DOI:10.1111/j.1365-2303.2010.00780.

x
Atypical squamous cells and low-grade squamous intraepithelial
lesion in cervical cytology: cytohistological correlation and
implication for management in a low-resource setting
N. Gupta*, R. Srinivasan*, R. Nijhawan*, A. Rajwanshi*, P. Dey*, V. Suri and
L. Dhaliwal
*Department of Cytology and Gynecological Pathology and Department of Obstetrics and Gynecology, Postgraduate Institute of
Medical Education and Research, Chandigarh, India
Accepted for publication 12 April 2010
N. Gupta, R. Srinivasan, R. Nijhawan, A. Rajwanshi, P. Dey, V. Suri and L. Dhaliwal
Atypical squamous cells and low-grade squamous intraepithelial lesion in cervical cytology:
cytohistological correlation and implication for management in a low-resource setting
Objectives: To perform an audit of all cervical smears reported as atypical squamous cells (ASC) and low-grade
squamous intraepithelial lesion (LSIL) as in the Bethesda system (TBS) 2001, and determine their histological
follow-up and outcome when available, in order to dene the threshold for colposcopic referral.
Material and methods: A total of 25 203 cervical smears were screened over a period of 3 years (January
2006 December 2008) and all ASC and LSIL smears were reviewed with the corresponding histological follow-
up. All cervical intraepithelial neoplasia (CIN) grade 2 lesions and above (CIN2+) were considered as clinically
signicant lesions for analysis.
Results: Out of 25 203 cervical smears, 424 (1.7%) were reported as ASC and 113 (0.4%) as LSIL. Additionally,
three were reported as atypical cells, not otherwise specied. The ASC : SIL ratio was 2.18 : 1. Follow-up
histology was available in 153 (36.8%) of the ASC cases and revealed CIN2+ lesions in 22 (14.4%). Follow-up
histology was available in 50 (44.2%) of LSIL cases and revealed clinically signicant abnormalities in ve (10%),
all of which were CIN2. CIN3 1 and invasive squamous carcinomas were seen in 5.9% and 1.4%, respectively, of
cases of ASC, and not seen in LSIL. Reclassication of ASC smears into ASC-US (ASC-undetermined signicance)
and ASC-H (ASC- high grade SIL not excluded) revealed ASC-H in 2.6% of all ASC smears, with a clinically
signicant outcome in 45.4%.
Conclusion: In a low-resource setting where human papillomavirus testing is unaffordable, the threshold for
colposcopic referral and follow-up histology should be ASC rather than SIL.
Keywords: Cervical smear, atypical squamous cells, squamous intraepithelial lesion, invasive cervical cancer,
ASC-US, ASC, LSIL, ASC-H, cytohistological correlation
Introduction
The Bethesda system (TBS) for reporting cervical
cytology introduced the term atypical squamous cells
of undetermined signicance (ASC-US) to cover the
broad zone separating normal cytomorphology from
denitive squamous intraepithelial lesions (SIL),
while a separate category is used for ASC in which
high-grade SIL cannot be excluded (ASC-H); the two
categories include cases previously classied as AS-
CUS.
1
2 Atypical squamous cells is dened as an
epithelial cell abnormality that exceeds those abnor-
malities attributable to a benign reactive process but
falls short of a diagnosis of SIL. Therefore, ASC in fact
reects the limitation of the technique and the
interpretative abilities of cytoscreeners and cytopa-
thologists to categorize a particular lesion into a
denite category of SIL. It is a common abnormal
C Y T
7 8 0
B
Dispatch: 24.6.10 Journal: CYT CE: Chandra
Journal Name Manuscript No. Author Received: No. of pages: 6 PE: Ramyaa
-
Correspondence:
Dr Radhika Srinivasan, Additional Professor, Department of
Cytology and Gynecological Pathology, Postgraduate Insti-
tute of Medical Education and Research, Chandigarh, India
160012
Tel.: +91 172 2755119; Fax: +91 172 2744401;
E-mail: drsradhika@gmail.com
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cytology result, and a substantial proportion of
clinically signicant lesions representing high-grade
disease, dened as cervical intraepithelial neoplasia
(CIN) grade 2 (CIN2) or CIN3 or even invasive cancer,
occurs among women presenting with this equivocal
interpretation. Likewise, there are dilemmas in the
management of patients with a diagnosis of ASC.
2
Follow-up with repeat cytological examination,
human papillomavirus (HPV) DNA testing and imme-
diate colposcopy are the three options that have been
tested for triaging women with low grade lesions in
different settings, including the ASCUS-LSIL Triage
study,
3,4
and have formed the basis of recommenda-
