be misinter-

preted as atypia, and as a result these cases require careful

evaluation. In these instances, knowledge of the patient’s his-

tory is important to avoid overcalling these benign findings as

dysplasia. Sampling error, such as failure to adequately sample

the transformation zone and sampling of the non-representative

areas during colposcopy, are the most common factors that

produce discrepant results between Pap smears and their cor-

responding subsequent biopsy. Close follow-up of cytology

cases diagnosed as ASC-H is recommended.

Conflict of interest We declare that we have no conflict of interest.

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