tions for the management of these women.
5
However,
HPV testing may not be affordable by many women in
developing countries and hence there is a need to
develop suitable strategies in such settings as well. It
has also been debated whether the threshold for
colposcopy should be ASC-US or LSIL, and Sodhani et
al
6
, in a study from India, have recommended LSIL as
the threshold for colposcopy. Development of a
suitable management strategy requires knowledge of
the outcome in such women. Hence in this study, we
performed an audit of all smears reported as ASC and
LSIL, as dened in TBS 2001, over a 3-year period.
The frequency of this diagnosis, rate of follow-up and
the outcome based on correlation with histopathology
were determined. We compared the outcome of
ASCUS and LSIL smears to ascertain the colposcopic
threshold in our setting where HPV testing cannot be
afforded by most women.
Material and methods
A total of 25 203 cervical smears were screened over a
period of 3 years (January 2006 December 2008) in
the Department of Cytology and Gynecological
Pathology, Postgraduate Institute of Medical Educa-
tion and Research, Chandigarh, India. All smears
were conventional smears and no liquid-based prep-
aration was used. All cases of ASC and LSIL smears
were reviewed with the corresponding histological
follow-up. The cytohistological correlation was
recorded for patients with a subsequent histological
tissue examination (i.e. cervical biopsy, conization,
endocervical and or endometrial curettage and hys-
terectomy specimens). The results of the most repre-
sentative of these procedures and with the most serious
histological diagnosis were recorded. All CIN2 and
above lesions (CIN2+) were considered as clinically
signicant.
The smears were collected by trained gynaecologists
or residents-in-training using the Ayres spatula. The
smears were xed in 95% ethyl alcohol, transported
to the laboratory and stained with a standard Papa-
nicolaou stain. In our institution, all the smears are
initially screened by pathology residents-in-training
and all negative as well as positive smears are doubly
screened and diagnosed by the reporting cytopathol-
ogist. The quality assurance monitors were the cyto-
histological correlations with detection of epithelial
abnormality in the biopsy. During the observation
period, the ASC diagnoses in our series were not
qualied further during routine reporting; however,
upon review by two observers (NG and SR), the cases
of ASC were reclassied as those representing ASC-US
(of uncertain signicance) and ASC-H (a high-grade
lesion cannot be excluded) and their outcomes were
compared.
Results
A total of 25 203 conventional cervical smears were
examined during the 3-year observation period. Out
of 25 203 smears, 424 were reported as ASC and 113
as LSIL, constituting 1.7% and 0.4%, respectively.
Additionally, three cases were reported as atypical
cells, unqualied. Follow-up histological data were
available in 156 (36.5%) of those with a cytological
report of ASC or atypical cells, unqualied, and in 50
(44.2%) cases of LSIL. During the same period, 83
smears were reported as HSIL (0.3%), giving an
ASC : SIL ratio of 2.18 : 1.
Atypical squamous cells
Follow-up histology in ASC cases is shown in Table 1.
Overall, clinically signicant cervico-vaginal lesions
were seen in 22 cases (14.4%) of ASC. Clinically
signicant squamous abnormalities were present in 19
(12.4%) cases and included CIN2 (5, 3.3%), CIN3 (9,
5.9%), with associated vulval carcinoma in two cases,
squamous cell carcinoma (SCC) of the cervix (4,
2.6%), and vaginal squamous cell carcinoma (1,
0.7%). Non-squamous cervical abnormalities
included two cases of adenocarcinoma and one case
of small cell carcinoma of the cervix. In addition,
malignant endometrial lesions had been reported
cytologically as atypical cells, unqualied; these
included two cases of endometrial adenocarcinoma
and one of carcinosarcoma. These three cases were
excluded from further analysis.
N. Gupta et al. 2
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The clinical outcome of ASC was analysed with
respect to age and type of lesion. In women less than
35 years of age, ve of 54 clinically signicant
abnormalities were all of squamous cell type com-
pared with 17 of 99 in women over 35 years, among
whom the three non-squamous cell lesions were
found; the difference between age groups was not
statistically signicant (chi-square test, P = 0.094)
(Table 2). Cytology smears initially reported as ASC
were reclassied by two cytopathologists (NG and SR)
as ASC-US and ASC-H. Eleven smears of all ASC
smears were reclassied as ASC-H or ASC-carcinoma
not excluded. Clinically signicant outcome was seen
in 12% (17 142) cases of smears reclassied as ASC-
US as compared with 45.5% (5 11 cases) in the group
ASC-H; the difference was highly signicant (Fishers
exact test, P value 0.010; Table 2).
Incidental ndings in cases of benign histological
outcome included endocervical and endometrial pol-
yps and one case of papillary serous carcinoma of the
ovary (with non-specic cervicitis in the resected
specimen).
Low-grade squamous intraepithelial lesion
Follow-up histology in 50 cases revealed benign
squamous abnormalities (45, 90%) with inadequate
biopsy (n = 5), normal histology (n = 2), chronic
cervicitis with squamous metaplasia (n = 28) and
CINI HPV related changes (n = 10) (Table 1). Clini-
cally signicant abnormalities were observed in ve
(10%) (Table 1) and all were CIN2. Outcome analysis
with respect to age revealed two cases in women less
than 35 years of age and three in women over
35 years; the difference was not signicant.
Discussion
The management of women with low-grade and
equivocal lesions in cervical cytology, which include
ASC and LSIL, remains a problem.
2
A proportion of
women with these changes in cervical smears have
clinically signicant lesions on follow-up whereas the
vast majority of them are innocuous and may there-
fore not require aggressive treatment. The ALTS study
examined three options of repeat cytology, HPV DNA
testing and immediate colposcopy,
3
on the basis of
which the American Society of Colposcopy and
Cervical Pathology has laid down guidelines for their
management.
4
This prompted us to critically examine
our data regarding the follow-up of women with low-
grade cytology to determine the most appropriate
management strategy in a low-resource setting.
A National Cancer Institute-sponsored workshop in
1992 concluded that a diagnosis of ASCUS could be
expected in approximately 5% of screened patients
and that, in general, an acceptable rate of ASCUS
should be two to three times the laboratorys rate of
SIL.
7
The overall rate of ASC in our study was 1.7%
Table 1. Comparison of histological outcome of ASC (n = 153) versus LSIL (n = 50)
Group Follow-up histology ASC cases (%) LSIL cases (%)
Inadequate biopsy 12 (7.8) 5 (10)
Benign squamous abnormalities Normal histology 15 (9.8) 2 (4)
Chronic cervicitis squamous metaplasia 89 (58.2) 28 (56)
CIN1 with HPV changes 15 (9.8) 10 (20)
CIN2 and CIN3 changes CIN2 5 (3.3) 5 (10)
CIN3 9* (5.9) 0
Cervicovaginal cancer Cervix SCC 4 (2.6) 0
Cervical adenocarcinoma 2 (1.3) 0
Small cell carcinoma of the cervix 1 (0.7) 0
Vagina SCC 1 (0.7) 0
*Two cases of CIN3 also had vulval squamous cell carcinoma.
Table 2. Outcome of women with respect to age and
subcategory of ASC
Groups
Total No.
of smears
Clinically
insignicant
on follow-up
Clinically
signicant
on follow-up
Fishers
exact
test,
P value
Age (years)
< 35
54 49 5 0.232
Age (years)
35
99 82 17
ASC-US 142 125 17 0.010
ASC-H 11 6 5
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with an ASC : SIL ratio of 2.18 : 1. The rate of ASC in
our study is comparatively lower than the corre-
sponding rate of the interim Bethesda guidelines and
that reported by Davey et al.;
8
however, the ratio of
ASC to SIL is within the acceptable range. Our lower
rate of ASC may be attributed to the fact that all
smears were signed out by trained expert cytopathol-
ogists. It has been documented in previous studies that
rescreening of smears by expert cytopathologists can
decrease the frequency of ASC as smears may be
downgraded to negative for intraepithelial lesion or
malignancy (NILM) or reactive changes or upgraded
to SIL. Thrall et al. reported that 32% smears were
downgraded to NILM upon review and 9.3%
upgraded to SIL.
9
The population screened included both clinically
symptomatic and asymptomatic women. The percent-
age of ASC cases that were clinically symptomatic was
about 54% and the percentage of symptomatic LSIL
cases was about 67%. Asymptomatic women screened
in the hospital mainly included women who came for
availing family planning services. Hence the observa-
tions and conclusions from this study may not be
relevant for decision making for planning of large
scale population-based screening programmes, where
the smears are initially screened by cytoscreeners.
The follow-up of ASC lesions can show diverse
histology ranging from normal histology to invasive
cancer. After histological follow-up, the rate of normal
and inammatory outcome in ASC cases was found to
be 68% in the present study, which is similar to
previous reports where 6070% of follow-up biopsies
after a diagnosis of ASCUS demonstrated no lesion or
showed inammatory changes.
1012
The rate of squa-
mous intraepithelial lesion or CIN after a diagnosis of
ASC was found to be 19% in the present study,
including almost equal proportions of CIN1 (9.8%)
and CIN2 3 (9.2%). There is a wide range of reported
frequency of CIN in follow-up biopsies of smears
classied as ASCUS or ASC-US. It can range from as
low as 7.7% to as high as 38%.
1117
Clinically
signicant lesions (dened as CIN2+) were observed
in 11.6% cases in the ALTS study,
3
which is compa-
rable to our observations. Similar observations have
been made in other studies.
15,17
We observed invasive
cancers of the cervix (including squamous carcinoma,
adenocarcinomas and others) in smears interpreted as
ASC in 5.2% (8 153) cases. The reported prevalence
of invasive cancer in women with ASC is 0.10.2%.
18
The explanation for the increased rate of invasive
cancers in the follow-up of women with a report of
ASC in our study is the absence of an organized
population-based screening programme for cervical
cancer in India; hence, it is not surprising to nd cases
of invasive cancer when opportunistic hospital-based
screening is performed. It was interesting to note that
only smears categorized as atypical squamous cells
and not as LSIL showed invasive cancers on follow-
up. An occasional adenocarcinoma and small cell
carcinoma was also interpreted as ASC. Generally
these were smears with very few atypical cells in a
haemorrhagic background, making further categori-
zation difcult. Pitman et al.
19
have also reported an
occasional case of endocervical adenocarcinoma
reported as ASC in their series of cases.
The recommendation by TBS 2001
1
to further
classify ASC smears as ASC-US and ASC-H is unfor-
tunately not adhered to in routine day-to-day report-
ing by many pathologists. Following reclassication of
all smears reported as ASCUS, 2.6% of smears were
reclassied as ASC-H and revealed a clinically signif-
icant outcome (CIN2+) in 45.4% (5 11 cases) com-
pared with 12% (17 142) cases of ASC-US. The
usefulness of ASC-H as a better predictor of CIN2+
compared with ASC-US is well documented in several
studies and we reiterate this observation.
20,21
There is
no doubt that a report of ASC-H warrants immediate
colposcopic evaluation and biopsy.
Follow-up of LSIL lesions revealed CIN2 in 10% of
cases with follow-up histology whereas nearly twice
this number had CIN1 with HPV-associated changes.
The prevalence of CIN2+ identied at initial colpos-
copy among women with LSIL was 1216% in various
studies,
3,15,22
which is comparable to our study.
Interestingly, we did not observe any case of invasive
cancer or even CIN3 in the follow-up of women with
a cytology smear report of LSIL, implying that the
cytomorphological features of LSIL are more likely to
represent CIN1 in the vast majority and CIN2 in a
minority of cases. Whereas the merging of CIN2 and
CIN3 into a single category of HSIL in TBS provides
better reproducibility of cytology reports and facili-
tates comparison across laboratories, it may be worth-
while to separate the two at least on histology. This is
because the outcome of CIN2 is in between the
innocuous CIN1, which almost always regresses, and
CIN3, which has a high rate of progression.
23
The
importance of making this distinction has also been
highlighted recently by others as well.
24
The ASCUS and LSIL Triage Study (ALTS) found
HPV DNA testing to be an effective strategy to triage
women with equivocal or ASCUS cytology.
3,4,23
On
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the other hand, for women with LSIL, this approach
was not satisfactory due to the high frequency of HPV
positivity.
26
The cost-effectiveness of HPV DNA testing
for triaging women with ASCUS was also highlighted
by another study.
27
However, this approach works
well in settings where HPV DNA testing is less
expensive than colposcopy. In our hospital as well as
several other government-aided hospitals across the
country where colposcopy is available, the latter is
eight to ten times less expensive. Colposcopy costs
approximately 100 Rupees (2 US$) whereas HPV
testing costs 800 Rupees (16US$). Hence it is impor-
tant to determine the threshold for colposcopy. In a
study by Sodhani et al.,
6
the sensitivity of
LSIL + cytology to detect CIN2+ was 91.5% (referral
load, 30.7%). While the sensitivity of ASCUS + cytol-
ogy threshold was almost the same (92.3%), the
referral load was much higher (42.2%), indicating
that the threshold for colposcopic referrals should be
LSIL cytology. From our data, while 10% of women
with LSIL had CIN2, 14.4% of those with ASC had
CIN2+ and 17 of 22 were found to have CIN3 or
cancer. If LSIL had been applied as the threshold for
colposcopic referral, all the CIN3+ cases would have
been missed unless cases of ASC-H had been identi-
ed. This implies that the threshold for colposcopic
referral should be ASC and not LSIL in a low-resource
setting such as ours where HPV testing is not afford-
able. A few reports in the Chinese language literature
have also recommended colposcopy for all women
with ASCUS and above smears.
28,29
Further 3 , in most
centres across India, a cervical smear is a once-in-a-
lifetime test for women who attend the gynaecology
outpatient department of a city hospital for various
reasons. Follow-up rates are abysmal and the call-
recall system practically non-existent. In such a
scenario, the gynaecologists also would like to play it
safe and advise colposcopy in all women with an
abnormal cytology tests (ASCUS and above) rather
than advise a repeat cytology test and lose the patient
to follow-up. Follow-up histological data in the
present study were available only in 36.5% cases
with cytological report of ASC or atypical cells,
unqualied, and in 44.2% cases of LSIL, which re-
enforces the message of a low threshold for referral.
Cytopathologists should be aware that ASC reports
should be audited carefully and not overused in order
to avoid unnecessary colposcopy and further man-
agement. It is also strongly recommended that cytop-
athologists must subclassify ASC smears as ASC-US or
ASC-H, with immediate colposcopy recommended for
the latter. It is hoped that this study along with
experience from other centres will help evolve appro-
priate management strategies for women with a
cervical smear report of ASC in developing countries
with limited resources.
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26. Schiffman M, Solomon D. Findings to date from the
ASCUS-LSIL Triage Study (ALTS). Arch Pathol Lab Med
2003;127:9469.
27. Kulasingam SL, Kim JJ, Lawrence WF et al. Cost-
effectiveness analysis based on the atypical squamous
cells of undetermined signicance low grade squamous
intraepithelial lesion. Triage Study (ALTS). J Natl Cancer
Inst 2006;98:823.
28. Liu J, Song XH, Wang QX. Clinical signicance of
atypical squamous cells and low grade squamous intra-
epithelial lesions in cervical smear. Zhonghua Yi Xue Za
Zhi 2007;87:17646.
29. Wang Y. Comment on diagnosis of atypical squamous
cells using new the Bethesda system 2001. Zhonghua Fu
Chan Ke Za Zhi 2004;39:279.
N. Gupta et al. 6
Cytopathology 2010 2010 Blackwell Publishing Ltd
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Author Query Form
Journal: CYT
Article: 780
Dear Author,
During the copy-editing of your paper, the following queries arose. Please respond to these by marking up your
proofs with the necessary changes/additions. Please write your answers on the query sheet if there is insufcient space
on the page proofs. Please write clearly and follow the conventions shown on the attached corrections sheet. If
returning the proof by fax do not write too close to the papers edge. Please remember that illegible mark-ups may
delay publication.
Many thanks for your assistance.
Query
reference
Query Remarks
1 AUTHOR: Does this sentence read correctly now? CIN3 and
invasive squamous carcinomas were seen in 5.9% and 1.4%,
respectively, of cases of ASC, and not seen in LSIL
2 AUTHOR: Does this sentence read correctly now? Atypical
squamous cells is dened as an epithelial cell abnormality that
exceeds those abnormalities attributable to a benign reactive
process but falls short of a diagnosis of SIL.
3 AUTHOR: Does this read correctly now? Further, in most
centres across India, a cervical smear is a once-in-a-lifetime
test for women who attend the gynaecology outpatient
department of a city hospital for various reasons.
4 AUTHOR: Please provide the volume number for reference [5].
5 AUTHOR: Please provide the page range for reference [10].
6 AUTHOR: Reference [25] has not been cited in the text. Please
indicate where it should be cited; or delete from the Reference
List and renumber the References in the text and Reference
List.


Page 1 of 3

USING E-ANNOTATION TOOLS FOR ELECTRONIC PROOF CORRECTION
Required Software
Adobe Acrobat Professional or Acrobat Reader (version 7.0 or above) is required to e-annotate PDFs.
Acrobat 8 Reader is a free download: http://www.adobe.com/products/acrobat/readstep2.html
Once you have Acrobat Reader 8 on your PC and open the proof, you will see the Commenting Toolbar (if it
does not appear automatically go to Tools>Commenting>Commenting Toolbar). The Commenting Toolbar
looks like this:

If you experience problems annotating files in Adobe Acrobat Reader 9 then you may need to change a
preference setting in order to edit.
In the Documents category under Edit Preferences, please select the category Documents and
change the setting PDF/A mode: to Never.

Note Tool For making notes at specific points in the text
Marks a point on the paper where a note or question needs to be addressed.

Replacement text tool For deleting one word/section of text and replacing it
Strikes red line through text and opens up a replacement text box.

Cross out text tool For deleting text when there is nothing to replace selection
Strikes through text in a red line.


How to use it:
1. Right click into area of either inserted
text or relevance to note
2. Select Add Note and a yellow speech
bubble symbol and text box will appear
3. Type comment into the text box
4. Click the X in the top right hand corner
of the note box to close.

How to use it:
1. Select cursor from toolbar
2. Highlight word or sentence
3. Right click
4. Select Replace Text (Comment) option
5. Type replacement text in blue box
6. Click outside of the blue box to close

How to use it:
1. Select cursor from toolbar
2. Highlight word or sentence
3. Right click
4. Select Cross Out Text



Page 2 of 3

Approved tool For approving a proof and that no corrections at all are required.


Highlight tool For highlighting selection that should be changed to bold or italic.
Highlights text in yellow and opens up a text box.

Attach File Tool For inserting large amounts of text or replacement figures as a files.
Inserts symbol and speech bubble where a file has been inserted.


Pencil tool For circling parts of figures or making freeform marks
Creates freeform shapes with a pencil tool. Particularly with graphics within the proof it may be useful to use
the Drawing Markups toolbar. These tools allow you to draw circles, lines and comment on these marks.










How to use it:
1. Click on the Stamp Tool in the toolbar
2. Select the Approved rubber stamp from
the standard business selection
3. Click on the text where you want to rubber
stamp to appear (usually first page)

How to use it:
1. Select Highlighter Tool from the
commenting toolbar
2. Highlight the desired text
3. Add a note detailing the required change

How to use it:
1. Select Tools > Drawing Markups > Pencil Tool
2. Draw with the cursor
3. Multiple pieces of pencil annotation can be grouped together
4. Once finished, move the cursor over the shape until an arrowhead appears
and right click
5. Select Open Pop-Up Note and type in a details of required change
6. Click the X in the top right hand corner of the note box to close.
How to use it:
1. Click on paperclip icon in the commenting toolbar
2. Click where you want to insert the attachment
3. Select the saved file from your PC/network
4. Select appearance of icon (paperclip, graph, attachment or
tag) and close